Proximity interaction analysis

Abstract

The present disclosure relates to methods for assessing the identity and spatial relationship between a polypeptide and a portion in a sample. In some embodiments, the polypeptide and the portion are both parts of a larger polypeptide, and the methods of the present invention can be used to assess the identity and spatial relationship between the polypeptide and the portion in the same polypeptide or protein. In other embodiments, the polypeptide and the portion belong to different molecules, and the methods can be used to assess the identity and spatial relationship between the polypeptide and the portion in different molecules (e.g., protein-protein complexes, protein-DNA complexes, or protein-RNA complexes).

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Patent Application No. 62 / 726,933 filed on September 4, 2018, U.S. Provisional Patent Application No. 62 / 726,959 filed on September 4, 2018, and U.S. Provisional Patent Application No. 62 / 812,861 filed on March 1, 2019, the disclosures and contents of which are incorporated by reference in their entirety for all purposes.

[0003] Submit sequence listing as ASCII text file

[0004] The following submissions on ASCII text files are incorporated herein by reference in their entirety: Computer Readable Form (CRF) of the Sequence Listing (File Name: 4614-2000940_SeqList_ST25_20190829; Recording Date: August 29, 2019; Size: 1021 bytes). Technical Field

[0005] The present disclosure relates to methods for assessing the identity and spatial relationship between a polypeptide and a moiety in a sample. In certain embodiments, the polypeptide and the moiety are both parts of a larger polypeptide, and the methods of the present invention can be used to assess the identity and spatial relationship between the polypeptide and the moiety in the same polypeptide or protein. In other embodiments, the polypeptide and the moiety belong to different molecules, and the methods can be used to assess the identity and spatial relationship between the polypeptide and the moiety in different molecules (e.g., protein-protein complexes, protein-DNA complexes or protein-RNA complexes). Background Art

[0006] Protein plays a key role in cell and organism physiology. Proteomics is the study of protein on a global level, including measuring protein abundance, protein interactions and protein modifications. These protein measurements illustrate how proteins are used in cells, tissues and organisms. In addition, the identification of protein markers in tissues or body fluids such as blood or plasma can be used as a prognosis or diagnostic assay reflecting a specific disease or condition state, and provides a means for monitoring disease or condition progression. The measurement of protein in plasma is particularly useful because blood soaks most of the tissues in the body, and potential protein biomarkers are collected from cells and tissues throughout the body. The main challenge in proteomics is that the overall analysis of proteins is difficult, and current tools are largely inadequate. In addition, the most common method of proteomics analysis, i.e., bottom-up peptide sequencing using mass spectrometry, first digests intact polypeptides into peptides, which are then analyzed in LC-MS / MS. Digestion of polypeptides into peptides destroys protein-protein interactions, and destroys the single-molecule information of the precise combination identity of post-translational modifications (PTMs) on a given molecule, i.e., destroys protein form information. Top-down mass spectrometry has been used to resolve proteoforms, but still has many limitations (Kilpatrick and Kilpatrick 2017). Therefore, a robust technique is needed that preserves information about protein-protein interactions and about single-molecule proteoforms (especially about the combination of PTMs for a given molecule).

[0007] Therefore, there remains a need in the art for improved techniques related to assessing or analyzing the identity and spatial relationships between polypeptides and moieties in a sample. The present disclosure satisfies these and other needs.

[0008] Various aspects of the invention will be readily understood with reference to the following detailed description. To this end, various references are set forth herein that describe in more detail certain background information, processes, compounds and / or compositions, and each is incorporated herein by reference in its entirety. Summary of the invention

[0009] This summary is not intended to be used to limit the scope of the claimed subject matter. Other features, details, utilities, and advantages of the claimed subject matter will become apparent from the detailed description including those aspects disclosed in the accompanying drawings and appended claims.

[0010] In one aspect, the present disclosure provides a method for assessing the identity and spatial relationship between a polypeptide and a portion in a sample, the method comprising: a) forming a connection structure between a site of the polypeptide in the sample and a site of a portion in the sample, the connection structure comprising a polypeptide tag associated with the site of the polypeptide and a portion tag associated with the site of the portion, wherein the polypeptide tag and the portion tag are associated; b) transferring information between the associated polypeptide tag and the portion tag or connecting the associated polypeptide tag and the portion tag to form a shared unique molecular identifier (UMI) and / or barcode; c) breaking the linkage structure by dissociating the polypeptide from the portion and the polypeptide tag from the portion tag while maintaining the association between the polypeptide and the polypeptide tag and maintaining the association between the portion and the portion tag; and d) evaluating at least a partial sequence of the polypeptide tag and the polypeptide, and evaluating at least a partial identity of the portion tag and the portion; wherein the polypeptide tag and the evaluated portion of the portion tag comprise a shared unique molecular identifier (UMI) and / or barcode that indicates spatial proximity of a site of the polypeptide and a site of the portion in the sample.

[0011] In one aspect, the present disclosure provides a method for assessing the identity and spatial relationship between a polypeptide and a moiety in a sample, the method comprising: a) providing a preassembled structure, the preassembled structure comprising a shared unique molecular identifier (UMI) and / or barcode in the middle portion, the UMI and / or barcode flanked on one side by a polypeptide tag and on the other side by a moiety tag (moiety b) forming a connection structure between a site of a polypeptide in a sample and a site of a certain part in the sample by associating the polypeptide tag of the preassembled structure with the site of the polypeptide and associating the partial tag of the preassembled structure with the site of the part; c) destroying the connection structure by dissociating the polypeptide from the part and the polypeptide tag from the partial tag while maintaining the association between the polypeptide and the polypeptide tag and maintaining the association between the certain part and the partial tag; and d) evaluating at least a partial sequence of the polypeptide tag and the polypeptide, and evaluating at least a partial identity of the partial tag and the part; wherein the evaluated portions of the polypeptide tag and the partial tag comprise a shared unique molecular identifier (UMI) and / or barcode that indicates the spatial proximity of the site of the polypeptide and the site of the part in the sample.

[0012] Also provided herein are methods for assessing the identity and spatial relationship between a polypeptide and a moiety in a sample, the method comprising: a) forming a junction structure between a site of a polypeptide in the sample and a site of a moiety in the sample, the junction structure comprising a polypeptide tag associated with the site of the polypeptide and a moiety tag associated with the site of the moiety, wherein the polypeptide tag and the moiety tag are associated; b) transferring information between the associated polypeptide tag and the moiety tag to form a shared unique molecular identifier (UMI) and / or barcode, wherein the shared UMI and / or barcode is formed as a separate record polynucleotide; c) disrupting the junction structure by dissociating the polypeptide from the moiety and dissociating the polypeptide tag from the moiety tag while maintaining the association between the polypeptide and the polypeptide tag and maintaining the association between the moiety and the moiety tag; d) assessing at least a partial sequence of the polypeptide tag and the polypeptide, and assessing at least a partial identity of the moiety tag and the moiety; and e) assessing the separate record polynucleotide to establish a spatial relationship between the site of the polypeptide and the site of the moiety.

[0013] In some embodiments, the principles of the present methods and compositions can be applied, or can be adapted for application, to polypeptide analysis assays known in the art or in related applications. For example, the principles of the present methods and compositions may be applied or may be adapted for application to compositions, kits and methods disclosed and / or claimed in U.S. Provisional Patent Application Nos. 62 / 330,841, 62 / 339,071, 62 / 376,886, 62 / 579,844, 62 / 582,312, 62 / 583,448, 62 / 579,870, 62 / 579,840, 62 / 582,916, International Patent Application Publication Nos. WO 2019 / 089836, WO 2019 / 089846, WO 2019 / 089851, and International Patent Application No. PCT / US2017 / 030702 (published as WO2017 / 192633 A1). BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Non-limiting embodiments of the present invention are described by way of example with reference to the accompanying drawings, which are schematic and are not intended to be drawn to scale. For illustrative purposes, not every component is marked in each figure, nor are every component not necessary for a person of ordinary skill in the art to understand the present invention shown in each embodiment of the present invention.

[0015] Figure 1An exemplary workflow for association by proximity labeling is shown. The proximity of peptide regions within a polypeptide or between associated proteins can be recorded and, after digestion into peptide fragments and ProteoCode sequencing (see, e.g., U.S. Provisional Patent Application Nos. 62 / 330,841, 62 / 339,071, 62 / 376,886, 62 / 579,844, 62 / 582,312, 62 / 583,448, 62 / 579,870, 62 / 579,840, and 62 / 582,916, International Patent Application Publication Nos. WO 2019 / 089836, WO 2019 / 089846, WO 2019 / 089851, and International Patent Application No. PCT / US2017 / 030702, which is filed under WO 2017 / 192633 A1 discloses that shared UMIs can be used to map "neighboring peptides". (A) A protein sample including a protein complex having a P polypeptide and an M portion (in this case, another polypeptide) is labeled using a DNA tag. (B) Neighboring DNA tags (within a polypeptide and between P and M polypeptide units) are allowed to interact and exchange information. In the example shown, primer extension is used to transfer information between neighboring tags or from one tag to another. (C) The protein complex is dissociated and reactive amino acid residues such as cysteine ​​and lysine are capped. (D) Denatured polypeptides are digested using an endoprotease such as trypsin. (E) The resulting peptide fragments contain various types of fragments, including peptides labeled with neighboring recorded tags (rTags) containing shared UMI information, peptides labeled with recorded tags (w / o shared UMI information), and unlabeled peptides. (F) The rTag-labeled peptides are immobilized on a suitable sequencing substrate for ProteoCode peptide sequencing. (G) ProteoCode peptide sequencing is completed to determine neighboring associated peptides by identifying shared UMI sequences.

[0016] Figure 2Exemplary formats and designs of proximity coding tags are shown. (A) DNA proximity coding tags for double-sided proximity extension coding. (B) DNA proximity coding tags for single-sided proximity extension coding. (C) DNA proximity coding tags for proximity ligation coding. (D) DNA proximity coding tags for proximity ligation (alternating with exogenous UMI sequences). (E) DNA tags containing UMIs are attached to P (or M). Complementary primers at the 3' portion of the DNA tag hybridize with the DNA tag attached to P. The complementary tag contains an optional UMI and a conjugation functional element (BP-benzophenone in the example shown). The BP element is attached to the M region, and a subsequent primer extension step transfers the UMI information. Similar sequences in the event of hybridization or ligation and subsequent functional conjugation with M can be used in schemes BD. (F) Schematic diagram of multi-point attachment. The DNA tags can be pre-hybridized before conjugation to the PM complex, or can be conjugated first and then hybridized. The information is transferred from the P tag to the two M tags by primer extension. Other methods can also be used, including ligation, double-stranded and single-stranded ligation.

[0017] Figure 3 Exemplary proximity encoding of macromolecules and macromolecular complexes by DNA tagging and proximity extension is illustrated. (A) DNA tags with embedded barcodes / UMIs are attached to polypeptide molecules. Proximity extension between adjacent DNA tags results in unidirectional or bidirectional information transfer between tags (depending on the tag design). The end result is that adjacent DNA tag sites share UMI / barcode information. The polypeptide is then cleaved into peptide fragments, many of which are labeled with DNA tags containing proximity UMI information (B). (B) Protein complexes can be labeled with UMI / barcode DNA tags, allowing the tags to exchange information through proximity extension. The dotted line illustrates an extended DNA tag containing shared UMI / barcode information. The shared UMI information can then be used to reconstruct the identity of the interacting proteins (i.e., A interacts with B).

[0018] Figure 4 Illustrated are exemplary proximity encoding of macromolecules and macromolecular complexes by DNA crosslinking with a DNA crosslinker containing a UMI / barcode. (A) A DNA crosslinker containing a UMI / barcode sequence and benzophenone (BP) for coupling to a polypeptide backbone. The BP DNA crosslinker crosslinks two adjacent sites on a polypeptide. BP is shown for illustrative purposes (Park, Koh et al. 2016), but any chemical conjugation reagent that reacts with a peptide backbone or amino acid side chains can be used (Hermanson 2013). After cleavage into peptides, a subset of peptides are labeled with adjacent DNA tags that share UMI information. (B) A DNA crosslinker with a UMI is used to label adjacent sites in a protein complex. After labeling, adjacent proteins contain DNA tags that share UMI information.

[0019] Figure 5 An exemplary sequence design of a proximal DNA crosslinker is shown. Boxes P and M show attachment to the P polypeptide and M moiety, respectively, and should be understood to be present throughout the description. (A) Design of a DNA tag that can be proximal extended and formatted to serve as a "record tag" for downstream ProteoCode peptide / protein analysis. (B) The tag shown uses BP to mark the peptide site, but any chemically reactive group of the peptide backbone or peptide amino acid residue can be used. The sequence structure of the double-stranded DNA crosslinker is shown to have different sequence elements for conversion into a record tag. F1-Forward primer sequence with built-in restriction enzyme (RE) site, Sp1=Spacer 1 for priming, Sp2=Spacer 2 for priming, UMI=Unique Molecular Identifier, abbreviation represents complement sequence. The double-stranded DNA crosslink tag is constructed by annealing two oligonucleotides, one containing the UMI and the other capable of priming on the UMI oligonucleotide. The primer extension step writes the UMI to the other strand, generating a dsDNA crosslink tag. Restriction enzyme digestion can be used to remove the region of the crosslinked tag to prepare it in a "record tag" format. (C) After immobilizing the DNA-tagged peptides on the sequencing substrate, Sp1 and Sp2 sequences can be converted into Sp sequences (recording the tag structure) for NGPS sequencing analysis.

[0020] Figure 6 Shown is a design for direct chemical immobilization or hybridization / connection of immobilized DNA tags on sequencing substrates. The joint between the DNA tag and the peptide can be attached to the 5' end (A) or attached to the DNA (B) by internal connection. In the example shown in CE, an internal joint is used to achieve effective hybridization of the 5' phosphorylated end of the DNA tag with the DNA hairpin capture probe on the sequencing substrate. (CE) The peptide with the attached DNA tag is annealed to the sequencing substrate by a fixed DNA capture probe. After annealing, the DNA record tag is connected to the surface capture probe.

[0021] Figure 7An exemplary workflow for association by proximity labeling is shown. (A) A protein sample including a protein complex having a P, polypeptide, and an M, moiety (in this case another polypeptide) is labeled using DNA tags. (B) The proximity DNA tags (within the polypeptide and between the P and M polypeptide units) are allowed to interact. In the example shown, primer extension is used to transfer information between the polypeptide tag and the moiety tag to produce a separated transcript polynucleotide. (C) The protein complex is dissociated, and active amino acid residues such as cysteine ​​and lysine are optionally capped. (D) The denatured polypeptide is digested using an endoprotease. (E) The resulting peptide fragments contain various types of fragments, including peptides labeled with proximity transcript tags (rTags) containing shared UMI information, peptides labeled with transcript tags (w / o shared UMI information), unlabeled peptides, and separated transcript polynucleotides. (F) The separated transcript polynucleotides are collected and analyzed, and the rTag-labeled peptides are immobilized on a suitable sequencing substrate for ProteoCode peptide sequencing. (G) ProteoCode peptide sequencing was performed to identify neighboring associated peptides by identifying the consensus UMI sequences.

[0022] FIG8 depicts a ligation-based proximity cycle. The polypeptide and moiety are labeled with a DNA tag, which is used for primer extension to generate a double-stranded DNA tag product ( Figures 8A-8B ). The connection thermal cycle generates a record that provides information about the proximity of the polypeptide to the moiety ( Figure 8C-8D ).

[0023] Figures 9A-9C Depicted is the production of a record polynucleotide separated from a polypeptide tag and from one or more moiety tags. In an exemplary embodiment, the polypeptide is spatially adjacent to a first moiety (M1) and a second moiety (M2). Two or more separated record polynucleotides are formed in a paired connection structure, which indicates that P is spatially adjacent to M1 and M2. In addition, further separated record polynucleotides are formed between M1 and M3 or M2 and M4, indicating that M1 and M3 and M2 and M4 are spatially adjacent. In some embodiments, spatially adjacent polypeptides and one or more moieties (e.g., P-M1-M3) are indicated by indirect or overlapping information from one or more separated record polynucleotides ( Fig. 9C ).

[0024] Figures 10A-10B An exemplary model system for labeling proximity molecules and protein analysis is depicted. Fig. 10A(Top left) Three molecules are shown in schematic form: DNA 1, DNA 2, and a peptide (K(biotin)GSGSK(N3)GSGSRFAGVAMPGAEDDVVGSGS-K(N3)-NH2 as specified in SEQ ID NO:1). These components are used in Example 7 to construct a model connection structure between the site of the polypeptide and the site of the portion. The 5' end of DNA1 consists of a 24nt sequence designed to hybridize with DNA1', which is a complementary capture sequence attached to the beads. UMI-1 is a randomized sequence used as a unique molecular identifier; sp is a spacer sequence used to connect the capping sequence and the coding sequence that enables NGS sequencing; "U" represents a uracil base, which can be cut to remove the downstream PEG linker-sp'-UMI-1'-OL' sequence after information transfer from DNA 1 to DNA2. This part is used for information transfer from DNA 1 to DNA 2 and / or to form a connection structure between DNA 1 and DNA 2. The removal after the transfer eliminates the complementarity generated between DNA 1 and DNA 2 due to the information transfer, allowing the DNA 1-part and the DNA2-peptide complex to be separated under mild conditions after trypsin cleavage. This enables trypsin cleavage and subsequent hybridization and connection of the DNA 2-peptide complex with the DNA 2' capture sequence to be carried out under mild, homogeneous conditions. The OL' sequence at the 3' end of DNA 1 is complementary to the OL at the 3' end of DNA 2, allowing the polymerase to extend DNA 2 using DNA1 as a template. Replication terminates at the PEG linker. The 5' end of DNA 2 consists of a 24nt sequence designed to hybridize with DNA 2', which is a complementary capture sequence attached to the beads. The peptide contains a single phenylalanine (F) immediately downstream of a single trypsin cleavage site. In this way, trypsin treatment can produce two subpeptides. For the purpose of illustration, these are referred to as model peptides containing F at the amino terminus in Example 1, and model parts containing biotin connected to lysine (K) at the N terminus. DNA 1 and DNA 2 each contain DBCO (not shown in the schematic diagram) so that it can be connected to the N3 (azide) part in the peptide by a suitable method such as click chemistry, as shown in the upper middle figure. The upper right figure and the lower left figure show beads containing DNA 1 and DNA 2 capture sequence mixtures (not distinguished in the figure). In the lower left figure, the DNA 1-DNA 2-peptide complex captured on the beads by the DNA1 capture sequence is shown. By temporarily blocking the DNA 2 'capture sequence in this capture step, the capture by DNA 1 is completed instead of by DNA 2. After the capture complex, information transfer occurs by intramolecular extension (i.e. in a single DNA 1-DNA 2-peptide complex), as shown in the following middle figure. In the lower right figure, USER cutting and washing remove the complementary region produced by intramolecular extension from DNA 1.This enables the peptide-DNA 2 fragment to be released under mild conditions after trypsinization.

[0025] Fig. 10B For continuity purposes, the Fig. 10A . Fig. 10B Shown in the top middle are moiety-DNA 1 and peptide-DNA 2 complexes captured by their respective DNA 1' and DNA 2' capture sequences attached to a solid support. The top right and bottom middle panels show the encoding process for evaluating polypeptide sequences and moieties, where seqA and seqB recognize moiety (biotin, "B") and peptide (phenylalanine, "F") binders, respectively. The bottom right panel shows the capping step, which uses the sp sequence to add R1, the capping sequence, to enable subsequent sequence analysis by NGS. DETAILED DESCRIPTION

[0026] In order to provide a thorough understanding of the present disclosure, many specific details are set forth in the following description. These details are provided for illustrative purposes, and the claimed subject matter can be implemented according to the claims without some or all of these specific details. It should be understood that other embodiments can be used and structural changes can be made without departing from the scope of the claimed subject matter. It should be understood that the various features and functions described in one or more separate embodiments are not limited to the specific embodiments in which they are described in terms of their applicability. Whether or not such an embodiment is described, and whether or not such features are presented as part of the described embodiment, they can be applied to one or more other embodiments of the present disclosure individually or in some combination. For the sake of clarity, technical materials known in the technical field related to the claimed subject matter are not described in detail, so as not to unnecessarily obscure the claimed subject matter.

[0027] All publications, including patent documents, scientific papers and databases, mentioned in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication was individually incorporated by reference. Citation of these publications or documents is not intended to be an admission that any of them are relevant prior art, nor does it constitute any admission as to the contents or date of these publications or documents.

[0028] All headings are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading unless so specified.

[0029] Unless otherwise indicated, the implementation of the provided embodiments will employ conventional techniques and descriptions of organic chemistry, polymer technology, molecular biology (including recombinant techniques), cell biology, biochemistry, and sequencing techniques, which are within the skill of those skilled in the art. These conventional techniques include polypeptide and protein synthesis and modification, polynucleotide and / or oligonucleotide synthesis and modification, polymer array synthesis, hybridization and connection of polynucleotides and / or oligonucleotides, hybridization detection, and nucleotide sequencing. The specific description of suitable techniques can be referenced to the examples herein. However, other equivalent routine procedures may certainly be used. Such routine techniques and descriptions can be found in standard laboratory manuals, such as Green et al., Eds., Genome Analysis: A Laboratory Manual Series (Volumes I-IV) (1999); Weiner, Gabriel, Stephens, Eds., Genetic Variation: A Laboratory Manual (2007); Dieffenbach, Dveksler, Eds., PCR Primer: A Laboratory Manual (2003); Bowtell and Sambrook, DNA Microarrays: A Molecular Cloning Manual (2003); Mount, Bioinformatics: Sequence and Genome Analysis (2004); Sambrook and Russell, Condensed Protocols from Molecular Cloning: A Laboratory Manual (2003); Manual (2006); and Sambrook and Russell, Molecular Cloning: A Laboratory Manual (2002) (all from Cold Spring Harbor Laboratory Press); Ausubel et al., eds., Current Protocols in Molecular Biology (1987); T. Brown ed., Essential Molecular Biology (1991), IRL Press; Goeddel ed., Gene Expression Technology (1991), Academic Press; A. Bothwell et al. eds., Methods for Cloning and Analysis of Eukaryotic Genes (1990), Bartlett Publ.; M. Kriegler, Gene Transfer and Expression (1990), Stockton Press; R. Wu et al., eds., Recombinant DNA Methodology (1989), Academic Press; M. McPherson et al., PCR: A Practical Approach (1991), IRL Press at Oxford University Press; Stryer, Biochemistry (4th Ed.) (1995), WH Freeman, New York NY; Gait, Oligonucleotide Synthesis: A Practical Approach (2002), IRL Press, London; Nelson and Cox, Lehninger, Principles of Biochemistry (2000) 3rd Ed., WH Freeman Pub., New York, NY; Berg, et al., Biochemistry (2002) 5th Ed., WH Freeman Pub., New York, NY, all of which are incorporated herein by reference in their entirety.

[0030] Provided herein are methods and approaches for assessing the spatial relationship between a polypeptide and one or more parts in a sample. In some embodiments, the method provided also includes macromolecular analysis, identification and / or sequencing. In some embodiments, the spatial relationship between a polypeptide and a part is assessed by forming a connecting structure between the site of the polypeptide in the sample and the site of the part in the sample. In some embodiments, the connecting structure comprises a polypeptide tag associated with the site of the polypeptide and a partial tag associated with the site of the part, wherein the polypeptide tag and the partial tag are associated. In some embodiments, the method further comprises assessing polypeptide tags and partial tags. In some cases, the sequence (e.g., partial sequence (partial sequence)) and the identity (e.g., partial sequence or identity) of a part (moiety) are determined using multiple macromolecular binding assays to determine the sequence of the polypeptide tag. In some embodiments, the information from the macromolecular binding assay is converted into a nucleic acid molecule library in conjunction with the assay for reading out by next generation sequencing.

[0031] Existing methods for determining molecular interactions occurring in biological systems include imaging and microscopy techniques, e.g. Or fluorescence resonance energy transfer (FRET) technology.Other biochemical assays measuring protein interactions include yeast two-hybrid assays, affinity purification assays, mass spectrometry and co-immunoprecipitation techniques.However, there is still a need for improved techniques for assessing the spatial interactions of high-throughput macromolecules (e.g., polypeptides or polynucleotides), and more than one interaction between various molecules that can detect and can also provide the identity / sequence of molecules in samples, and the products, related methods and kits needed to achieve these.In certain embodiments, it is necessary to assess the identity of molecules and assess accurate, sensitive and / or high-throughput spatial relationships.In certain embodiments, the method provided allows assessment, analysis and / or sequencing that overcomes constraints, to achieve accurate, sensitive and / or high-throughput assessment of the spatial relationship between molecules and the identity (e.g., sequence) of molecules.

[0032] In some cases, the provided method allows the identification of adjacent molecules without the need for specific binding reagents, so as to detect molecular targets that require information about spatial interactions. In some instances, the provided method for assessing spatial proximity does not require a specific target binding portion, such as an antibody or its binding fragment to bind to a specific molecular target. In certain embodiments, the disclosure provides, in part, methods for analyzing the proximity of molecules (e.g., proteins, polypeptides, parts, etc.), for assessing interactions between molecules, and / or for mapping interactions between two or more molecules. In certain embodiments, the provided method comprises connecting polypeptide tags and partial tags that can bind to a variety of polypeptides and parts. In certain embodiments, exemplary advantages of the provided method include the ability to assess the interactions of many adjacent molecules (e.g., polypeptides and parts) in a sample.

[0033] In some embodiments, the target polypeptide is a part of a larger polypeptide, and the part is also a part of the same larger polypeptide. In some embodiments, the method provided is used to analyze a polypeptide and a part that are all a part of a larger polypeptide, and the analysis can be used for applications in sequencing. In some embodiments, the method includes assessing at least a partial sequence of the polypeptide and the part. In some cases, the sequence information of the polypeptide and the part can be used to identify peptide sequence matches. In some instances, the method provided allows to increase confidence and / or accuracy of sequencing applications (including mapping the sequence to the polypeptide).

[0034] In some embodiments, the provided methods can provide the benefit that shorter and / or less accurate sequences can be used than longer and / or more accurate sequences that may be required using methods for identifying proteins without information of neighboring molecules. In some embodiments, the provided methods can be used with physical partitioning. In some embodiments, the provided methods allow networks to be constructed using proximity information such that physical partitioning is not required.

[0035] definition

[0036] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present disclosure belongs. If the definitions set forth in this section are contrary to or inconsistent with the definitions set forth in the patents, applications, published applications, and other publications incorporated herein by reference, the definitions set forth in this section take precedence over the definitions incorporated herein by reference.

[0037] As used herein, the singular forms "a," "an," and "the" include plural references unless otherwise indicated. Thus, for example, reference to "a peptide" includes one or more peptides or mixtures of peptides. Furthermore, unless otherwise indicated or obvious from the context, as used herein, the term "or" should be understood to be inclusive and encompass both "or" and "and."

[0038] As used herein, the term "macromolecule" encompasses large molecules composed of smaller subunits. Examples of macromolecules include, but are not limited to, peptides, polypeptides, proteins, nucleic acids, carbohydrates, lipids, macrocyclic compounds. Macromolecules also include chimeric macromolecules, which are composed of a combination of two or more types of macromolecules covalently linked together (e.g., peptides linked to nucleic acids). Macromolecules may also include "macromolecular assemblies," which are composed of non-covalent complexes of two or more macromolecules. Macromolecular assemblies can be composed of the same type of macromolecule (e.g., protein-protein) or two different types of macromolecules (e.g., protein-DNA).

[0039] As used herein, the term "polypeptide" includes peptides and proteins, and refers to a molecule comprising a chain of two or more amino acids connected by peptide bonds. In some embodiments, the polypeptide comprises 2 to 50 amino acids, for example, having more than 20-30 amino acids. In some embodiments, the peptide does not contain secondary, tertiary or higher structures. In some embodiments, the polypeptide is a protein. In some embodiments, the protein comprises 30 or more amino acids, for example, having more than 50 amino acids. In some embodiments, in addition to the primary structure, the protein also comprises secondary, tertiary or higher structures. The amino acids of the polypeptide are most typically L-amino acids, but can also be D-amino acids, modified amino acids, amino acid analogs, amino acid mimetics, or any combination thereof. The polypeptide can be naturally occurring, synthetically produced, or recombinantly expressed. The polypeptide can be synthetically produced, isolated, recombinantly expressed, or produced by a combination of the above methods. The polypeptide can also contain other groups that modify the amino acid chain, for example, functional groups added by post-translational modification. The polymer can be linear or branched, it can contain modified amino acids, and it can be interrupted by non-amino acids. The term also encompasses an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component.

[0040] As used herein, the term "amino acid" refers to an organic compound comprising an amine group, a carboxylic acid group, and a side chain specific to each amino acid, which is used as a monomer subunit of a peptide. Amino acids include 20 standard, naturally occurring or typical amino acids and non-standard amino acids. Standard natural amino acids include alanine (A or Ala), cysteine ​​(C or Cys), aspartic acid (D or Asp), glutamic acid (E or Glu), phenylalanine (F or Phe), glycine (G or Gly), histidine (H or His), isoleucine (I or Ile), lysine (K or Lys), leucine (L or Leu), methionine (M or Met), asparagine (N or Asn), proline (P or Pro), glutamine (Q or Gln), arginine (R or Arg), serine (S or Ser), threonine (T or Thr), valine (V or Val), tryptophan (W or Trp), and tyrosine (Y or Tyr). Amino acids can be L-amino acids or D-amino acids. Non-standard amino acids can be modified amino acids, amino acid analogs, amino acid mimetics, non-standard proteinogenic amino acids, or naturally occurring or chemically synthesized non-proteinogenic amino acids. Examples of non-standard amino acids include, but are not limited to, selenocysteine, pyrrolysine and N-formylmethionine, β-amino acids, homologous amino acids, proline and pyruvate derivatives, 3-substituted alanine derivatives, glycine derivatives, ring-substituted phenylalanine and tyrosine derivatives, linear core amino acids, N-methyl amino acids.

[0041] As used herein, the term "post-translational modification" refers to modifications that occur on a peptide after the peptide has been translated by the ribosome. The post-translational modification can be a covalent chemical modification or an enzymatic modification. Examples of post-translational modifications include, but are not limited to, acylation, acetylation, alkylation (including methylation), biotinylation, butyrylation, carbamylation, carbonylation, deamidation, deiminiation, dinaphthylamine formation, disulfide bridge formation, eliminylation, flavin attachment, formylation, γ-carboxylation, glutamylation, glycylation, glycosylation, glypiation, heme C attachment, hydroxylation, hypusine formation, iodination, prenylation, lipidation, lipoylation, propionylation, methylation, myristoylation, oxidation, palmitoylation, pegylation, phosphoesterification, phosphorylation, prenylation, propionylation, retinylidene Schiff base formation, S-glutathionylation, S-nitrosylation, S-sulfenylation, selenoylation, succinylation, sulfation, ubiquitination, and C-terminal amidation. Post-translational modifications include modifications of the amino terminal and / or carboxyl terminal of the peptide. Modifications of the terminal amino group include, but are not limited to, deamination, N-low alkyl, N-di-low alkyl and N-acyl modifications. Modifications of the terminal carboxyl group include, but are not limited to, amide, low alkyl amide, dialkyl amide and low alkyl ester modifications (e.g., wherein the low alkyl is C1-C4 alkyl). Post-translational modifications also include, for example but not limited to, modifications of the above-mentioned amino acids falling between the amino and carboxyl terminals. The term post-translational modifications can also include modifications of peptides comprising one or more detectable tags.

[0042] As used herein, the term "binding agent" refers to a nucleic acid molecule, peptide, polypeptide, protein, carbohydrate or small molecule that binds, associates, combines, recognizes or combines with a polypeptide or a component or feature of a polypeptide. The binding agent can form a covalent association or a non-covalent association with a polypeptide or a component or feature of a polypeptide. The binding agent can also be a chimeric binding agent, composed of two or more types of molecules, such as a nucleic acid molecule-peptide chimeric binding agent or a carbohydrate-peptide chimeric binding agent. The binding agent can be a naturally occurring, synthetically produced or recombinantly expressed molecule. The binding agent can bind to a single monomer or subunit of a polypeptide (e.g., a single amino acid of a polypeptide) or to multiple connected subunits of a binding polypeptide (e.g., a dipeptide, a tripeptide, a higher peptide, a polypeptide or a protein molecule of a long peptide). The binding agent can bind to a linear molecule or a molecule with a three-dimensional structure (also called a conformation). For example, an antibody binding agent can bind to a linear peptide, polypeptide or protein, or to a conformational peptide, polypeptide or protein. The binding agent can bind to an N-terminal peptide, a C-terminal peptide, or an intermediate peptide of a peptide, polypeptide or protein molecule. The binding agent can bind to the N-terminal amino acid, C-terminal amino acid or intermediate amino acid of the peptide molecule. Compared with unmodified or labeled amino acids, the binding agent can preferably bind to chemically modified or labeled amino acids (e.g., amino acids functionalized by reagents containing any of the compounds of formula (I)-formula (VII) as described in International Patent Application No. WO 2019 / 089846). For example, compared with amino acids without the said part, the binding agent can preferably bind to acetyl moieties, cbz moieties, amidino moieties, aminoguanidine moieties, dansyl moieties, phenylthiocarbamoyl (PTC) moieties, dinitrophenyl (DNP) moieties, sulfonyl nitrophenyl (SNP) moieties, etc. The binding agent can bind to post-translational modifications of the peptide molecule. The binding agent can exhibit selective binding to components or features of the polypeptide (e.g., the binding agent can selectively bind to one of 20 possible natural amino acid residues and bind to the other 19 natural amino acid residues with very low affinity or not at all). The binding agent may exhibit less selective binding, where the binding agent is able to bind to multiple components or features of a polypeptide (e.g., the binding agent may bind to two or more different amino acid residues with similar affinity). The binding agent comprises a coding tag, which may be attached to the binding agent via a linker.

[0043] As used herein, the term "fluorophore" refers to a molecule that absorbs electromagnetic energy at one wavelength and re-emits energy at another wavelength. A fluorophore can be a molecule or a portion of a molecule including fluorescent dyes and proteins. Additionally, a fluorophore can be chemically, genetically, or otherwise linked or fused to another molecule to produce a molecule that has been "labeled" with the fluorophore.

[0044] As used herein, the term "joint" refers to one or more of nucleotides, nucleotide analogs, amino acids, peptides, polypeptides, or non-nucleotide chemical groups used to connect two molecules. The joint can be used to combine a binding agent and a coding tag, a recording tag and a polypeptide, a polypeptide and a solid support, a recording tag and a solid support, etc. In certain embodiments, the joint connects two molecules by an enzymatic reaction or a chemical reaction (e.g., click chemistry).

[0045] As used herein, the term "ligand" refers to any molecule or moiety attached to a compound described herein. A "ligand" may refer to one or more ligands attached to a compound. In some embodiments, a ligand is a side group or a binding site (e.g., a site to which a binding agent binds).

[0046] As used herein, the term "proteome" may include a whole set of proteins, polypeptides or peptides (including conjugates or complexes thereof) expressed at a certain time by the genome, cells, tissues or organisms of any organism. In one aspect, it is a protein expressed in a cell or organism of a given type under given conditions, at a given time. Proteomics is the study of proteomes. For example, a "cellular proteome" may include a collection of proteins found in a specific cell type under a specific set of environmental conditions (e.g., exposed to hormone stimulation). The complete proteome of an organism may include proteins from a complete set of all various cellular proteomes. A proteome may also include the collection of proteins in certain subcellular biological systems. For example, all proteins in a virus may be referred to as a viral proteome. As used herein, the term "proteome" includes subsets of the proteome, including but not limited to the kinome; secretome; receptorome (e.g., GPCRome); immunoproteasome; nutritional proteome; subsets of the proteome defined by post-translational modification (e.g., phosphorylation, ubiquitination, methylation, acetylation, glycosylation, oxidation, lipidation and / or nitrosylation), such as the phosphoproteomics (e.g., phosphotyrosine-proteome, tyrosine-kinome and tyrosine-phosphorylation), glycoproteome, etc.; subsets of the proteome associated with tissues or organs, developmental stages, or physiological or pathological conditions; subsets of the proteome associated with cellular processes such as cell cycle, differentiation (or dedifferentiation), cell death, aging, cell migration, transformation or metastasis; or any combination thereof. As used herein, the term "proteomics" refers to the quantitative analysis of the proteome, for example, the proteome within a cell, the proteome within a tissue, and the proteome within a body fluid, as well as the corresponding spatial distribution of the proteome within a cell and within a tissue. In addition, proteomic studies include the dynamic state of the proteome, which changes continuously over time with biological functions and defined biological or chemical stimuli.

[0047] As used herein, the term "non-cognate binder" refers to a binder that cannot bind to a polypeptide feature, component, or subunit being measured with low affinity in a specific binding cycle reaction compared to a "cognate binder", which binds to the corresponding polypeptide feature, component, or subunit with high affinity. For example, if a tyrosine residue of a peptide molecule is measured in a binding reaction, non-cognate binders are those binders that bind to the tyrosine residue with low affinity or do not bind to the tyrosine residue at all, so that under conditions suitable for transferring the coded tag information from the cognate binder to the record tag, the non-cognate binder cannot transfer the coded tag information to the record tag. Alternatively, if a tyrosine residue of a peptide molecule is measured in a binding reaction, non-cognate binders are those agents that bind to the tyrosine residue with low affinity or do not bind to the record tag at all, so that under conditions suitable for embodiments involving extended coded tags rather than extended record tags, the record tag information cannot be effectively transferred to the coded tag.

[0048] The terminal amino acid at one end of the peptide chain with a free amino group is referred to herein as the "N-terminal amino acid" (NTAA). The terminal amino acid at the other end of the chain with a free carboxyl group is referred to herein as the "C-terminal amino acid" (CTAA). The amino acids that make up the peptide can be numbered sequentially, where the length of the peptide is "n" amino acids. As used herein, NTAA is considered to be the nth amino acid (also referred to herein as the "nth NTAA"). Using this nomenclature, the next amino acid is the n-1th amino acid, followed by the n-2th amino acid, and so on for the length of the peptide from the N-terminus to the C-terminus. In certain embodiments, NTAA, CTAA, or both can be functionalized with chemical moieties.

[0049] As used herein, the term "barcode" refers to a nucleic acid molecule of about 2 to about 30 bases (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 bases) that provides a unique identifier tag or origin information for a polypeptide, a binding agent, a binding agent set from a binding cycle, a sample polypeptide, a sample set, a polypeptide within a compartment (e.g., a droplet, a bead, or an isolated location), a collection of polypeptides within a compartment, a polypeptide fraction, a collection of polypeptide fractions, a spatial region or a collection of spatial regions, a polypeptide library, or a library of binding agents. A barcode can be an artificial sequence or a naturally occurring sequence. In certain embodiments, each barcode within a barcode population is different. In other embodiments, a portion of the barcodes in the barcode population are different, for example, at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% of the barcodes in the barcode population are different. The barcode population can be randomly generated or non-randomly generated. In certain embodiments, the barcode population is an error correction barcode. The barcodes can be used to computationally deconvolute multiplexed sequencing data and identify sequence reads originating from a single polypeptide, sample, library, etc. The barcodes can also be used to deconvolute a collection of polypeptides that have been distributed into small compartments to enhance mapping. For example, instead of mapping peptides back to a proteome, the peptides are mapped back to their origin protein molecules or protein complexes.

[0050] A “sample barcode,” also called a “sample tag,” identifies which sample a peptide came from.

[0051] "Spatial barcodes" identify regions of 2D or 3D tissue sections from which peptides are derived. Spatial barcodes can be used for molecular pathology on tissue sections. Spatial barcodes allow multiplex sequencing of multiple samples or libraries from tissue sections.

[0052] As used herein, the term "coding tag" refers to a polynucleotide of any suitable length, such as about 2 bases to about 100 bases, including any nucleic acid molecule including 2 and 100 and integers therebetween, which contains identification information of its associated binding agent. "Coding tags" can also be made of "sequenceable polymers" (see, e.g., Niu et al., 2013, Nat. Chem. 5: 282-292; Roy et al., 2015, Nat. Commun. 6: 7237; Lutz, 2015, Macromolecules 48: 4759-4767; each of which is incorporated by reference in its entirety). The coding tag may include an encoder sequence, which may be optionally flanked by a spacer on one side or by a spacer on each side. The coding tag may also consist of an optional UMI and / or an optional binding cycle-specific barcode. The coding tag may be single-stranded or double-stranded. A double-stranded coding tag may include a blunt end, an overhang, or both. The coding tag may refer to a coding tag directly attached to a binding agent, a complementary sequence hybridized to a coding tag directly attached to a binding agent (e.g., for double-stranded coding tags), or the coding tag information in an extended record tag. In certain embodiments, the coding tag may further comprise a binding cycle-specific spacer or barcode, a unique molecular identifier, a universal priming site, or any combination thereof.

[0053] As used herein, the term "encoder sequence" or "encoder barcode" refers to a nucleic acid molecule of about 2 bases to about 30 bases (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 bases) length, providing identification information of its associated binding agent. The encoder sequence can uniquely identify the binding agent to which it is associated. In certain embodiments, the encoder sequence provides identification information of its associated binding agent and the binding cycle using the binding agent. In other embodiments, the encoder sequence is combined with a separate binding cycle-specific barcode within the coding tag. Alternatively, the encoder sequence can identify the binding agent to which it is associated as a member of a group comprising two or more different binding agents. In some embodiments, this level of recognition is sufficient for analytical purposes. For example, in some embodiments of binding agents related to amino acids, it may be sufficient to know that a peptide contains one of two possible amino acids at a specific position without explicitly identifying the amino acid residue at the position. In another example, a common encoder sequence is used for a polyclonal antibody, which comprises a mixture of antibodies that recognize more than one epitope of a protein target, and has different specificities. In other embodiments, in the case where the encoder sequence recognizes a group of possible binding agents, a sequential decoding method can be used to generate a unique identification of each binding agent. This is achieved by changing the encoding sequence of a given binding agent in repeated binding cycles (see Gunderson et al., 2004, Genome Res.14:870-7). When combined with the encoding information from other cycles, the partial identification encoding tag information from each binding cycle is a unique identifier generated for the binding agent, for example, a specific combination of encoding tags rather than a separate encoding tag (or encoder sequence) is provided to generate unique identification information for the binding agent. Preferably, the encoding sequence in the binding agent library has the same or similar number of bases.

[0054] As used herein, the terms "binding cycle-specific tag," "binding cycle-specific barcode," or "binding cycle-specific sequence" refer to a unique sequence used to identify a library of binders used within a particular binding cycle. The binding cycle-specific tag may comprise a length of about 2 bases to about 8 bases (e.g., 2, 3, 4, 5, 6, 7, or 8 bases). The binding cycle-specific tag may be incorporated into the encoding tag of the binder as part of a spacer sequence, part of an encoder sequence, part of a UMI, or as a separate component within the encoding tag.

[0055] As used herein, the term "spacer" (Sp) refers to a nucleic acid molecule of about 1 base to about 20 bases (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 bases) in length, which is present at the end of a record tag or a coding tag. In certain embodiments, the spacer sequence is flanked by the coding sequence of the coding tag at one or both ends. After the binding agent binds to the polypeptide, the complementary spacer sequences on the associated coding tag and the record tag are annealed separately, allowing the binding information to be transferred to the record tag, coding tag or double tag construct by primer extension reaction or connection. Sp' refers to a spacer sequence complementary to Sp. Preferably, the spacer sequences within the binding agent library have the same number of bases. A common (shared or identical) spacer can be used in the binding agent library. The spacer sequence may have a "cycle-specific" sequence to track the binding agent used in a specific binding cycle. The spacer sequence (Sp) can be constant in all binding cycles, specific to a particular class of polypeptides, or specific to the number of binding cycles. The polypeptide class-specific spacer allows the encoded tag information of the homologous binder present in the extended record tag in the completed binding / extension cycle to be annealed to the encoded tag of another binder, which recognizes the same class of polypeptides through the class-specific spacer in the subsequent binding cycle. Only the sequential binding of the correct homologous pairs can produce interactive spacer elements and effective primer extension. The spacer sequence may contain a sufficient number of bases to anneal with the complementary spacer sequence in the record tag to initiate a primer extension (also called polymerase extension) reaction, or provide a "splint" for the ligation reaction, or mediate a "sticky end" ligation reaction. The spacer sequence may contain a smaller number of bases than the encoder sequence in the coding tag.

[0056] As used herein, the term "recording tag" refers to a moiety, such as a chemically coupled moiety, a nucleic acid molecule, or a sequenceable polymer molecule (see, e.g., Niu et al., 2013, Nat. Chem. 5:282-292; Roy et al., 2015, Nat. Commun. 6:7237; Lutz, 2015, Macromolecules 48:4759-4767; each of which is incorporated by reference in its entirety), that can transfer identification information of a coding tag to a coding tag, or that can transfer identification information (e.g., UMI information) about a macromolecule associated with a recording tag from a coding tag to a coding tag. The identification information can include any information that characterizes a molecule, such as information about a sample, a fraction, a partition, a spatial location, an interacting neighboring molecule, a cycle number, and the like. In addition, the presence of UMI information can also be classified as identification information. In certain embodiments, after a binding agent binds to a polypeptide, information from a coding tag attached to the binding agent can be transferred to a recording tag associated with the polypeptide while the binding agent is bound to the polypeptide. In other embodiments, after the binding agent binds to the polypeptide, the information from the record tag associated with the polypeptide can be transferred to the coding tag connected to the binding agent, while the binding agent is bound to the polypeptide. The recoded tag can be directly connected to the polypeptide, connected to the polypeptide through a multifunctional linker, or bound to the polypeptide by means of its proximity (or co-localization) on the solid support. The record tag can be connected through its 5' end or 3' end or internal site, as long as the connection is compatible with the method for transferring the coding tag information to the record tag, and vice versa. The record tag can also include other functional components, such as a universal priming site, a unique molecular identifier, a barcode (e.g., a sample barcode, a graded object barcode, a spatial barcode, a compartment label, etc.), a spacer sequence complementary to the spacer sequence of the coding tag, or any combination thereof. In the embodiment where the coding tag information is transferred to the record tag using polymerase extension, the spacer sequence of the record tag is preferably located at the 3' end of the record tag.

[0057] As used herein, the term "primer extension", also referred to as "polymerase extension", refers to a reaction catalyzed by a nucleic acid polymerase (e.g., DNA polymerase) in which a nucleic acid molecule (e.g., an oligonucleotide primer, a spacer sequence) annealed to a complementary strand is extended by the polymerase using the complementary strand as a template.

[0058] As used herein, the term "unique molecular identifier" or "UMI" refers to a nucleic acid molecule of about 3 to about 40 bases (3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 bases) in length that provides a unique identifier tag for each polypeptide or binder attached to the UMI. Polypeptide UMIs can be used to computationally deconvolute sequencing data from multiple extended transcripts to identify extended transcripts derived from a single polypeptide. Polypeptide UMIs can be used to accurately count starting polypeptide molecules by collapsing NGS reads to unique UMIs. Binder UMIs can be used to identify each single molecule binder that binds to a specific polypeptide. For example, a UMI can be used to identify the number of individual binding events of a binder specific for a single amino acid that occurred for a particular peptide molecule. It should be understood that when both a UMI and a barcode are referenced in the context of a binder or polypeptide, the barcode refers to identification information of the individual binder or polypeptide other than the UMI (e.g., sample barcode, compartment barcode, binding cycle barcode).

[0059] As used herein, the term "universal priming site" or "universal primer" or "universal priming sequence" refers to a nucleic acid molecule that can be used for library amplification and / or sequencing reactions. Universal priming sites can include, but are not limited to, priming sites (primer sequences) for PCR amplification, flow slot adapter sequences that anneal to complementary oligonucleotides on the flow slot surface (which promote bridge amplification in some next-generation sequencing platforms), sequencing priming sites, or combinations thereof. Universal priming sites can be used for other types of amplification, including amplifications that are typically used in conjunction with next-generation digital sequencing. For example, extended record tag molecules can be cyclized, and universal priming sites are used for rolling circle amplification to form DNA nanoballs that can be used as sequencing templates (Drmanac et al., 2009, Science 327: 78-81). Alternatively, record tag molecules can be directly cyclized and sequenced by polymerase extension from universal priming sites (Korlach et al., 2008, Proc. Natl. Acad. Sci. 105: 1176-1181). The term "forward" when used in the context of a "universal priming site" or "universal primer" may also be referred to as "5'" or "sense". The term "reverse" when used in the context of a "universal priming site" or "universal primer" may also be referred to as "3'" or "antisense".

[0060] As used herein, the term "extended record tag" refers to a record tag to which the information of the coding tag (or its complementary sequence) of at least one binding agent has been transferred after the binding agent binds to the polypeptide. The information of the coding tag can be transferred to the record tag directly (e.g., ligation) or indirectly (e.g., primer extension). The information of the coding tag can be transferred to the record tag enzymatically or chemically. The extended record tag may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200 or more binding agent information of the coding tag. The base sequence of the extended record tag may reflect the binding time and order of the binding agent identified by its coding tag, may reflect the partial binding order of the binding agent identified by the coding tag, or may not reflect the binding order of any binding agent identified by the coding tag. In certain embodiments, the encoded tag information present in the extended record tag represents the analyzed polypeptide sequence with at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity. In certain embodiments where the extended record tag does not represent the analyzed polypeptide sequence with 100% identity, the error may be due to off-target binding of the binding agent, or due to a "missed" binding cycle (e.g., because the binding agent cannot bind to the polypeptide during the binding cycle because the primer extension reaction failed), or both.

[0061] As used herein, the term "extended coding tag" refers to a coding tag to which the information of at least one recording tag (or its complementary sequence) is transferred after the binding agent that binds the coding tag binds to the macromolecule, and the recording tag is associated. The information of the recording tag can be transferred to the coding tag directly (e.g., connection) or indirectly (e.g., primer extension). The information of the recording tag can be transferred enzymatically or chemically. In certain embodiments, the extended coding tag contains the information of the recording tag, reflecting a binding event. As used herein, the term "double tag" or "double tag construct" or "double tag molecule" refers to a nucleic acid molecule, and the information of at least one recording tag (or its complementary sequence) and at least one coding tag (or its complementary sequence) is transferred to the nucleic acid molecule after binding to the binding agent connected to the coding tag, the coding tag is bound to the binding agent, and the recording tag is bound to the polypeptide (see, for example, Figure 11B of International Patent Application Publication No. WO 2017 / 192633). Recording tag and polypeptide The information of the recording tag and the coding tag can be transferred to the double tag indirectly (e.g., primer extension). The information of the recording tag can be transferred enzymatically or chemically. In certain embodiments, the dual tag comprises a UMI of a recording tag, a compartment tag of a recording tag, a universal priming site of a recording tag, a UMI of an encoding tag, a coding sequence of an encoding tag, a combined cycle-specific barcode, a universal priming site of an encoding tag, or any combination thereof.

[0062] As used herein, the terms "solid support," "solid surface," "solid substrate," "sequencing substrate," or "substrate" refer to any solid material, including porous and non-porous materials, to which macromolecules (e.g., peptides) can be directly or indirectly bound by means known in the art, including covalent and non-covalent interactions, or any combination thereof. The solid support can be two-dimensional (e.g., a planar surface) or three-dimensional (e.g., a gel matrix or beads). The solid support can be any support surface, including, but not limited to, beads, microbeads, arrays, glass surfaces, silicon surfaces, plastic surfaces, filters, membranes, nylon, silicon crystal chips, fluidic chips, flow channels, biochips including signal transduction electronic devices, channels, microtiter wells, ELISA plates, rotating interferometry disks, nitrocellulose membranes, nitrocellulose-based polymer surfaces, polymer matrices, nanoparticles, or microspheres. The material for solid support includes but is not limited to acrylamide, agarose, cellulose, nitrocellulose, glass, gold, quartz, polystyrene, polyethylene vinyl acetate, polypropylene, polymethacrylate, polyethylene, polyethylene oxide, polysilicate, polycarbonate, Teflon, fluorocarbon, nylon, silicone rubber, polyanhydride, polyglycolic acid, polylactic acid, polyorthoester, functionalized silane, polypropyl fumarate, collagen, glycosaminoglycan, polyamino acid, dextran or any combination thereof.Solid support also includes film, membrane, bottle, dish, fiber, braided fiber, shaped polymer, such as tube, particle, bead, microsphere, microparticle or any combination thereof.For example, when solid surface is bead, bead can include but is not limited to ceramic bead, polystyrene bead, polymer bead, methyl styrene bead, agarose bead, acrylamide bead, solid core bead, porous bead, paramagnetic bead, glass bead or controlled pore bead.Bead can be spherical or irregular shape. Bead or support can be porous.The size of bead can be nanometer (for example, 100nm) to millimeter (for example, 1mm).In certain embodiments, the size range of bead is about 0.2 micron to about 200 microns, or about 0.5 micron to about 5 microns.In certain embodiments, bead can be about 1 μm, 1.5 μm, 2 μm, 2.5 μm, 2.8 μm, 3 μm, 3.5 μm, 4 μm, 4.5 μm, 5 μm, 5.5 μm, 6 μm, 6.5 μm, 7 μm, 7.5 μm, 8, 8.5 μm, 9 μm, 9.5 μm, 10 μm, 10.5 μm, 15 μm, or 20 μm diameter.In certain embodiments, "bead" solid support can refer to single bead or multiple beads.In certain embodiments, solid surface is nanoparticle.In certain embodiments, the size range of nanoparticles is a diameter from about 1 nm to about 500 nm, for example, between about 1 nm and about 20 nm, between about 1 nm and about 50 nm, between about 1 nm and about 100 nm, between about 10 nm and about 50 nm, between about 10 nm and about 100 nm, between about 10 nm and about 200 nm, between about 50 nm and about 100 nm, between about 50 nm and about 150 nm, between about 50 nm and about 200 nm, between about 100 nm and about 200 nm, or between about 200 nm and about 500 nm. In some embodiments, the diameter of nanoparticles can be about 10 nm, about 50 nm, about 100 nm, about 150 nm, about 200 nm, about 300 nm or about 500 nm. In some embodiments, the diameter of nanoparticles is less than about 200 nm.

[0063] As used herein, the term "nucleic acid molecule" or "polynucleotide" refers to a single-stranded or double-stranded polynucleotide containing deoxyribonucleotides or ribonucleotides linked by 3'-5' phosphodiester bonds, and polynucleotide analogs. Nucleic acid molecules include, but are not limited to, DNA, RNA, and cDNA. Polynucleotide analogs can have connections other than the standard phosphodiester bonds found in natural polynucleotides, and optionally have modified sugar groups other than ribose or deoxyribose. Polynucleotide analogs contain bases that can form hydrogen bonds with standard polynucleotide bases by Watson-Crick base pairing, wherein the analog backbone presents the bases in a manner that allows hydrogen bonding between the oligonucleotide analog molecule and the bases in the standard polynucleotide in a sequence-specific manner. Examples of polynucleotide analogs include, but are not limited to, xenogeneic nucleic acids (XNA), bridged nucleic acids (BNA), glycol nucleic acids (GNA), peptide nucleic acids (PNAs), γPNA, morpholino polynucleotides, locked nucleic acids (LNAs), threose nucleic acids (TNA), 2'-O-methyl polynucleotides, 2'-O-alkylribosyl substituted polynucleotides, phosphorothioate polynucleotides, and borophosphate polynucleotides. Polynucleotide analogs can have purine or pyrimidine analogs, including, for example, 7-deazapurine analogs, 8-halopurine analogs, 5-halopyrimidine analogs, or universal base analogs that can pair with any base, including hypoxanthine, nitroazoles, isoquinolone analogs, azole carboxamides and aromatic triazole analogs, or base analogs with additional functionality, such as a biotin group for affinity binding. In some embodiments, the nucleic acid molecule or oligonucleotide is a modified oligonucleotide. In some embodiments, the nucleic acid molecule or oligonucleotide is a DNA with pseudo-complementary bases, a DNA with protected bases, an RNA molecule, a BNA molecule, an XNA molecule, an LNA molecule, a PNA molecule, a PNA molecule or a morpholino DNA, or a combination thereof. In some embodiments, the nucleic acid molecule or oligonucleotide is backbone-modified, sugar-modified or nucleobase-modified. In some embodiments, the nucleic acid molecule or oligonucleotide has a nucleobase protecting group, such as Alloc, an electrophilic protecting group, such as thiranes, an acetyl protecting group, a nitrobenzyl protecting group, a sulfonate protecting group, or a conventional base-labile protecting group.

[0064] As used herein, "nucleic acid sequencing" refers to determining the order of nucleotides in a nucleic acid molecule or a sample of nucleic acid molecules.

[0065] As used herein, "next generation sequencing" refers to a high-throughput sequencing method that allows parallel sequencing of millions to billions of molecules. The example of the next generation sequencing method includes sequencing by synthesis, connection sequencing, hybridization sequencing, Polony sequencing, ion semiconductor sequencing and pyrophosphate sequencing. By connecting primers to a solid substrate and a complementary sequence to a nucleic acid molecule, the nucleic acid molecule can be hybridized with the solid substrate by primers, and then amplified by using polymerase to produce multiple copies in discrete regions on the solid substrate (these groups are sometimes referred to as polymerase clones). Therefore, in the sequencing process, the nucleotides at a specific position can be sequenced multiple times (for example, hundreds or thousands of times)-this coverage depth is referred to as "deep sequencing". The example of high-throughput nucleic acid sequencing technology includes the platform provided by Illumina, BGI, Qiagen, Thermo-Fisher and Roche, including parallel bead arrays, synthesis sequencing, connection sequencing, capillary electrophoresis, electronic microchips, "biochips", microarrays, parallel microchips and single molecule array formats as reviewed by Service (Science 311: 1544-1546, 2006).

[0066] As used herein, "single molecule sequencing" or "third generation sequencing" refers to a new generation sequencing method, in which the reading from the single molecule sequencing instrument is generated by sequencing a single DNA molecule. Unlike the next generation sequencing method that relies on amplification to clone many DNA molecules in parallel for sequencing in a phased approach, single molecule sequencing examines a single DNA molecule and does not require amplification or synchronization. Single molecule sequencing includes methods that require pausing the sequencing reaction after each base incorporation ("rinse and scan" cycle) and methods that do not require stopping between reading steps. Examples of single molecule sequencing methods include single molecule real-time sequencing (Pacific Biosciences), nanopore-based sequencing (Oxford Nanopore), double-stranded interruption nanopore sequencing, and direct imaging DNA with advanced microscopes.

[0067] As used herein, "analyzing" a polypeptide means that all or part of the components of a polypeptide are quantified, characterized, distinguished, or a combination thereof. For example, analyzing a peptide, polypeptide, or protein includes determining all or part of the amino acid sequence (continuous or non-continuous) of a peptide. Analyzing a polypeptide also includes partial identification of the components of a polypeptide. For example, partially identifying the amino acids in a polypeptide protein sequence can identify the amino acids in a protein as belonging to a possible subset of amino acids. The analysis usually starts with analyzing the nth NTAA and then proceeds to the next amino acid (i.e., n-1, n-2, n-3, etc.) of the peptide. This is achieved by eliminating the nth NTAA, thereby converting the n-1st amino acid of the peptide into an N-terminal amino acid (referred to herein as "the (n-1)th NTAA"). Analyzing the peptide may also include determining the presence and frequency of post-translational modifications on the peptide, which may include or not include information about the order of post-translational modifications on the peptide. Analyzing a peptide may also include determining the presence and frequency of an epitope in a peptide, which may include or not include information about the order or position of an epitope in a peptide. Analyzing a peptide may include combining different types of analysis, such as obtaining epitope information, amino acid sequence information, post-translational modification information, or any combination thereof.

[0068] As used herein, the term "compartment" refers to a physical area or volume that separates or isolates a subset of polypeptides from a polypeptide sample. For example, a compartment can separate a single cell from other cells, or separate a subset of a sample proteome from the rest of the sample's proteome. The compartment can be an aqueous compartment (e.g., a microfluidic droplet), a solid compartment (e.g., a Picotiter well or microtiter well on a plate, a tube, a vial, a gel bead), a bead surface, a separation region within or on a porous bead. The compartment can contain one or more beads to which the polypeptide can be fixed.

[0069] As used herein, the term "compartment tag" or "compartment barcode" refers to a single-stranded or double-stranded nucleic acid molecule of about 4 bases to about 100 bases (including 4 bases, 100 bases and any integer therebetween) containing identification information of a component (e.g., the proteome of a single cell) within one or more compartments (e.g., a microfluidic droplet or bead surface). The compartment barcode identifies a subset of polypeptides in a sample that have been separated from multiple (e.g., millions to billions) compartments into the same physical compartment or compartment group. Therefore, even after the components are brought together, the compartment tag can be used to distinguish between components from one or more compartments with the same compartment tag and components in another compartment with a different compartment tag. By labeling proteins and / or peptides within each compartment or within a group of two or more compartments with a unique compartment tag, peptides derived from the same protein, protein complex, or cells within a single compartment or compartment group can be identified. The compartment tag comprises a barcode and an optional universal primer, and the barcode is optionally flanked by a spacer sequence on one or both sides. The spacer sequence may be complementary to the spacer sequence of the record tag, so that the compartment tag information can be transferred to the record tag. The compartment tag may also include a universal priming site, a unique molecular identifier (for providing identification information of the peptide connected thereto), or both, particularly for embodiments in which the compartment tag includes a record tag for the peptide analysis method described below. The compartment tag may include a functional portion for coupling the peptide (e.g., aldehyde, NHS, mTet, alkyne, etc.). Alternatively, the compartment tag may include a peptide, which includes a recognition sequence for a protein ligase to allow the compartment tag to be connected to the target peptide. The compartment may include a single compartment tag, a plurality of identical compartment tags reserved for optional UMI sequences, or two or more different compartment tags. In certain embodiments, each compartment includes a unique compartment tag (one-to-one mapping). In other embodiments, multiple compartments from a larger compartment group include the same compartment tag (many-to-one mapping). The compartment tag may be connected to a solid support within a compartment (e.g., a bead) or to the surface of the compartment itself (e.g., the surface of a picotiter well). Alternatively, the compartment tag may be free in the solution within the compartment.

[0070] As used herein, the term "partitioning" refers to assigning (e.g., randomly assigning) unique barcodes to subsets of polypeptides from a population of polypeptides within a sample. In certain embodiments, partitioning can be achieved by assigning polypeptides into compartments. A partition can consist of polypeptides within a single compartment or polypeptides within multiple compartments from a group of compartments.

[0071] As used herein, "partition tag" or "partition barcode" refers to a single-stranded or double-stranded nucleic acid molecule comprising about 4 bases to about 100 bases (including 4 bases, 100 bases and any integer therebetween) that contains identification information for a partition. In certain embodiments, a partition tag for a polypeptide refers to the same compartment tag generated by partitioning a polypeptide into compartments labeled with the same barcode.

[0072] As used herein, the term "fractionate" refers to a subset of polypeptides in a sample that has been selected from the rest of the sample or organelle using a physical or chemical separation method, such as fractionation by size, hydrophobicity, isoelectric point, affinity, etc. Separation methods include HPLC separation, gel separation, affinity separation, cell fractionation, organelle fractionation, tissue fractionation, etc. Fluid fluidity, magnetism, electrical current, mass, density and similar physical properties can also be used for separation.

[0073] As used herein, the term "fractionator barcode" refers to a single-stranded or double-stranded nucleic acid molecule comprising about 4 bases to about 100 bases (including 4 bases, 100 bases, and any integer therebetween) that contains identification information for a polypeptide in a fractionator.

[0074] I. Methods for Proximity Interaction Analysis by Forming Linked Structures

[0075] In one aspect, the present disclosure provides a method for assessing the identity and spatial relationship between a polypeptide and a portion in a sample, the method comprising: a) forming a junction structure between a site of a polypeptide in the sample and a site of a portion in the sample, the junction structure comprising a polypeptide tag associated with the site of the polypeptide and a portion tag associated with the site of the portion, wherein the polypeptide tag and the portion tag are associated; b) transferring information between the associated polypeptide tag and the portion tag or linking the associated polypeptide tag and the portion tag to form a shared unique molecular identifier (UMI) and / or barcode; c) disrupting the junction structure by dissociating the polypeptide from the portion and dissociating the polypeptide tag from the portion tag while maintaining the association between the polypeptide and the polypeptide tag and maintaining the association between the portion and the portion tag; and d) assessing at least a partial sequence of the polypeptide tag and the polypeptide, and assessing at least a partial identity of the portion tag and the portion; wherein the assessed portions of the polypeptide tag and the portion tag comprise a shared unique molecular identifier (UMI) and / or barcode indicating the spatial proximity of the site of the polypeptide and the site of the portion in the sample.

[0076] Also provided herein are methods for assessing the identity and spatial relationship between a polypeptide and a moiety in a sample, comprising: a) forming a junction structure between a site of a polypeptide in the sample and a site of a moiety in the sample, the junction structure comprising a polypeptide tag associated with the site of the polypeptide and a moiety tag associated with the site of the moiety, wherein the polypeptide tag and the moiety tag are associated; b) transferring information between the associated polypeptide tag and the moiety tag to form a shared unique molecular identifier (UMI) and / or barcode, wherein the shared UMI and / or barcode is formed as a separate record polynucleotide; c) disrupting the junction structure by dissociating the polypeptide from the moiety and dissociating the polypeptide tag from the moiety tag while maintaining the association between the polypeptide and the polypeptide tag and maintaining the association between the moiety and the moiety tag; d) assessing at least a partial sequence of the polypeptide tag and the polypeptide, and assessing at least a partial identity of the moiety tag and the moiety; and e) assessing the separate record polynucleotide to establish a spatial relationship between the site of the polypeptide and the site of the moiety. In some embodiments, step e) establishes a spatial relationship between the site of the polypeptide and two or more sites of the moiety or two or more moieties. In some embodiments, the separated record polynucleotide is released from the polypeptide tag and / or the moiety tag.

[0077] Any suitable moiety can be used in the present method. For example, the moiety can be an atom, an inorganic moiety, an organic moiety, or a complex thereof. The organic moiety can be an amino acid, a polypeptide (e.g., a peptide or protein), a nucleoside, a nucleotide, a polynucleotide (e.g., an oligonucleotide or nucleic acid), a vitamin, a monosaccharide, an oligosaccharide, a carbohydrate, a lipid, and a complex thereof. In some embodiments, the moiety can comprise a polypeptide. In other embodiments, the moiety can comprise a polynucleotide.

[0078] In certain embodiments, polypeptide and / or part have three-dimensional structure.In certain embodiments, polypeptide and part belong to different molecules, and this method can be used to assess the identity and spatial relationship between polypeptide and part in different molecules (for example, protein-protein complex, protein-DNA complex or protein-RNA complex).Macromolecular assembly can be composed of the same type of macromolecule (for example protein-protein) or two or more different types of macromolecule (for example protein-DNA).In other embodiments, polypeptide and part belong to the same macromolecule.

[0079] A. Exemplary Tags and Components

[0080] Any suitable polypeptide tag can be used in the present method. For example, the polypeptide tag can be an atom, an inorganic part, an organic part or a complex thereof. The organic part can be an amino acid, a polypeptide (e.g., a peptide or protein), a nucleoside, a nucleotide, a polynucleotide (e.g., an oligonucleotide or a nucleic acid), a vitamin, a monosaccharide, an oligosaccharide, a carbohydrate, a lipid and a complex thereof. In some embodiments, the polypeptide tag can comprise a polynucleotide.

[0081] Any suitable moiety label can be used in the present method. For example, the moiety tag can be an atom, an inorganic moiety, an organic moiety or a complex thereof. The organic moiety can be an amino acid, a polypeptide (e.g., a peptide or protein), a nucleoside, a nucleotide, a polynucleotide (e.g., an oligonucleotide or nucleic acid), a vitamin, a monosaccharide, an oligosaccharide, a carbohydrate, a lipid and a complex thereof. In some embodiments, the moiety tag can comprise a polynucleotide.

[0082] Both the polypeptide tag and the partial tag may comprise polynucleotides. In some embodiments, the polypeptide tag comprises a UMI and / or a barcode. In some embodiments, the partial tag comprises a UMI and / or a barcode. In some embodiments, the polypeptide tag comprises a first polynucleotide and the partial tag comprises a second polynucleotide, the first and second polynucleotides comprise complementary sequences, and the polypeptide tag and the partial tag are associated via the complementary sequence. In some embodiments, the sequence and the complementary sequence comprise a palindromic sequence. In some embodiments, the polypeptide tag and / or the partial tag do not comprise a palindromic sequence.

[0083] In some embodiments, polypeptide tags and part tags are used to generate separated record polynucleotides. In some embodiments, the separated record polynucleotides are or comprise DNA or RNA molecules. In some embodiments, the separated record polynucleotides comprise information about one or more polypeptides and / or one or more parts.

[0084] In some embodiments, the polypeptide tag and the separated record polynucleotide comprise complementary sequences. In some embodiments, the polypeptide tag and the separated record polynucleotide are associated by complementary sequences. In some embodiments, the partial tag and the separated record polynucleotide comprise complementary sequences. In some cases, the partial tag and the separated record polynucleotide are associated by complementary sequences.

[0085] In some embodiments, the polypeptide tag and the partial tag each comprise one or more nucleic acid chains arranged into a double-stranded palindromic region, a double-stranded barcode region, and / or a primer binding region. In some cases, the polypeptide tag and the partial tag comprise the following in the order listed: palindromic region-barcode region-primer binding region. In some embodiments, the polypeptide tag and the partial tag each comprise a hairpin structure having a partially double-stranded primer binding region, a double-stranded barcode region, a double-stranded palindromic region, and a single-stranded loop region containing a target binding portion. In some embodiments, the molecule that terminates polymerization is located between the double-stranded palindromic region and the loop region.

[0086] In some embodiments, the partial tag and / or polypeptide tag comprises one or more nucleic acid chains arranged into a double-stranded palindrome, a double-stranded barcode region, and / or a primer binding region. In some embodiments, the tag is arranged to form a hairpin structure, which is a single segment of continuous nucleotides that folds and forms a double-stranded region called a "stem" and a single-stranded region called a "loop". A double-stranded region is formed when the nucleotides of two regions of the same nucleic acid base pair with each other (intramolecular base pairing principle).

[0087] In some embodiments, the polypeptide tag and / or partial tag comprises two parallel nucleic acid chains (e.g., as two separate nucleic acids, or as a continuously folded hairpin structure). One of the chains is referred to as the "complementary chain" and the other chain is referred to as the "displacement chain". The complementary chain typically contains a primer binding region, or at least a single-stranded fragment of a primer binding region, where the primer binds (e.g., hybridizes, etc.). The complementary chain and the displacement chain are bound to each other at least through a double-stranded barcode region and through a double-stranded palindromic region. As described herein, the "displacement chain" is a chain that is initially displaced by a newly generated half-record, and when the displacement chain "rebinds" to the complementary chain, it sequentially displaces the newly generated half-record.

[0088] Two nucleic acids or two nucleic acid regions are "complementary" to each other if they base pair or bind to each other through Watson-Crick interactions (also known as hybridization) to form a double-stranded nucleic acid molecule. As used herein, "binding" refers to an association between at least two molecules due to, for example, electrostatic, hydrophobic, ionic and / or hydrogen bonding interactions under physiological conditions.

[0089] A "double-stranded region" of a nucleic acid refers to a region of a nucleic acid (e.g., DNA or RNA) containing two parallel nucleic acid chains that are bound to each other by hydrogen bonds between complementary purines (e.g., adenine and guanine) and pyrimidines (e.g., thymine, cytosine, and uracil) to form a double helix. In some embodiments, the two parallel nucleic acid chains that form the double-stranded region are part of adjacent nucleic acid chains. For example, polypeptide tags and partial tags can include or be attached to a hairpin structure.

[0090] A "double-stranded palindromic region" refers to a region of a nucleic acid (eg, cDNA or RNA) that is identical to a nucleotide sequence reading 5' (5 primers) to 3' (3 primers) on one strand or 5' to 3' on the complementary strand that forms a duplex therewith.

[0091] In some embodiments, the palindromic sequence allows for the connection of polypeptide tags and partial tags that are adjacent to each other. Polymerase extension of the primer bound to the primer binding region produces a "half transcript," which refers to a newly generated nucleic acid chain. The generation of the half transcript displaces one strand of the polypeptide or partial tag, referred to as the "displaced strand." The displaced strand sequentially displaces a portion of the half transcript (by binding to its "complementary strand"), starting from the 3' end, such that the 3' end of the half transcript containing the palindromic sequence is bound to another half transcript similarly displaced from an adjacent barcoded nucleic acid.

[0092] In some embodiments, the length of the double-stranded palindromic region is 4-10 nucleotide base pairs. That is, in some embodiments, the double-stranded palindromic region may include 4-10 consecutive nucleotides bound to 4-10 respectively complementary nucleotides. For example, the double-stranded palindromic region can have a length of 4, 5, 6, 7, 8, 9 or 10 nucleotide base pairs. In some embodiments, the double-stranded palindromic region can have a length of 5 or 6 nucleotide base pairs. In some embodiments, the double-stranded palindromic region is longer than 10 nucleotide base pairs. For example, the double-stranded palindromic region can have a length of 4-50 nucleotide base pairs. In some embodiments, the length of the double-stranded palindromic region is 4-40, 4-30 or 4-20 nucleotide base pairs.

[0093] The double-stranded palindrome may contain guanine (G), cytosine (C), adenine (A) and / or thymine (T). In some embodiments, the percentage of G and C nucleotide base pairs (G / C) relative to A and T nucleotide base pairs (A / T) is greater than 50%. For example, the percentage of G / C relative to A / T in the double-stranded palindrome may be 50% to 100%. In some embodiments, the percentage of G / C relative to A / T is greater than 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%.

[0094] In some embodiments, the double-stranded palindrome region may include an even number of nucleotide base pairs, although the double-stranded palindrome region of the present disclosure is not limited thereto. For example, the double-stranded palindrome region may include 4, 6, 8, or 10 nucleotide base pairs. Alternatively, the double-stranded palindrome region may include 5, 7, or 9 nucleotide base pairs.

[0095] In a plurality of polypeptides and partial tags, typically, the double-stranded palindromic region is identical for each tag in the plurality of tags, such that polypeptide tags proximal to the partial tags can bind to each other through the resulting half transcript containing the palindromic sequence. However, in some embodiments, the double-stranded palindromic region may be identical only in a subset of polypeptides / partial tags, such that two different subsets contain two different double-stranded palindromic regions.

[0096] "Primer binding region" refers to a region of a nucleic acid (e.g., DNA or RNA) comprising a partial tag or polypeptide tag, where a single-stranded primer (e.g., DNA or RNA primer) binds to initiate replication. The primer binding region can be a single-stranded region or a partially double-stranded region, and a partially double-stranded region refers to a region containing both single-stranded fragments and double-stranded fragments. The primer binding region can contain any combination of nucleotides in a random or rationally designed order. In some embodiments, the length of the primer binding region is 4 to 40 nucleotides (or nucleotide base pairs, or a combination of nucleotides and nucleotide base pairs, depending on the single-stranded and / or double-stranded nature of the primer binding region). For example, the primer binding region can have a length of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 nucleotides (and / or entire nucleotide base pairs). In certain embodiments, the primer binding region can have a length of 4 to 10, 4 to 15, 4 to 20, 4 to 25, 4 to 30, 4 to 35 or 4 to 40 nucleotides (and / or nucleotide base pairs). In certain embodiments, the primer binding region is longer than 40 nucleotides. For example, the primer binding region can have a length of 4 to 100 nucleotides. In some embodiments, the primer binding region has a length of 4 to 90, 4 to 80, 4 to 70, 4 to 60, or 4 to 50 nucleotides.

[0097] In some embodiments, the primer binding region is designed to accommodate the binding of more than one (e.g., 2 or 3 different) primers. "Primer" is a single-stranded nucleic acid used as a starting point for nucleic acid synthesis. Polymerase adds nucleotides to the primer to produce a new nucleic acid chain. The primers disclosed herein are designed to be complementary to and bind to the primer binding region of a polypeptide tag or a partial tag. Therefore, the primer length and composition (e.g., nucleotide composition) depend at least in part on the length and composition of the primer binding region of the polypeptide or partial tag. In some embodiments, the primer has a length of 4 to 40 nucleotides. For example, the primer can have a length of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 nucleotides. In some embodiments, the primer may have a length of 4 to 10, 4 to 15, 4 to 20, 4 to 25, 4 to 30, 4 to 35, or 4 to 40 nucleotides.

[0098] In order to, for example, limit binding to primers that meet their geometric criteria, there may be primers connected in pairs or other combinations (e.g., triplets of any geometric shape or more). The rigid double-stranded connection shown enforces minimum and maximum distances between the partial tag and the polypeptide tag. The double-stranded "ruler" domain can be of any length (e.g., 2 to 100 nucleotides, or more), and can optionally include the barcode itself, which connects the two halves through information content as long as they are separated during processing. In some embodiments, the double-stranded ruler domain is a complex structure, such as a 2-, 3-, or 4-DNA helical bundle, a DNA nanostructure, such as a DNA origami structure, or other structures that increase or change the hardness / rigidity of the ruler, and the double-stranded ruler domain enforces a typical distance between the partial tag and the polypeptide tag that can generate a record.

[0099] "Strand displacement polymerase" refers to a polymerase that can displace downstream nucleic acids (e.g., DNA) encountered during nucleic acid synthesis. Different polymerases can have varying degrees of displacement activity. Examples of strand displacement polymerases include, but are not limited to, Bst large fragment polymerase (e.g., New England Biolabs (NEB) #M0275, phi 29 polymerase (e.g., NEB #M0269), Deep VentR polymerase, Klenow fragment polymerase, and modified Taq polymerase. Other strand displacement polymerases are contemplated.

[0100] In some embodiments, the primer comprises at least one nucleotide mismatch relative to the single-stranded primer binding region. Such mismatches can be used to promote displacement of the complementary strand of the half-record from the partial tag and / or polypeptide tag. In some embodiments, the primer comprises at least one artificial linker.

[0101] In some embodiments, primer extension by displacement polymerase (bound to the primer binding site) is usually terminated by the presence of a molecule or modification that terminates polymerization. Therefore, in some embodiments, the partial tag and / or polypeptide tag may include a molecule or modification that terminates polymerization. The molecule or modification that terminates polymerization ("inhibitor" or "blocker") is usually located in the double-stranded region of the partial tag or polypeptide tag, adjacent to the double-stranded palindromic region, so that the polymerization termination primer extends through the double-stranded palindromic region. For partial or polypeptide tags arranged in the form of hairpins, the molecule or modification that terminates polymerization can be located between the double-stranded palindromic region and the hairpin loop. In some embodiments, the molecule that terminates polymerization is a synthetic non-DNA linker, for example, a triethylene glycol spacer, such as Int spacer 9 (iSp9), C3 spacer or spacer 18 (Integrated DNA Technologies, IDT). It should be understood that any non-natural linker that terminates polymerization by a polymerase can be used, as provided herein. Other non-limiting examples of such molecules and modifications include three-carbon linkage ( / iSpC3 / ), (IDT), ACRYDITE TM (IDT), Adenylated, Azide, Digoxigenin (NHS ester), Cholesteryl-TEG (IDT), I-LINKER TM (IDT), and 3-cyanovinylcarbazole (CNVK) and its variants. Typically, but not always, short linkers (e.g., iSp9) result in faster reaction times.

[0102] In some embodiments, the molecules that terminate polymerization are single or paired non-natural nucleotide sequences, such as iso-dG and iso-dC (IDT), which are chemical variants of cytosine and guanine, respectively. Iso-dC will base pair (hydrogen bond) with iso-dG, but not with dG. Similarly, Iso-dG will base pair with Iso-dC, but not with dC. By incorporating these nucleotides in pairs on opposite sides of the hairpin, at the inhibitor position, the polymerase will stop because no complementary nucleotides are added at this position in the solution.

[0103] In some embodiments, the efficiency of performing "inhibitor" or "blocker" modifications is increased by reducing the dNTP concentration in the reaction (eg, from 200 μM) to 100 μM, 10 μM, 1 μM, or lower.

[0104] Molecules or modifications comprising a termination of polymerization generally produce a "bump" in the double-stranded region of the moiety tag or polypeptide tag (e.g., the stem region of a hairpin structure) because the molecule or modification is not paired. Thus, in some embodiments, the moiety and / or polypeptide tag is designed to include a single nucleotide (e.g., thymine), at least two of the same nucleotide (e.g., a thymine dimer (TT) or trimer (TTT)), or a non-natural modification, as opposed to the molecule or modification.

[0105] In some aspects, to prevent the polymerase from extending the end (e.g., 5' or 3' end) of the partial tag and / or polypeptide tag, a poly-T sequence (e.g., a sequence of 2, 3, 4, 5, 7, 8, 9 or 10 thymine nucleotides) can be used. Alternatively, a synthetic base (e.g., reverse dT) or other modification can be added to the end (e.g., 5' or 3' end) of the tag to prevent undesired polymerization of the tag. Other termination molecules (molecules that prevent the extension of the 3' end that is not intended to be extended) include, but are not limited to, iso-dG and iso-dC or other non-natural nucleotides or modifications.

[0106] In some embodiments, the generation of half-record replaces a chain of a partial tag or polypeptide tag. The replaced chain replaces a part of the half-record from the 3' end in turn. In some embodiments, this replacement of half-record is promoted by a "double-stranded replacement zone" adjacent to a molecule or modification that terminates polymerization. In embodiments where the partial tag and / or polypeptide tag has a hairpin structure, the double-stranded replacement zone can be located between the molecule or modification that terminates polymerization and the hairpin loop. The double-stranded replacement zone can contain any combination of nucleotides in a random or rationally designed order. In some embodiments, the length of the double-stranded replacement zone is 2-10 nucleotide base pairs. For example, the double-stranded replacement zone can have a length of 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotide base pairs. In some embodiments, the double-stranded palindromic zone can have a length of 5 or 6 nucleotide base pairs. In some embodiments, the double-stranded palindromic zone can contain only a combination of C and G nucleotides.

[0107] In some embodiments, the displacement of half-record can also be promoted by modifying the reaction conditions. For example, some self-circulation reactions may include thiophosphate nucleotides (2'-deoxynucleoside α-thiol 2'-deoxynucleoside α-thiol triphosphate products, Trilink Biotechnology) to replace natural soluble dNTPs for new chain generation. These are not as stable as natural dNTPs in hybridization, and cause the interaction between half-record and stem to weaken. They can be used in any combination (for example, thiophosphate A with natural T, C and G bases, or other combinations or ratios of mixtures). Other such chemical modifications can be performed to weaken half-record pairing and promote displacement.

[0108] In some embodiments, part of the tag and / or the polypeptide tag itself can be modified, in some embodiments using non-natural nucleotides, which are used to replace to enhance the hairpin structure. In such an embodiment, the displacement polymerase that produces the semi-record can still open and replicate the stem, but during the strand displacement, the stem sequence re-hybridization is energetically superior to the semi-record hybridization using the stem template. Non-limiting examples of non-natural nucleotides include 5-methyl dC (5-methyldeoxycytidine; when replacing dC, each nucleotide insertion increases the melting temperature of the nucleic acid by up to 5°C), 2,6-diaminopurine (each insertion increases the melting temperature by up to 1-2°C), Super T (5-hydroxybutyl)-2'-deoxyuridine also increases the melting temperature of nucleic acids), and / or locked nucleic acids (LNA). They can appear in either or both strands of the hairpin structure.

[0109] In some embodiments, non-natural nucleotides can be used to introduce mismatches between the new half transcript sequence and the stem. For example, if isoG nucleotides are present in the template strand of the stem, in some cases, the polymerase will mistakenly add one of the soluble nucleotides that can be used to extend the half transcript, and in doing so, a "bump" is generated between the new half transcript and the stem, very similar to the bump (included in the primer). In some respects, it will serve the same purpose of weakening the half transcript-template interaction and promoting displacement.

[0110] In some embodiments, a portion of the tag and / or polypeptide tag is arranged to form a hairpin structure, which is a single segment of continuous nucleotides that folds and forms a double-stranded region called a "stem" and a single-stranded region called a "loop". In some embodiments, the double-stranded loop region has a length of 3-50 nucleotides. For example, the single-stranded loop region can have a length of 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides. In some embodiments, the single-stranded loop region can have a length of 3 to 10, 3 to 15, 3 to 20, 3 to 25, 3 to 30, 3 to 35, 3 to 40, 3 to 45, or 3 to 50 nucleotides. In some embodiments, the single-stranded loop region is longer than 50 nucleotides. For example, the single-stranded loop region can have a length of 3 to 200 nucleotides. In some embodiments, the single-stranded loop region has a length of 3 to 175, 3 to 150, 3 to 100, or 3 to 75 nucleotides. In some embodiments, the loop region includes a smaller region of intramolecular base pairing. In some embodiments, the hairpin loop allows flexibility in the orientation of the moiety tag and / or polypeptide tag relative to the target binding moiety. That is, the loop generally allows the moiety tag or polypeptide tag to occupy a variety of positions and angles relative to the target binding moiety, thereby allowing continuous interaction with a large number of adjacent tags (e.g., attached to other targets).

[0111] In some embodiments, the partial tag and / or polypeptide tag comprises at least one locked nucleic acid (LNA) nucleotide or other modified base. LNA pairs or other modified base pairs can be used as stronger (or weaker) base pairs in the double-stranded region of the partial tag and / or polypeptide tag, thereby biasing the strand displacement reaction. In some embodiments, at least one LNA molecule is located on the complementary strand of the tag, between the double-stranded barcode region and the single-stranded primer binding region.

[0112] Partial labeling and / or polypeptide labeling can be DNA, such as D-type DNA and L-type DNA and RNA, and various modifications thereof. Nucleic acid modifications include base modifications, sugar modifications, and backbone modifications. Non-limiting examples of such modifications are provided below.

[0113] Examples of modified nucleic acids (e.g., DNA variants) that can be used according to the present disclosure include, but are not limited to, L-DNA (a backbone enantiomer of DNA, known in the literature), peptide nucleic acid (PNA) bisPNA clamps, pseudo-complementary PNA, locked nucleic acids (LNA), and the above-mentioned co-nucleic acids, such as DNA-LNA co-nucleic acids. Therefore, the present disclosure contemplates nanostructures comprising DNA, RNA, LNA, PNA, or a combination thereof. It should be understood that the nucleic acids used in the methods and compositions of the present disclosure can be homogeneous or heterogeneous in nature. As an example, the nucleic acids can be completely DNA in nature, or they can be composed of DNA and non-DNA (e.g., LNA) monomers or sequences. Therefore, any combination of nucleic acid elements can be used. Nucleic acid modifications can make nucleic acids more stable and / or less susceptible to degradation under certain conditions. For example, in some embodiments, the nucleic acids are nuclease-resistant.

[0114] Also provided herein are multiple partial tags and polypeptide tags. "Multiple" includes at least two tags. In some embodiments, the multiple tags include 2 to 2 million tags (e.g., unique tags). For example, the multiple tags can include 100, 500, 1000, 5000, 10000, 100000, 1000000 or more tags. The present disclosure is not limited in this respect.

[0115] B. Information Transfer

[0116] The information between the associated polypeptide tags and the partial tags can be transferred in any suitable manner to form a shared UMI and / or barcode. In some embodiments, the information between the associated polypeptide tags and the partial tags can be transferred to a separate recording polynucleotide (e.g., Figure 7 C) In some embodiments, the isolated record polynucleotide is a newly formed polypeptide comprising a shared UMI and / or barcode.

[0117] In some embodiments, transferring information between an associated polypeptide tag and a partial tag comprises extending both a first polynucleotide of the polypeptide tag and a second polynucleotide of the partial tag to form a shared UMI and / or barcode. In other embodiments, transferring information between an associated polypeptide tag and a partial tag comprises extending one of the first polynucleotide of the polypeptide tag and the second polynucleotide of the partial tag to form a shared UMI and / or barcode. In some embodiments, the polypeptide tag comprises a double-stranded polynucleotide, and the partial tag comprises a double-stranded polynucleotide, and transferring information between an associated polypeptide tag and a partial tag comprises connecting the double-stranded polynucleotide to form a shared UMI and / or barcode. The shared UMI and / or barcode may comprise a sequence of the double-stranded polynucleotide. The shared UMI and / or barcode may also comprise a sequence of one of the double-stranded polynucleotides. In some embodiments, transferring information between an associated polypeptide tag and a partial tag comprises extending the polypeptide tag and the partial tag, and then performing a ligation reaction to form a double-stranded separated record polynucleotide, which record polynucleotide comprises information from the polypeptide tag and the partial tag (e.g., a shared UMI and / or barcode).

[0118] In some embodiments, the shared UMI and / or barcode comprises information about one or more polypeptides and / or one or more moieties.

[0119] In some embodiments, the information transfer between the associated polypeptide tag and the partial tag can be mediated by a polymerase (e.g., a polymerase, a DNA polymerase, an RNA polymerase, or a reverse transcriptase). In other embodiments, the information transfer between the associated polypeptide tag and the partial tag can be mediated by a ligase (e.g., a DNA ligase, an ssDNA ligase (e.g., Circligase), a dsDNA ligase, or an RNA ligase). In other embodiments, the information transfer between the associated polypeptide tag and the partial tag can be mediated by a topoisomerase. In other embodiments, the information transfer between the associated polypeptide tag and the partial tag can be mediated by chemical connection. In some embodiments, the information transfer between the associated polypeptide tag and the partial tag can be mediated by extension and / or connection.

[0120] In the connection structure, the polypeptide tag and the partial tag can be associated in any suitable manner. In some embodiments, the connection structure between the polypeptide tag and the partial tag and their respective polypeptides and parts can be connected using a covalent cross-linking method as described by Schenider et al., as well as Holding (Holding 2015, Schneider, Belsom et al., 2018) in cross-linked mass spectrometry for proteomics applications. In some embodiments, in the connection structure, the polypeptide tag and the partial tag can be stably or covalently associated. In other embodiments, in the connection structure, the polypeptide tag and the partial tag can be instantaneously associated. The association between the polypeptide tag and the partial tag can change over time or the execution of the method of the present invention. Before and after the information transfer between the polypeptide tag and the partial tag, the association between the polypeptide tag and the partial tag can be different. For example, in the connection structure, the polypeptide tag and the partial tag can be instantaneously associated before the information transfer between the polypeptide tag and the partial tag. After the information transfer between the polypeptide tag and the partial tag, the association between the polypeptide tag and the partial tag can become more stable. In other embodiments, in the connection structure, the polypeptide tag and the partial tag can be directly associated. In other embodiments, in a linked structure, the polypeptide tag and the moiety tag can be indirectly associated, for example, via a linker or a UMI between the polypeptide tag and the moiety tag.

[0121] In some of any of the embodiments provided, in the connection structure, the polypeptide tag and the separated record polynucleotide are directly associated. In some of any of the embodiments provided, in the connection structure, the partial tag and the separated record polynucleotide are directly associated. In some embodiments, in the connection structure, the polypeptide tag and the partial tag can be associated by the separated record polynucleotide. In some embodiments, the connection structure formed between the polypeptide tag and the partial tag via the separated record polynucleotide is instantaneous. In some embodiments, the separated record polynucleotide is formed by the extension between the polypeptide tag and / or the partial tag. In some embodiments, the separated record polynucleotide comprises a sequence complementary to the polypeptide tag and / or the partial tag. In some embodiments, the separated record polynucleotide is formed by connection. In some embodiments, the separated record polynucleotide is formed by the connection of the polypeptide tag and / or the partial tag.

[0122] When forming a connection structure, any suitable number of polypeptide tags can be associated with a suitable number of sites of a polypeptide. For example, in forming a connection structure, a single polypeptide tag can be associated with a single site of a polypeptide, a single polypeptide tag can be associated with multiple sites of a polypeptide, or multiple polypeptide tags can be associated with multiple sites of a polypeptide. Similarly, in forming a connection structure, any suitable number of partial tags can be associated with a suitable number of partial sites. For example, in forming a connection structure, a single partial tag can be associated with a single site of a part, a single partial tag can be associated with multiple sites of a part, or multiple partial tags can be associated with multiple sites of a part.

[0123] In some embodiments, the information transfer between the associated polypeptide tag and the partial tag uses cyclic annealing, extension and connection. For example, in some cases, the polypeptide tag and the partial tag are used as templates to produce double-stranded DNA tags (e.g., using primer extension). In some embodiments, the double-stranded DNA tags (e.g., polypeptide tags and partial tags) are connected. In some embodiments, the DNA tag is or comprises a separated record polynucleotide. In some embodiments, the separated record polynucleotide is further PCR amplified.

[0124] In some embodiments, the information transferred between the associated polypeptide tags and the partial tags to the separated record polynucleotide can be mediated by a polymerase (e.g., DNA polymerase, RNA polymerase or reverse transcriptase). In some embodiments, the transfer is based on an "auto-cycling" reaction (see, e.g., Schaus et al., Nat Comm (2017) 8: 696; U.S. Patent Application Publication No. US2018 / 0010174, and International Patent Application Publication No. WO 2018 / 017914 and No. WO 2017 / 143006). In some embodiments of repeated auto-cycling of separated record polynucleotides, the reaction is carried out at 37° C. or about 37° C. in the presence of a displacement polymerase. The polypeptide tags and partial tags associated with the polypeptide and the partial tags are barcoded, respectively, and are designed so that in the presence of a displacement polymerase and a universal soluble primer, the partial tags and / or polypeptide tags guide the automatic cycling process of repeating the recording of the adjacent tags. In some specific embodiments, an automated cyclic method for transferring information includes 1) application of paired primer exchange hairpins as polypeptide or partial tags, wherein individual extensions are made to bound half transcripts, 2) strand displacement and 3' palindromic domain hybridization, and 3) half transcript extension to separate transcript polynucleotides.

[0125] In some further embodiments, the method includes, in a first step, a soluble universal primer binds to each of the polypeptide tag and the partial tag at a common single-stranded primer binding region, a displacement polymerase extends the primer through the barcode region and the palindromic region to a molecule or modification (e.g., a synthetic non-DNA linker) that terminates polymerization, thereby generating a "half-record", which refers to a newly generated nucleic acid chain). Secondly, the half-record is partially displaced from the barcoded polypeptide or partial tag by a "strand displacement" mechanism (see, e.g., Yurke et al., Nature 406:605-608, 2000; and Zhang et al., Nature Chemistry 3:103-113, 2011, each of which is incorporated herein by reference), and adjacent half-records hybridize to each other through the 3' palindromic region. Third, the half-record is extended through the barcode region and the primer binding region, releasing a soluble, separated record polynucleotide, which includes information from the polypeptide tag and the partial tag. Polypeptide tags and moiety tags associated with the same or other molecular pairs (other polypeptide-moiety pairs or interactions) undergo similar cycles to form separate record polynucleotides.

[0126] In some embodiments, when the cyclic reaction terminates, the separated record polynucleotides are collected, prepared, amplified, analyzed and / or sequenced (e.g., using parallel next generation sequencing technology). In some embodiments, the separated record polynucleotides are sequenced, thereby generating sequencing data. In some embodiments, the separated record polynucleotides are collected and modified. In some embodiments, the separated record polynucleotides are collected and attached (e.g., cascaded). In some embodiments, the method includes connecting the separated record polynucleotides collected before evaluating the separated record polynucleotides. For example, in some embodiments, the cascade is mediated by a ligase or Gibson assembly. In some embodiments, any suitable technology or program is used to analyze, evaluate or sequence the separated record polynucleotides connected. For example, the cascaded separated record polynucleotides are sequenced in strings. In some embodiments, the cascaded polynucleotides are sequenced using nanopore sequencing.

[0127] In some embodiments, the separated recorded polynucleotides are evaluated, and the shared unique molecular identifier (UMI) and / or barcode indicating that the site of the polypeptide and the site of the portion are close in space. In some embodiments, the sequence data represents the spatial configuration of the macromolecule, and in some cases represents the connectivity and / or interaction of the macromolecule. In some embodiments, the method further comprises reconstruction and / or statistical analysis. In some embodiments, the sequencing data provides information about the interaction of two or more molecules.

[0128] In other embodiments, the information transfer between the associated polypeptide tags and the partial tags to the separated record polynucleotides can be mediated by a ligase, such as a DNA ligase, an ssDNA ligase (e.g., Circligase), a dsDNA ligase, or an RNA ligase. In other embodiments, the information transfer between the associated polypeptide tags and the partial tags to the separated record polynucleotides can be mediated by a topoisomerase. In other embodiments, the information transfer between the associated polypeptide tags and the partial tags can be mediated by chemical connection. In some embodiments, the information transfer between the associated polypeptide tags and / or partial tags and the separated record polynucleotides can be mediated by extension and / or connection.

[0129] In some embodiments, the methods form a plurality of separate record polypeptides between more than one site of the polypeptide tag and the moiety or more than one moiety.

[0130] In some embodiments, the connection structure is formed between the site of the polypeptide and one or more sites of the part or between the polypeptide tag and one or more parts. In some embodiments, one or more connection structures are formed between the site of the polypeptide and two or more sites of the part or two or more parts. In some embodiments, the connection structure is formed between the site of the polypeptide and 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more sites of the part or between the site of the polypeptide and 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more parts. In some embodiments, the sites of the parts each belong to different polypeptides or proteins. In some embodiments, the sites of these parts are each different sites on the polypeptide. In some instances, a connection structure is formed between the site of the polypeptide and the site of part 1, between the site of the polypeptide and the site of part 2, between the site of the polypeptide and the site of part 3, and so on. In some embodiments, the same site of the polypeptide can form a connection structure with more than one site on the part or with more than one part in a paired manner (see, e.g. Figures 9A-9C ). In some embodiments, a first connection structure is formed between the polypeptide and the first portion (M1), dissociates, and a second or subsequent connection structure is formed between the polypeptide and the second or subsequent portion (M2). In some embodiments, overlapping UMIs and / or barcodes indicate that the polypeptide forms a connection structure with M1 and M2. In some embodiments, information from two or more shared UMIs and / or barcodes indicates that the site of the polypeptide and the site of each portion M1 and M2 are spatially close. In some instances, indirect or overlapping paired information from two or more separate recorded polynucleotides indicates spatial proximity information of a polypeptide having two or more portions ( Fig. 9C ).

[0131] Transfer information between associated polypeptide tags and partial tags or connect associated polypeptide tags and partial tags to form any number of shared unique molecular identifiers (UMIs) and / or barcodes. For example, transfer information between associated polypeptide tags and partial tags or connect associated polypeptide tags and partial tags to form a single shared unique molecular identifier (UMI) and / or barcode. A single shared unique molecular identifier (UMI) and / or barcode may contain any suitable substance or sequence. In some embodiments, a single shared unique molecular identifier (UMI) and / or barcode may be formed by combining multiple sequences from polypeptide tags and / or partial tags, for example, multiple UMIs and / or barcodes. In some examples, the shared UMI and / or barcode is a composite tag or composite UMI that contains a sequence of UMIs and / or barcodes of polypeptide tags and a sequence of UMIs and / or barcodes of partial tags. In another example, transferring information between or linking the associated polypeptide tags and the moiety tags can form multiple shared unique molecular identifiers (UMIs) and / or barcodes.

[0132] The UMI may comprise any suitable substance or sequence. In some embodiments, the UMI has a probability of occurring multiple times by chance in a sample that is appropriate or sufficiently low. In other embodiments, the UMI comprises a polynucleotide comprising about 3 nucleotides to about 40 nucleotides. The nucleotides in the UMI polynucleotide may or may not be consecutive. In other embodiments, the polynucleotide in the UMI comprises a degenerate sequence. In other embodiments, the polynucleotide in the UMI does not comprise a degenerate sequence. In other embodiments, the UMI comprises a nucleic acid, an oligonucleotide, a modified oligonucleotide, a DNA molecule, a DNA with pseudo-complementary bases, a DNA with protected bases, an RNA molecule, a BNA molecule, an XNA molecule, an LNA molecule, a PNA molecule, a γPNA molecule, or a morpholino DNA, or a combination thereof. The DNA molecule may be backbone-modified, sugar-modified, or nucleobase-modified. The DNA molecule may also have a nucleobase protecting group, such as Alloc, an electrophilic protecting group, such as thiarane, an acetyl protecting group, a nitrobenzyl protecting group, a sulfonate protecting group, or a traditional base-labile protecting group, which includes Ultramild reagents.

[0133] The polypeptide tag and the partial tag may be dissociated from each other using any suitable technique or procedure. For example, if the polypeptide tag and the partial tag are associated with each other via a polypeptide-polypeptide, polypeptide-polynucleotide, or polynucleotide-polynucleotide interaction, the polypeptide tag and the partial tag may be dissociated from each other using any technique or procedure suitable for disrupting such polypeptide-polypeptide, polypeptide-polynucleotide, or polynucleotide-polynucleotide interaction. In some embodiments, in the connection structure, the shared UMI and / or barcode comprises a complementary polynucleotide hybrid, and dissociating the polypeptide tag from the partial tag comprises denaturing the complementary polynucleotide hybrid.

[0134] The polypeptide and part can be dissociated from each other using any suitable technique or procedure. For example, if the polypeptide and part are associated with each other via polypeptide-polypeptide or polypeptide-polynucleotide interactions, the polypeptide and part can be dissociated from each other using any technique or procedure suitable for destroying such polypeptide-polypeptide or polypeptide-polynucleotide interactions. In some embodiments, the polypeptide and part are all part of a larger polypeptide, and the polypeptide is dissociated from the part including fragmenting the larger polypeptide into peptide fragments. Any suitable technique or procedure can be used to fragment the larger polypeptide. For example, the larger polypeptide fragments can be digested with a protease into peptide fragments. Any suitable protease can be used. For example, a protease can be an exopeptidase such as an aminopeptidase or a carboxypeptidase. In another example, a protease can be an endopeptidase or an endoproteinase, such as trypsin, LysC, LysN, ArgC, chymotrypsin, pepsin, thermolysin, papain or elastase. (See, for example, Switzar, Giera et al., 2013). In some embodiments, the evaluation of at least a portion of the sequence of the polypeptide and at least a portion of the identity of the part is performed after the polypeptide and part are dissociated from each other. For example, the dissociated polypeptides and portions can be used in peptide or polypeptide sequencing assays (e.g., degradation-based polypeptide sequencing assays by constructing extended record tags). In some cases, the dissociated polypeptides and portions can be used in assays involving cyclic removal of terminal amino acids.

[0135] The method of the present invention can be used to assess the identity and spatial relationship between a polypeptide and a portion in a sample, regardless of whether the polypeptide and the portion belong to the same molecule. For example, the target polypeptide and the portion can belong to two different molecules. In another example, the target polypeptide and the portion can be parts of the same molecule.

[0136] In some embodiments, the target polypeptide is part of a larger polypeptide, and the part is also part of the same larger polypeptide. The part can be any suitable substance or a complex thereof. For example, the part can comprise an amino acid or a polypeptide. The part of the amino acid or polypeptide can comprise one or more modified amino acids. Exemplary modified amino acids include glycosylated amino acids, phosphorylated amino acids, methylated amino acids, acylated amino acids, hydroxyproline or sulfated amino acids. The glycosylated amino acid can comprise an N-linked or O-linked glycosyl moiety. The phosphorylated amino acid can be phosphotyrosine, phosphoserine or phosphothreonine. The acylated amino acid can comprise a farnesyl, myristoyl or palmitoyl moiety. The sulfated amino acid can be part of a sulfotyrosine or a disulfide bond.

[0137] In other embodiments, the portion can be a portion of a molecule that is bound, complexed, or immediately adjacent to a polypeptide in a sample. The portion can be any suitable substance or a complex thereof. For example, the portion can be an atom, an amino acid, a polypeptide, a nucleoside, a nucleotide, a polynucleotide, a vitamin, a monosaccharide, an oligosaccharide, a carbohydrate, a lipid, or a complex thereof. In a specific embodiment, the portion comprises an amino acid or a polypeptide. The portion of amino acids or polypeptides may comprise one or more modified amino acids. Exemplary modified amino acids include glycosylated amino acids, phosphorylated amino acids, methylated amino acids, acylated amino acids, hydroxyproline, or sulfated amino acids. Glycosylated amino acids may comprise N-linked or O-linked glycosyl moieties. Phosphorylated amino acids may be phosphotyrosine, phosphoserine, or phosphothreonine. Acylated amino acids may comprise farnesyl, myristoyl, or palmitoyl moieties. Sulfated amino acids may be part of sulfotyrosine or a disulfide bond.

[0138] In some embodiments, the polypeptide and the moiety may belong to two different proteins in the same protein complex. In other embodiments, the moiety may be a portion of a polynucleotide molecule (e.g., a DNA or RNA molecule) that is bound, complexed, or in close proximity to a polypeptide in a sample.

[0139] Any suitable technique or procedure can be used to evaluate the at least partial sequence of a polypeptide tag, a partial tag, a polypeptide, and / or at least partial identity of the portion. For example, if the polypeptide tag, the portion, and / or the partial tag comprises a polypeptide and / or a polynucleotide, any suitable technique or procedure for evaluating the identity or sequence of the polypeptide and / or polynucleotide can be used. Similarly, any suitable technique or procedure for evaluating a polypeptide can be used to evaluate at least a partial sequence of a polypeptide.

[0140] In some embodiments, the polypeptide tag and / or partial tag comprises a polypeptide, and the polypeptide tag and / or partial tag can be evaluated using a binding assay (e.g., an immunoassay). Exemplary immunoassays include enzyme-linked immunosorbent assay (ELISA), immunoblotting, immunoprecipitation, radioimmunoassay (RIA), immunostaining, latex agglutination, indirect hemagglutination assay (IHA), complement fixation, indirect immunofluorescence assay (IFA), turbidimetry, flow cytometry, surface plasmon resonance (SPR), chemiluminescence assay, lateral flow immunoassay, u-capture assay, inhibition assay, and affinity assay.

[0141] In some embodiments, the polypeptide tag and / or partial tag comprises a polynucleotide, such as DNA or RNA. Before or simultaneously with the evaluation, the polynucleotide can be amplified. The polynucleotides in the polypeptide tag and / or partial tag can be amplified using any suitable technique or procedure. For example, the polynucleotide can be amplified using polymerase chain reaction (PCR), strand displacement amplification (SDA), transcription-mediated amplification (TMA), ligase chain reaction (LCR), nucleic acid sequence-based amplification (NASBA), primer extension, rolling circle amplification (RCA), autonomous sequence replication system (3SR), or loop-mediated isothermal amplification (LAMP) procedures.

[0142] Any suitable technique or procedure can be used to assess at least a portion of the sequence of the polypeptide or at least a portion of the identity of the portion. If the portion comprises a polypeptide, at least a portion of the sequence of both the polypeptide and the portion can be assessed by any suitable polypeptide sequencing technique or procedure. For example, at least a portion of the sequence of both the polypeptide and the portion can be assessed by N-terminal amino acid analysis, C-terminal amino acid analysis, Edman degradation and mass spectrometry. In some embodiments, at least a portion of the sequence of one or both of the polypeptide and the portion can be assessed by using an associated binding agent (e.g., a mixed population of antibodies or monoclonal antibodies) that binds or recognizes at least a portion of the macromolecule. In another example, at least a portion of the sequence of both the polypeptide and the portion can be evaluated by techniques or procedures disclosed and / or claimed in U.S. Provisional Patent Application Nos. 62 / 330,841, 62 / 339,071, 62 / 376,886, 62 / 579,844, 62 / 582,312, 62 / 583,448, 62 / 579,870, 62 / 579,840, and 62 / 582,916, and International Patent Application No. PCT / US2017 / 030702 (published as WO 2017 / 192633 A1). In some embodiments, prior to evaluating at least a portion of the sequence of the polypeptide and / or at least a portion of the identity of the portion, the polypeptide and the portion are dissociated from each other and fixed to a support. In some aspects, the assessment of at least partial identity of at least a portion of a sequence or portion of a polypeptide is performed using a method that includes or uses DNA and / or DNA encoding.

[0143] In some embodiments, at least a partial sequence of a polypeptide is evaluated using a procedure comprising the following steps: a1) providing a polypeptide and an associated polypeptide tag as a record tag; b1) contacting the polypeptide with a first binding agent capable of binding to the polypeptide, wherein the first binding agent comprises a first coding tag having identification information about the first binding agent; c1) transferring the information of the first coding tag to the record tag to generate a first order extended record tag; d1) analyzing the first order extended record tag. This step a1) may further comprise providing a polypeptide bound to a solid support and an associated polypeptide tag. The method may further comprise contacting the polypeptide with a second (or higher order) binding agent, the second (or higher order) binding agent comprising a second (or higher order) binding moiety capable of binding to the polypeptide and a coding tag having identification information about the second (or higher order) binding agent, transferring the information of the second (or higher order) coding tag to the first order extended record tag to generate a second order (or higher order) extended record tag, and analyzing the second order (or higher order) extended record tag.

[0144] In some embodiments, at least a partial sequence of a polypeptide is evaluated using a procedure comprising the following steps: a1) providing a polypeptide and an associated polypeptide tag as a record tag; b1) contacting the polypeptide with a first binding agent capable of binding to the N-terminal amino acid (NTAA) of the polypeptide, wherein the first binding agent comprises a first coding tag having identification information about the first binding agent; c1) transferring the information of the first coding tag to the record tag to generate a first order extended record tag; d1) analyzing the extended record tag. The method may further comprise providing a polypeptide and an associated polypeptide tag bound to a solid support. The method also comprises contacting the target polypeptide with a second (or higher order) binding agent comprising a second (or higher order) coding tag, the second (or higher order) coding tag having identification information about the second (or higher order) binding agent, wherein the second (or higher order) binding agent is capable of binding to an NTAA other than the NTAA of the polypeptide. The contact between the polypeptide and the second (or higher order) binding agent may be performed in any suitable manner. For example, after the polypeptide is contacted with the first binding agent, the polypeptide may then be contacted with the second (or higher order) binding agent. In another example, contacting the polypeptide with a second (or higher order) binding agent can occur simultaneously with contacting the polypeptide with a first binding agent.

[0145] In some embodiments, at least a portion of the sequence of a polypeptide is evaluated using a procedure comprising the following steps: a1) providing a polypeptide and an associated polypeptide tag as a record tag; b1) contacting the polypeptide with a first binding agent capable of binding to the N-terminal amino acid (NTAA) of the polypeptide, wherein the first binding agent comprises a first encoding tag having identification information about the first binding agent; c1) transferring the information of the first encoding tag to the record tag to generate a first order extended record tag; d1) removing the NTAA to expose a new NTAA of the target polypeptide; e1) contacting the polypeptide with a second (or higher order binding agent); The method comprises contacting a first extended recording tag with a second (or higher order) binding agent, wherein the second (or higher order) binding agent comprises a second (or higher order) coding tag having identification information about the second (or higher order) binding agent, wherein the second (or higher order) binding agent is capable of binding to the new NTAA, wherein the second (or higher order) binding agent comprises a second coding tag having identification information about the second (or higher order) binding agent; f1) transferring the information of the second (or higher order) coding tag to the first extended recording tag to generate a second (or higher order) extended recording tag; g1) analyzing the second (or higher order) extended recording tag. Steps d1)-g1) can be repeated one or more times. The method can further include providing a polypeptide and an associated polypeptide tag bound to a solid support.

[0146] In some embodiments, at least a partial sequence of a polypeptide is evaluated using a procedure comprising the following steps: a1) providing a polypeptide and an associated polypeptide tag as a record tag; b1) modifying the N-terminal amino acid (NTAA) of the polypeptide, for example, using a chemical agent; c1) contacting the polypeptide with a first binding agent capable of binding to the modified NTAA, wherein the first binding agent comprises a first coding tag having identification information about the first binding agent; d1) transferring the information of the first coding tag to the record tag to generate a first order extended record tag; e1) analyzing the first order extended record tag. This step a1) may further comprise providing a polypeptide bound to a solid support and an associated polypeptide tag. The method also comprises contacting the polypeptide with a second (or higher order) binding agent comprising a second (or higher order) coding tag, the second (or higher order) coding tag having identification information about the second (or higher order) binding agent, wherein the second (or higher order) binding agent is capable of binding to a modified NTAA other than the modified NTAA of step b1). The contact between the polypeptide and the second (or higher order) binding agent may be performed in any suitable manner. For example, after the polypeptide is contacted with the first binding agent, the polypeptide can then be contacted with the second (or higher order) binding agent. In another example, the polypeptide can be contacted with the second (or higher order) binding agent at the same time as the polypeptide is contacted with the first binding agent.

[0147] In some embodiments, analysis of the first order and / or second order (or higher order) extension record tags also evaluates the polypeptide tags.

[0148] In some embodiments, the portion comprises a portion of a polypeptide, and at least partial identity or sequence of the portion is evaluated using a procedure comprising the following steps: a2) providing a portion of a polypeptide and an associated portion tag as a record tag; b2) contacting the portion of the polypeptide with a first binding agent capable of binding to the portion of the polypeptide, wherein the first binding agent comprises a first coding tag having identification information about the first binding agent; c2) transferring information of the first coding tag to the record tag to generate a first order extended record tag; d2) analyzing the first order extended record tag. The method may further comprise contacting the portion of the polypeptide with a second (or higher order) binding agent, the second (or higher order) binding agent comprising a second (or higher order) binding moiety capable of binding to the portion of the polypeptide and a coding tag having identification information about the second (or higher order) binding agent, transferring information of the second (or higher order) coding tag to the first order extended record tag to generate a second order (or higher order) extended record tag, and analyzing the second order (or higher order) extended record tag.

[0149] In some embodiments, at least partial identity or sequence of a portion is evaluated using a procedure comprising the following steps: a2) providing a partial polypeptide and an associated partial tag as a record tag; b2) contacting the partial polypeptide with a first binding agent capable of binding to the N-terminal amino acid (NTAA) of the partial polypeptide, wherein the first binding agent comprises a first coding tag having identification information about the first binding agent; c2) transferring the information of the first coding tag to the record tag to generate a first order extended record tag; d2) analyzing the extended record tag. The method may further comprise providing a partial polypeptide and an associated polypeptide tag bound to a solid support. The method also comprises contacting the partial polypeptide with a second (or higher order) binding agent comprising a second (or higher order) coding tag, the second (or higher order) coding tag having identification information about the second (or higher order) binding agent, wherein the second (or higher order) binding agent is capable of binding to an NTAA other than the NTAA of the polypeptide. The contact between the partial polypeptide and the second (or higher order) binding agent may be performed in any suitable manner. For example, after the partial polypeptide is contacted with the first binding agent, the partial polypeptide can then be contacted with the second (or higher order) binding agent. In another example, the partial polypeptide contacting with the second (or higher order) binding agent can occur simultaneously with the partial polypeptide contacting with the first binding agent.

[0150] In some embodiments, at least a portion of the sequence of a partial polypeptide is evaluated using a procedure comprising the following steps: a2) providing a partial polypeptide and an associated partial tag as a record tag; b2) contacting the partial polypeptide with a first binding agent capable of binding to the N-terminal amino acid (NTAA) of the partial polypeptide, wherein the first binding agent comprises a first coding tag having identification information about the first binding agent; c2) transferring the information of the first coding tag to the record tag to generate a first sequence extension record tag; d2) removing the NTAA to expose a new NTAA of the partial polypeptide; e2) contacting the partial polypeptide with a second (or more The method further comprises contacting a first (or higher order) binding agent with a second (or higher order) binding agent, the second (or higher order) binding agent comprising a second (or higher order) coding tag having identification information about the second (or higher order) binding agent, wherein the second (or higher order) binding agent is capable of binding to the new NTAA, wherein the second (or higher order) binding agent comprises a second coding tag having identification information about the second (or higher order) binding agent; f2) transferring the information of the second (or higher order) coding tag to the first extended recording tag to generate a second (or higher order) extended recording tag; g2) analyzing the second (or higher order) extended recording tag. Steps d2)-g2) can be repeated one or more times. The method can further comprise providing a polypeptide and an associated polypeptide tag bound to a solid support.

[0151] In some embodiments, at least a partial sequence of a partial polypeptide is evaluated using a procedure comprising the following steps: a2) providing a partial polypeptide and an associated partial tag as a record tag; b2) modifying the N-terminal amino acid (NTAA) of the partial polypeptide, for example, using a chemical agent for modification; c2) contacting the partial polypeptide with a first binding agent capable of binding to the modified NTAA, wherein the first binding agent comprises a first coding tag having identification information about the first binding agent; d2) transferring the information of the first coding tag to the record tag to generate a first order extended record tag; e2) analyzing the first order extended record tag. This step a2) may further comprise providing a polypeptide attached to a solid support and an associated partial tag. The method also comprises contacting the partial polypeptide with a second (or higher order) binding agent comprising a second (or higher order) coding tag, the second (or higher order) coding tag having identification information about the second (or higher order) binding agent, wherein the second (or higher order) binding agent is capable of binding to a modified NTAA other than the modified NTAA of step b2). The contact between the partial polypeptide and the second (or higher order) binding agent may be performed in any suitable manner. For example, after the partial polypeptide is contacted with the first binding agent, the partial polypeptide can then be contacted with the second (or higher order) binding agent. In another example, the partial polypeptide contacting with the second (or higher order) binding agent can occur simultaneously with the partial polypeptide contacting with the first binding agent.

[0152] In some embodiments, the methods described herein use a binding agent that can bind to a macromolecule (e.g., a polypeptide or portion). The binding agent can be any molecule that can bind to a component or feature of a polypeptide (e.g., a peptide, polypeptide, protein, nucleic acid, carbohydrate, small molecule, and the like). The binding agent can be a naturally occurring, synthetically produced, or recombinantly expressed molecule. In some embodiments, the scaffold for the engineered binding agent can be from any species, such as human, non-human, transgenic. The binding agent can bind to a single monomer or subunit of a polypeptide (e.g., a single amino acid) or to multiple connected subunits of a polypeptide (e.g., a dipeptide, tripeptide, or higher order peptide of a longer polypeptide molecule), or to an epitope.

[0153] In certain embodiments, the binding agent can be designed to covalently bind. Covalent binding can be designed to be conditional or preferred when combined with the correct group. For example, NTAA and its homologous NTAA specific binding agent can be modified with reactive groups respectively, so that once the NTAA specific binding agent is combined with the homologous NTAA, a coupling reaction is carried out to produce a covalent connection between the two. The non-specific binding of the binding agent to other positions lacking the homologous reactive group will not result in covalent bonding. In certain embodiments, the polypeptide comprises a ligand capable of forming a covalent bond with the binding agent. In certain embodiments, the polypeptide comprises a functionalized NTAA, and the functionalized NTAA comprises a ligand group capable of covalently binding to the binding agent. The covalent bond between the binding agent and its target can be combined to allow more stringent washing for removing the binding agent of non-specific binding.

[0154] In certain embodiments, the binding agent can be a selective binding agent. As used herein, selective binding refers to the ability of a binding agent to preferentially bind to a specific ligand (e.g., amino acid or amino acid class) relative to binding to different ligands (e.g., amino acids or amino acids). Selectivity generally refers to the equilibrium constant of a reaction in which a ligand is replaced by another ligand in a complex with a binding agent. Typically, this selectivity is related to the spatial geometry of the ligand and / or the manner and degree to which the ligand is bound to the binding agent, such as by hydrogen bonds or van der Waals forces (non-covalent interactions) or by reversible or irreversible covalent attachment to the binding agent. It should also be understood that selectivity can be relative and opposite to absolute, and it can be affected by different factors including ligand concentration. Therefore, in one example, the binding agent selectively binds to one of the twenty standard amino acids. In some examples, the binding agent binds to an N-terminal amino acid residue, a C-terminal amino acid residue, or an internal amino acid residue.

[0155] In some embodiments, the binding agent is partially specific or selective. In some aspects, the binding agent preferentially binds one or more amino acids. In some examples, the binding agent can bind two or more of the twenty standard amino acids. For example, the binding agent can preferentially bind amino acids A, C and G relative to other amino acids. In some other examples, the binding agent can selectively or specifically bind more than one amino acid. In some aspects, the binding agent may also preferably have one or more amino acids at the second, third, fourth, fifth, etc. positions from the terminal amino acid. In some cases, the binding agent preferentially binds to the specific terminal amino acid and one or more penultimate amino acids. In some cases, the binding agent preferentially binds to one or more specific terminal amino acids and a penultimate amino acid. For example, the binding agent may preferentially bind AA, AC and AG, or the binding agent may preferentially bind AA, CA and GA. In some specific examples, binding agents with different specificities can share the same encoding tag. In some embodiments, the binding agent may show flexibility and variability of target binding preferences in some or all positions of the target. In some examples, the binding agent may have a preference for one or more specific target terminal amino acids and a flexible preference for the penultimate target. In some other examples, the binding agent may have a preference for one or more specific target amino acids at the penultimate amino acid position and a flexible preference for the target at the terminal amino acid position. In some embodiments, the binding agent is selective for a target comprising a terminal amino acid and other components of a macromolecule. In some examples, the binding agent is selective for a target comprising a terminal amino acid and at least a portion of a peptide backbone. In some specific examples, the binding agent is selective for a target comprising a terminal amino acid and an amide peptide backbone. In some cases, the peptide backbone comprises a native peptide backbone or a post-translational modification. In some embodiments, the binding agent exhibits allosteric binding.

[0156] In the practice of the methods disclosed herein, the ability of a binding agent to selectively bind to a feature or component of a macromolecule (e.g., a polypeptide) only needs to be sufficient to allow its encoded tag information to be transferred to a record tag associated with the polypeptide. Therefore, selectivity only needs to be relative to other binding agents exposed to the polypeptide. It should also be understood that the selectivity of the binding agent does not have to be absolutely specific to a particular amino acid, but can be selective for a class of amino acids, such as amino acids with polar or non-polar side chains, or amino acids with charged (positive or negative) side chains, or amino acids with aromatic side chains, or amino acids with side chains of certain classes or sizes, and similar amino acids. In some embodiments, the ability of a binding agent to selectively bind to a feature or component of a macromolecule is characterized by comparing the binding ability of the binding agent. For example, the binding ability of a binding agent to a target can be compared with the binding ability of a binding agent that binds to different targets, for example, a binding agent that is selective for a class of amino acids is compared with a binding agent that is selective for different classes of amino acids. In some instances, a binding agent that is selective for non-polar side chains is compared with a binding agent that is selective for polar side chains. In some embodiments, a binding agent that is selective for a feature, component, or one or more amino acids of a peptide exhibits at least 1X, at least 2X, at least 5X, at least 10X, at least 50X, at least 100X, at least 500X greater binding than a binding agent that is selective for a different feature, component, or one or more amino acids of the peptide.

[0157] In a specific embodiment, the binding agent has high affinity and high selectivity for macromolecules. In particular, a high binding affinity with a low dissociation rate can be effective for information transfer between the encoding tag and the recording tag. In certain embodiments, the binding agent has a Kd of about ≤500nM, <200nM, <100nM, <50nM, <10nM, <5nM, <1nM, <0.5nM or <0.1nM. In some cases, the binding agent has a Kd of about <100nM. In a specific embodiment, the binding agent is >10×, >100× or >1000× its Kd. d The concentration of is added to the polypeptide to allow it to bind to completion. For example, a detailed discussion of the binding kinetics of antibodies to single protein molecules is described in Chang et al., J Immunol Methods (2012) 378 (1-2): 102-115.

[0158] In certain embodiments, the binding agent can bind to NTAA, CTAA, intervening amino acids, dipeptides (sequences of two amino acids), tripeptides (sequences of three amino acids), or higher order peptides of a peptide molecule. In some embodiments, each binding agent in the binding agent library selectively binds to a specific amino acid, such as one of the twenty standard naturally occurring amino acids. Standard natural amino acids include alanine (A or Ala), cysteine ​​(C or Cys), aspartic acid (D or Asp), glutamic acid (E or Glu), phenylalanine (F or Phe), glycine (G or Gly), histidine (H or His), isoleucine (I or Ile), lysine (K or Lys), leucine (L or Leu), methionine (M or Met), asparagine (N or Asn), proline (P or Pro), glutamine (Q or Gln), arginine (R or Arg), serine (S or Ser), threonine (T or Thr), valine (V or Val), tryptophan (W or Trp), and tyrosine (Y or Tyr). In some embodiments, the binding agent binds unmodified or natural amino acids. In some examples, the binding agent binds modified or natural dipeptides (sequences of two amino acids), tripeptides (sequences of three amino acids) or higher order peptides of peptide molecules. In order to make natural or unmodified NTAA have high affinity, natural or unmodified NTAA have high specificity, or both, the binding agent can be engineered. In some embodiments, a binding agent can be developed by using phage display directed evolution with good affinity scaffolds.

[0159] In some embodiments, the binding agent may bind to a natural or unmodified or unlabeled terminal amino acid. In certain embodiments, the binding agent may bind to a modified or labeled terminal amino acid (e.g., a functionalized or modified NTAA). In some embodiments, the binding agent may bind to a chemically or enzymatically modified terminal amino acid. The modified or labeled NTAA may be an NTAA functionalized with one of PITC, 1-fluoro-2,4-dinitrobenzene (Sanger reagent, DNFB), benzyloxycarbonyl chloride or benzyloxycarbonyl chloride (Cbz-Cl), N-(benzyloxycarbonyloxy) succinimide (Cbz-OSu or Cbz-O-NHS), dansyl chloride (DNS-Cl, or 1-dimethylaminonaphthalene-5-sulfonyl chloride), 4-sulfonyl-2-nitrofluorobenzene (SNFB), an acetylation agent, a guanidation agent, a thioacylation agent, a thioacetylation agent, or a thiobenzylation agent. In some examples, the binding agent binds to an amino acid by contacting with an agent or using a method described in International Patent Publication No. WO 2019 / 089846. In some cases, the binding agent binds to an amino acid labeled with an amine modification agent.

[0160] In some embodiments, the binding agent is derived from a biological source, a natural source, a non-natural source, or a synthetic source. In some instances, the binding agent is derived from a de novo protein design (Huang et al., (2016) 537(7620):320-327). In some instances, the binding agent has a structure, sequence, and / or activity designed according to first principles. In certain embodiments, the binding agent can be an aptamer (e.g., a peptide aptamer, a DNA aptamer, or an RNA aptamer), a peptide, an amino acid binding protein or enzyme, an antibody or its specific binding fragment, an antibody binding fragment, an antibody mimetic, a peptide, a peptide mimetic, a protein, or a polynucleotide (e.g., DNA, RNA, peptide nucleic acid (PNA), gPNA, bridging nucleic acid (BNA), xenogeneic nucleic acid (XNA), glycerol nucleic acid (GNA), or threose nucleic acid (TNA), or a variant thereof).

[0161] Potential scaffolds that can be designed for use in generating binding agents for the methods described herein include: anticalins, lipocalins, amino acid tRNA synthetases (aaRS), ClpS, Adnectin TM , T cell receptor, zinc finger protein, thioredoxin, GST A1-1, DARPin, affimer, affitin, alphabody, avimer, Kunitz domain peptide, monomer, single domain antibody, EETI-II, HPSTI, intracellular antibody, PHD finger, V(NAR)LDTI, evibody, Ig(NAR), knottin, maxibody, microbody, new tumor suppressor protein, pVIII, amylase inhibitor peptide, VLR, protein A scaffold, MTI-II, ecotin, GCN 4, Im9, kunitz domain, PBP, trans-body, tetranectin, WW domain, CBM4-2, DX-88, GFP, iMab, Ldl receptor domain A, Min-23, PDZ domain, avian pancreatic polypeptide, charybdo toxin / 10Fn3, domain antibody (Dab), a2p8 ankyrin repeat, insect defense A peptide, designed AR protein, C-type lectin domain, staphylococcal nuclease, Src homology domain 3 (SH3), or Src homology domain 2 (SH2). In some embodiments, the binder is derived from an enzyme that binds one or more amino acids (e.g., an aminopeptidase). In certain embodiments, the binder can be derived from an anticalin or ATP-dependent Clp protease adapter protein (Clps).

[0162] In certain embodiments, the binding agent further comprises a coding tag containing identification information about the binding agent. The coding tag is a nucleic acid molecule of about 3 bases to about 100 bases, which provides unique identification information for its associated binding agent. The coding tag may comprise about 3 to about 90 bases, about 3 to about 80 bases, about 3 to about 70 bases, about 3 to about 60 bases, about 3 bases to about 50 bases, about 3 bases to about 40 bases, about 3 bases to about 30 bases, about 3 bases to about 20 bases, about 3 bases to about 10 bases, or about 3 bases to about 8 bases. In some embodiments, the coding tag is about 3 bases, 4 bases, 5 bases, 6 bases, 7 bases, 8 bases, 9 bases, 10 bases, 11 bases, 12 bases, 13 bases, 14 bases, 15 bases, 16 bases, 17 bases, 18 bases, 19 bases, 20 bases, 25 bases, 30 bases, 35 bases, 40 bases, 55 bases, 60 bases, 65 bases, 70 bases, 75 bases, 80 bases, 85 bases, 90 bases, 95 bases, or 100 bases. The coding tag can be composed of DNA, RNA, polynucleotide analogs, or a combination thereof. Polynucleotide analogs include PNA, gPNA, BNA, GNA, TNA, LNA, morpholino polynucleotides, 2'-O-methyl polynucleotides, alkylribosyl substituted polynucleotides, phosphorothioate polynucleotides, and 7-deazapurine analogs.

[0163] The coding tag comprises an encoder sequence, which provides identification information about the relevant bonding agent. The coding sequence is about 3 bases to about 30 bases, about 3 bases to about 20 bases, about 3 bases to about 10 bases, or about 3 bases to about 8 bases. In certain embodiments, the coding sequence is about 3 bases, 4 bases, 5 bases, 6 bases, 7 bases, 8 bases, 9 bases, 10 bases, 11 bases, 12 bases, 13 bases, 14 bases, 15 bases, 20 bases, 25 bases or 30 bases in length. In certain embodiments, the length of the encoder sequence determines the quantity of the unique encoder sequence that can be generated. Shorter coding sequences produce a smaller number of unique coding sequences, which may be useful when using a small amount of bonding agent. In a particular embodiment, one group of> 50 unique encoder sequences are used for bonding agent libraries.

[0164] In certain embodiments, every kind of unique binding agent in the binding agent library has a unique coding sequence.For example, 20 kinds of unique coding sequences can be used for the library of 20 kinds of binding agents in conjunction with 20 kinds of standard amino acids.Extra coding tag sequences can be used for identifying the amino acid (for example, post-translationally modified amino acid) of modification.In another example, 30 kinds of unique coding sequences can be used for the library of 30 kinds of binding agents in conjunction with 20 kinds of standard amino acids and 10 kinds of post-translationally modified amino acids (for example phosphorylated amino acids, acetylated amino acids, methylated amino acids).In other embodiments, two or more different binding agents can share the same encoder sequence.For example, two kinds of binding agents in conjunction with different standard amino acids can share the same coding sequence.

[0165] In certain embodiments, the coding tag also comprises a spacer sequence at one or both ends. The spacer sequence is about 1 base to about 20 bases, about 1 base to about 10 bases, about 5 bases to about 9 bases, or about 4 bases to about 8 bases. In some embodiments, the length of the spacer is about 1 base, 2 bases, 3 bases, 4 bases, 5 bases, 6 bases, 7 bases, 8 bases, 9 bases, 10 bases, 11 bases, 12 bases, 13 bases, 14 bases, 15 bases, or 20 bases. In some embodiments, the spacer within the coding tag is shorter than the coding sequence, for example, at least 1 base, 2 bases, 3 bases, 4 bases, 5 bases, 6 bases, 7 bases, 8 bases, 9 bases, 10 bases, 11 bases, 12 bases, 13 bases, 14 bases, 15 bases, 20 bases or 25 bases. In other embodiments, the spacer within the coding tag is the same length as the encoder sequence. In certain embodiments, the spacer is binding agent specific, so that the spacer from the previous binding cycle only interacts with the spacer from the suitable binding agent in the current binding cycle. An example is a homologous antibody pair containing a spacer sequence, which allows the transfer of information only when the two antibodies bind to the polypeptide sequentially. The spacer sequence can be used as a primer annealing site for a primer extension reaction, or a splint or sticky end in a ligation reaction. The 5' spacer on the coding tag optionally contains pseudo-complementary bases of the 3' spacer on the recording tag to increase T m (Lehoudden et al., 2008, Nucleic Acids Res. 36:3409-3419) In other embodiments, the encoding tags within the library of binders do not have a binding cycle-specific spacer sequence.

[0166] In some embodiments, the coding tags within the binding agent set share a common spacer sequence used in the assay (e.g., the entire binding agent library used in the multiple binding cycle method has a common spacer in its coding tag). In another embodiment, the coding tag comprises a binding cycle tag that identifies a specific binding cycle. In other embodiments, the coding tag within the binding agent library has a binding cycle-specific spacer sequence. In some embodiments, the coding tag comprises a binding cycle-specific spacer sequence. For example, the coding tag for the binding agent in the first binding cycle comprises a "cycle 1" specific spacer sequence, the coding tag for the binding agent in the second binding cycle comprises a "cycle 2" specific spacer sequence, and so on reaches "n" binding cycles. In a further embodiment, the coding tag for the binding agent in the first binding cycle comprises a "cycle 1" specific spacer sequence and a "cycle 2" specific spacer sequence, and the coding tag for the binding agent in the second binding cycle includes a "cycle 2" specific spacer sequence and a "cycle 3" specific spacer sequence, and so on to "n" binding cycles. In some embodiments, the spacer sequence comprises a sufficient number of bases to anneal to a complementary spacer sequence in a recording tag or an extended recording tag to initiate a primer extension reaction or a sticky end ligation reaction.

[0167] In some embodiments, the coding tags associated with the binding agents used to bind in the alternating cycles include different binding cycle specific spacer sequences. For example, the coding tags for the binding agents used in the first binding cycle include the "cycle 1" specific spacer sequence, the coding tags for the binding agents used in the second binding cycle include the "cycle 2" specific spacer sequence, the coding tags for the binding agents used in the third binding cycle also include the "cycle 1" specific spacer sequence, and the coding tags for the binding agents used in the fourth binding cycle include the "cycle 2" specific spacer sequence. In this way, each cycle does not require a cycle-specific spacer.

[0168] When a group of record tags is associated with a polypeptide, a cycle-specific spacer sequence can also be used to cascade the information of the coding tag to a single record tag. The first binding cycle transfers the information from the coding tag to a randomly selected record tag, and subsequent binding cycles can trigger only the extended record tag through a cycle-dependent spacer sequence. More specifically, the coding tag of the binder used in the first binding cycle includes a "cycle 1" specific spacer sequence and a "cycle 2" specific spacer sequence, and the coding tag of the binder used in the second binding cycle includes a "cycle 2" specific spacer sequence and a "cycle 3" specific spacer sequence, and so on to "n" binding cycles. The coding tag of the binder from the first binding cycle can anneal with the record tag through a complementary cycle 1 specific spacer sequence. When transferring the coding tag information to the record tag, the cycle 2 specific spacer sequence is located at the 3' end of the extended record tag at the end of the binding cycle 1. The coding tag of the binder from the second binding cycle can anneal with the extended record tag through a complementary cycle 2 specific spacer sequence. When the coding tag information is transferred to the extended record tag, the cycle 3 specific spacer sequence is located at the 3' end of the extended record tag at the end of the binding cycle 2, and so on to complete the "n" binding cycle. This embodiment stipulates that the transfer of binding information in a specific binding cycle between multiple binding cycles will only occur on the (extended) record tag that has undergone the previous binding cycle. However, sometimes the binding agent cannot bind to the homologous polypeptide. Oligonucleotides containing binding cycle specific spacers after each binding cycle can be used as a "tracking" step to keep the binding cycle synchronized, even if the binding cycle event fails. For example, if the homologous binding agent cannot bind to the polypeptide during binding cycle 1, then after binding cycle 1, an oligonucleotide containing cycle 1 specific spacers, cycle 2 specific spacers and "invalid" coding sequences is used to increase the tracking step. The "invalid" encoder sequence can lack an encoder sequence, or preferably, it is a specific barcode that clearly identifies the "invalid" binding cycle. The "invalid" oligonucleotide can anneal with the record tag through the cycle 1 specific spacer, and the cycle 2 specific spacer is transferred to the record tag. Thus, despite the failed binding cycle 1 event, binders from binding cycle 2 are still able to anneal to the extended record tag through the cycle 2 specific spacer. The "null" oligonucleotide marks binding cycle 1 as a failed binding event within the extended record tag.

[0169] In some embodiments, the coding tag comprises a cleavable or nickable DNA chain within the second (3') spacer sequence adjacent to the binding agent. For example, the 3' spacer may have one or more uracil bases, which can be cut by a uracil-specific excision reagent (USER). USER produces a single nucleotide gap at the position of uracil. In another example, the 3' spacer may comprise a recognition sequence for a cutting endonuclease, which hydrolyzes only one chain of the duplex. Preferably, the enzyme used to cleave or cut the 3' spacer sequence acts only on one DNA chain (the 3' spacer of the coding tag), so that the other chain within the duplex belonging to the (extended) recording tag remains intact. These embodiments are particularly useful in the analysis of native conformation proteins because it allows the non-denaturing removal of the binding agent from the (extended) recording tag after primer extension occurs, and leaves a single-stranded DNA spacer sequence on the extended recording tag that can be used for subsequent binding cycles.

[0170] In certain embodiments, the coding tag may further comprise a unique molecular identifier of the binding agent attached to the coding tag.

[0171] The coding tag may include a terminator nucleotide incorporated at the 3' end of the 3' spacer sequence. After the binding agent binds to the polypeptide and its corresponding coding tag and the record tag anneal through the complementary spacer sequence, primer extension can transfer information from the coding tag to the record tag, or transfer information from the record tag to the coding tag. Adding a terminator nucleotide at the 3' end of the coding tag can prevent the record tag information from transferring to the coding tag. It should be understood that for embodiments related to generating extended coding tags herein, it is preferred to include a terminator nucleotide at the 3' end of the record tag to prevent the coding tag information from being transferred to the record tag.

[0172] The coding tag can be a single-stranded molecule, a double-stranded molecule or a partially double-stranded molecule. The coding tag may include a flat end, a hanging end or the same. In some embodiments, the coding tag is partially double-stranded, which prevents the coding tag from annealing with the internal encoder and spacer sequence in the extended extension record tag. In some embodiments, the coding tag comprises a hairpin. In certain embodiments, the hairpin comprises a mutually complementary nucleic acid region connected by a nucleic acid chain. In some embodiments, the nucleic acid hairpin may further include a 3' and / or 5' single-stranded region extending from a double-stranded stem segment. In some instances, the hairpin comprises a single-stranded nucleic acid.

[0173] In some examples, the coding tag may include a terminator nucleotide incorporated at the 3' end of the 3' spacer sequence. After the binding agent binds to the macromolecule and its corresponding coding tag and recording tag anneal through the complementary spacer sequence, primer extension can transfer information from the coding tag to the recording tag, or transfer information from the recording tag to the coding tag. Adding a terminator nucleotide at the 3' end of the coding tag can prevent the recording tag information from transferring to the coding tag. It should be understood that for embodiments of the present invention involving the generation of extended coding tags, it is preferred to include a terminator nucleotide at the 3' end of the recording tag to prevent the coding tag information from being transferred to the recording tag.

[0174] The coding tag is connected to the binding agent directly or indirectly by any means known in the art, including covalent and non-covalent interactions. In certain embodiments, the coding tag can be connected to the binding agent enzymatically or chemically. In certain embodiments, the coding tag can be combined with the binding agent by connection. In other embodiments, the coding tag is connected to the binding agent by affinity binding (e.g., biotin and streptavidin). In some cases, the coding tag can be connected to the binding agent of non-natural amino acids, for example, by covalent interactions with non-natural amino acids.

[0175] In some embodiments, the binding agent is connected to the coding tag by SpyCatcher-SpyTag interaction. SpyTag peptide forms an irreversible covalent bond with SpyCatcher protein through spontaneous isopeptide connection, thereby providing a genetically encoded way to produce resistance and harsh peptide interactions (Zakeri et al., 2012, Proc. Natl. Acad. Sci. 109: E690-697; Li et al., 2014, J. Mol. Biol. 426: 309-317). The binding agent can be expressed as a fusion protein comprising SpyCatcher protein. In some embodiments, SpyCatcher protein is attached to the N-terminus or C-terminus of the binding agent. The SpyTag peptide can be coupled to the coding tag using standard conjugation chemistry (Bioconjugate Techniques, GT Hermanson, Academic Press (2013)). In some embodiments, the binding agent is connected to the coding tag using an enzyme-based strategy. In one example, a protein (eg, SpyLigase) is used to link the binding agent to the coding tag (Fierer et al., Proc Natl Acad Sci USA, 2014 Apr1; 111(13): E1176-E1181).

[0176] In other embodiments, the binding agent is connected to the coding tag by the SnoopTag-SnoopCatcher peptide-protein interaction. The SnoopTag peptide forms an isopeptide bond with the SnoopCatcher protein (Veggiani et al., Proc. Natl. Acad. Sci. USA, 2016, 113: 1202-1207). The binding agent can be expressed as a fusion protein comprising the SnoopCatcher protein. In some embodiments, the SnoopCatcher protein is attached to the N-terminus or C-terminus of the binding agent. The SnoopTag peptide can be coupled to the coding tag using standard conjugation chemistry.

[0177] In other embodiments, the binding agent is Protein fusion tags and their chemical ligands are bound to the coding tag. HaloTag is a modified haloalkane dehalogenase designed to covalently bind to a synthetic ligand (HaloTag ligand) (Los et al., 2008, ACS Chem. Biol. 3: 373-382). The synthetic ligand contains a chloroalkane linker attached to a variety of useful molecules. The formation of a covalent bond between HaloTag and a highly specific chloroalkane linker occurs rapidly under physiological conditions and is essentially irreversible.

[0178] In some cases, the binding agent is linked to a coding tag by using enzyme attachment (conjugation), such as sortase-mediated labeling (see, e.g., Jouro, Antos et al., Curr Protoc Protein Sci. (2009) CHAPTER 15: Unit-15.3; International Patent Publication No. WO2013003555). The sortase catalyzes a transpeptidase reaction (see, e.g., Falck et al., Antibodies (2018) 7(4): 1-19). In some aspects, the binding agent is modified with or linked to one or more N-terminal or C-terminal glycine residues.

[0179] In some embodiments, the binding agent is linked to the encoding tag using π-clamp mediated cysteine ​​bioconjugation (see, e.g., Zhang et al., Nat Chem. (2016) 8(2): 120-128).

[0180] In some embodiments, the binding agent is directly or indirectly connected to the multimerization domain. Therefore, provided herein are monomers, dimers and higher order (e.g., 3,4,5 or higher order) multimeric polypeptides comprising one or more binding agents. In some specific embodiments, the binding agent is dimeric. In some instances, two polypeptides of the present invention can be covalently or non-covalently attached to each other to form a dimer.

[0181] In some embodiments, analyzing first order and / or second order (or higher order) extended record tags also evaluates partial tags.

[0182] In some embodiments, the first order and / or the second order (or higher order) extension record tag comprises a polynucleotide, such as DNA or RNA, and the first order and / or the second order (or higher order) extension record tag is evaluated to evaluate at least a partial sequence of the polynucleotide and / or a portion of the polypeptide, and / or to evaluate the polypeptide tag and / or a partial tag. Any suitable technique or program can be used to evaluate the polynucleotide sequence. For example, the polynucleotide sequence can be evaluated using Maxam-Gilbert sequencing, chain termination, shotgun sequencing, bridge PCR, single molecule real-time sequencing, ion semiconductor (ion flow sequencing), synthetic sequencing, connection sequencing (SOLiD sequencing), chain termination (Sanger sequencing), massively parallel tag sequencing (MPSS), polony sequencing, 454 pyrophosphate sequencing, Illumina (Solexa) sequencing, DNA nanoball sequencing, heliscope single molecule sequencing, single molecule real-time (SMRT) sequencing, nanopore DNA sequencing, tunnel current DNA sequencing, hybridization sequencing, mass spectrometry sequencing, microfluidic Sanger sequencing, microscopy-based technology, RNAP sequencing or in vitro virus high-throughput sequencing.

[0183] The methods of the present invention can be used to assess any suitable type of spatial proximity between a polypeptide and a portion in a sample. In some embodiments, the polypeptide and the portion are both parts of a larger polypeptide. In some examples, the larger polypeptide has a primary protein structure, and the polypeptide and the portion are spatially adjacent to the primary protein structure. In some instances, the larger polypeptide has a secondary, tertiary and / or quaternary protein structure, and the polypeptide and the portion are spatially adjacent to the secondary, tertiary and / or quaternary protein structure.

[0184] In other embodiments, the polypeptide and the portion belong to two different molecules. For example, the polypeptide and the portion may belong to two different proteins in the same protein complex. In other examples, the portion may be a portion of a polynucleotide molecule (e.g., a DNA or RNA molecule) that is bound to, complexed with, or in close proximity to the polypeptide in the sample. In these embodiments, the methods of the invention can be used to assess any suitable type of spatial proximity between or among different molecules, for example, spatial proximity between or among different subunits in a protein complex, a protein-DNA complex, or a protein-RNA complex.

[0185] II. Methods for Proximity Interaction Analysis Using Preassembled Structures

[0186] In one aspect, the present disclosure provides a method for assessing the identity and spatial relationship between a polypeptide and a moiety in a sample, the method comprising: a) providing a preassembled structure, the preassembled structure comprising a shared unique molecular identifier (UMI) and / or barcode in a middle portion, the UMI and / or barcode flanked on one side by a polypeptide tag and on the other side by a moiety tag; b) forming a junction between a site of a polypeptide in a sample and a site of a moiety in the sample by associating the polypeptide tag of the preassembled structure with a site of the polypeptide and associating the moiety tag of the preassembled structure with a site of the moiety c) disrupting the connection structure by dissociating the polypeptide from the portion and the polypeptide tag from the portion tag while maintaining the association between the polypeptide and the polypeptide tag and maintaining the association between the portion and the portion tag; and d) evaluating at least a partial sequence of the polypeptide tag and the polypeptide, and evaluating at least a partial identity of the portion tag and the portion; wherein the polypeptide tag and the evaluated portions of the portion tag comprise a shared unique molecular identifier (UMI) and / or barcode that indicates the spatial proximity of a site of the polypeptide and a site of the portion in the sample.

[0187] Any suitable moiety can be used in the present method. For example, the moiety can be an atom, an inorganic moiety, an organic moiety, or a complex thereof. The organic moiety can be an amino acid, a polypeptide (e.g., a peptide or protein), a nucleoside, a nucleotide, a polynucleotide (e.g., an oligonucleotide or nucleic acid), a vitamin, a monosaccharide, an oligosaccharide, a carbohydrate, a lipid, and a complex thereof. In some embodiments, the moiety can comprise a polypeptide. In other embodiments, the moiety can comprise a polynucleotide.

[0188] Any suitable polypeptide tag can be used in the present method. For example, the polypeptide tag can be an atom, an inorganic part, an organic part or a complex thereof. The organic part can be an amino acid, a polypeptide (e.g., a peptide or protein), a nucleoside, a nucleotide, a polynucleotide (e.g., an oligonucleotide or a nucleic acid), a vitamin, a monosaccharide, an oligosaccharide, a carbohydrate, a lipid and a complex thereof. In some embodiments, the polypeptide tag can comprise a polynucleotide.

[0189] Any suitable moiety tag can be used in the present method. For example, the moiety tag can be an atom, an inorganic moiety, an organic moiety, or a complex thereof. The organic moiety can be an amino acid, a polypeptide (e.g., a peptide or protein), a nucleoside, a nucleotide, a polynucleotide (e.g., an oligonucleotide or nucleic acid), a vitamin, a monosaccharide, an oligosaccharide, a carbohydrate, a lipid, and a complex thereof. In some embodiments, the moiety tag can comprise a polynucleotide.

[0190] Both the polypeptide tag and the partial tag may comprise polynucleotides. In some embodiments, the polypeptide tag comprises a UMI and / or a barcode. In some embodiments, the partial tag comprises a UMI and / or a barcode. In some embodiments, the polypeptide tag comprises a first polynucleotide and the partial tag comprises a second polynucleotide, the first and second polynucleotides comprise complementary sequences, and the polypeptide tag and the partial tag are associated via the complementary sequences.

[0191] In some embodiments, the preassembled structure comprises one or more barcodes or one or more UMIs. In some examples, each preassembled structure comprises two barcodes. In some examples, each preassembled structure comprises two UMIs. In some embodiments, a relationship or association of two or more associated UMIs of each preassembled structure is established. In some embodiments, two or more associated UMIs of the preassembled structure are evaluated (e.g., sequenced) to establish a relationship or association of the UMIs with each other. In some cases, two or more UMIs are synthesized into a preassembled structure. In some cases, two or more UMIs are connected (directly or indirectly via a linker) to form a preassembled structure. In some embodiments, the preassembled structure is connected to the polypeptide and the portion, for example by connecting a DNA containing one UMI of the preassembled structure to the polypeptide and the portion, and connecting a DNA containing one UMI of the preassembled structure to the portion. In some cases, after the preassembled structure is connected to the polypeptide and the portion, the two or more UMIs of the preassembled structure dissociate from each other (while each UMI remains associated with the polypeptide or the portion). In some embodiments, the relationship or association of two or more associated UMIs of each preassembled structure is established before dissociating the UMIs from each other. In some embodiments, the evaluation of the two or more associated UMIs is performed prior to dissociating the two or more associated UMIs from each other. In some embodiments, the method includes dissociating the two or more UMIs of the preassembled structure and dissociating the polypeptide and the moiety.

[0192] In some embodiments, the preassembled structure includes a cleavable or nickable DNA strand (e.g., between a first UMI and a second UMI). For example, the preassembled structure may have one or more uracil bases that can be nicked by a uracil-specific excision reagent (USER).

[0193] In some embodiments, the preassembled structure comprises a complementary sequence of the UMI. In some embodiments, the preassembled structure comprises a single-stranded DNA, a double-stranded DNA complex, a DNA duplex, or a DNA hairpin. In some embodiments, the preassembled structure comprising the UMI is synthesized or generated by extending or ligating from a template UMI sequence in the preassembled structure to generate a complementary sequence of the UMI sequence in the preassembled structure.

[0194] In some embodiments, the method provides a pre-assembled structure comprising a DNA cross-linker comprising a UMI or barcode ( Figure 4 A-4B). In some examples, a polypeptide and a moiety that are labeled or attached to a DNA complex adjacent to a DNA complex (e.g., a DNA crosslinker) or a portion thereof are dissociated from each other. After the polypeptide and the moiety dissociate, the polypeptide remains attached to one strand of the DNA complex (e.g., a DNA crosslinker) comprising the UMI or barcode, and the moiety remains attached to at least a portion of the complementary strand of the DNA complex (e.g., a DNA crosslinker) comprising the UMI or barcode. Figure 5 A-5C). In some embodiments, the DNA complex (e.g., a DNA crosslinker (or a portion thereof) is linked to the polypeptide and moiety directly or indirectly (e.g., linked to an attached nucleic acid) via enzymatic (e.g., ligation) or chemical methods.

[0195] In the connection structure, the polypeptide tag and the partial tag may be associated in any suitable manner. In some embodiments, in the connection structure, the polypeptide tag and the partial tag may be stably associated. In other embodiments, in the connection structure, the polypeptide tag and the partial tag may be transiently associated. The association between the polypeptide tag and the partial tag may change over time or with the execution of the method of the present invention. In other embodiments, in the connection structure, the polypeptide tag and the partial tag may be directly associated. In other embodiments, in the connection structure, the polypeptide tag and the partial tag may be indirectly associated, for example, via a linker or UMI between the polypeptide tag and the partial tag. In some embodiments, the connection structure is formed by associating the polypeptide tag of a preassembled structure (e.g., a DNA crosslinker) with a site of a polypeptide and associating the partial tag of the preassembled structure with the site of the portion.

[0196] When forming a connection structure, any suitable number of polypeptide tags can be associated with a suitable number of sites of a polypeptide. For example, in forming a connection structure, a single polypeptide tag can be associated with a single site of a polypeptide, a single polypeptide tag can be associated with multiple sites of a polypeptide, or multiple polypeptide tags can be associated with multiple sites of a polypeptide. Similarly, in forming a connection structure, any suitable number of partial tags can be associated with a suitable number of partial sites. For example, in forming a connection structure, a single partial tag can be associated with a single site of a part, a single partial tag can be associated with multiple sites of a part, or multiple partial tags can be associated with multiple sites of a part.

[0197] The formed ligation structure may include any suitable number of shared unique molecular identifiers (UMIs) and / or barcodes. For example, the formed ligation structure may include a single shared unique molecular identifier (UMI) and / or barcode. In another example, the formed ligation structure may include multiple shared unique molecular identifiers (UMIs) and / or barcodes. In some examples, the shared UMI and / or barcode is a composite tag or composite UMI that includes a sequence of a UMI and / or a barcode of a polypeptide tag and a sequence of a UMI and / or a barcode of a partial tag.

[0198] The UMI and / or barcode may comprise any suitable substance or sequence. In some embodiments, the UMI has a probability of occurring multiple times by chance in a sample that is appropriate or sufficiently low. In other embodiments, the UMI comprises a polynucleotide comprising about 3 nucleotides to about 40 nucleotides. The nucleotides in the UMI polynucleotide may or may not be consecutive. In other embodiments, the polynucleotide in the UMI comprises a degenerate sequence. In other embodiments, the polynucleotide in the UMI does not comprise a degenerate sequence. In other embodiments, the UMI comprises a nucleic acid, an oligonucleotide, a modified oligonucleotide, a DNA molecule, a DNA with pseudo-complementary bases, a DNA with protected bases, an RNA molecule, a BNA molecule, an XNA molecule, an LNA molecule, a PNA molecule, a γPNA molecule, or a morpholino DNA, or a combination thereof. The DNA molecule may be backbone-modified, sugar-modified, or nucleobase-modified. The DNA molecule may also have a nucleobase protecting group, such as Alloc, an electrophilic protecting group, such as thiarane, an acetyl protecting group, a nitrobenzyl protecting group, a sulfonate protecting group, or a traditional base-labile protecting group, including Ultramild reagents.

[0199] The polypeptide tag and the partial tag may be dissociated from each other using any suitable technique or procedure. For example, if the polypeptide tag and the partial tag are associated with each other via a polypeptide-polypeptide, polypeptide-polynucleotide, or polynucleotide-polynucleotide interaction, the polypeptide tag and the partial tag may be dissociated from each other using any technique or procedure suitable for disrupting such polypeptide-polypeptide, polypeptide-polynucleotide, or polynucleotide-polynucleotide interaction. In some embodiments, in the connection structure, the shared UMI and / or barcode comprises a complementary polynucleotide hybrid, and dissociating the polypeptide tag from the partial tag comprises denaturing the complementary polynucleotide hybrid.

[0200] The polypeptide and part can be dissociated from each other using any suitable technique or procedure. For example, if the polypeptide and part are associated with each other via polypeptide-polypeptide or polypeptide-polynucleotide interactions, the polypeptide and part can be dissociated from each other using any technique or procedure suitable for destroying such polypeptide-polypeptide or polypeptide-polynucleotide interactions. In some embodiments, the polypeptide and part are all part of a larger polypeptide, and the polypeptide is dissociated from the part including fragmenting the larger polypeptide into peptide fragments. Any suitable technique or procedure can be used to fragment the larger polypeptide. For example, the larger polypeptide fragments can be digested with a protease into peptide fragments. Any suitable protease can be used. For example, a protease can be an exopeptidase such as an aminopeptidase or a carboxypeptidase. In another example, a protease can be an endopeptidase or an endoproteinase, such as trypsin, LysC, LysN, ArgC, chymotrypsin, pepsin, thermolysin, papain or elastase. (See, e.g., Switzar, Giera et al., 2013).

[0201] The method of the present invention can be used to assess the identity and spatial relationship between a polypeptide and a portion in a sample, regardless of whether the polypeptide and the portion belong to the same molecule. For example, the target polypeptide and the portion can belong to two different molecules. In another example, the target polypeptide and the portion can be parts of the same molecule.

[0202] In some embodiments, the target polypeptide is part of a larger polypeptide, and the part is also part of the same larger polypeptide. The part can be any suitable substance or a complex thereof. For example, the part can comprise an amino acid or a polypeptide. The part of the amino acid or polypeptide can comprise one or more modified amino acids. Exemplary modified amino acids include glycosylated amino acids, phosphorylated amino acids, methylated amino acids, acylated amino acids, hydroxyproline or sulfated amino acids. The glycosylated amino acid can comprise an N-linked or O-linked glycosyl moiety. The phosphorylated amino acid can be phosphotyrosine, phosphoserine or phosphothreonine. The acylated amino acid can comprise a farnesyl, myristoyl or palmitoyl moiety. The sulfated amino acid can be part of a sulfotyrosine or a disulfide bond.

[0203] In other embodiments, the portion can be a portion of a molecule that is bound, complexed, or immediately adjacent to a polypeptide in a sample. The portion can be any suitable substance or a complex thereof. For example, the portion can be an atom, an amino acid, a polypeptide, a nucleoside, a nucleotide, a polynucleotide, a vitamin, a monosaccharide, an oligosaccharide, a carbohydrate, a lipid, or a complex thereof. In a specific embodiment, the portion comprises an amino acid or a polypeptide. The portion of amino acids or polypeptides may comprise one or more modified amino acids. Exemplary modified amino acids include glycosylated amino acids, phosphorylated amino acids, methylated amino acids, acylated amino acids, hydroxyproline, or sulfated amino acids. Glycosylated amino acids may comprise N-linked or O-linked glycosyl moieties. Phosphorylated amino acids may be phosphotyrosine, phosphoserine, or phosphothreonine. Acylated amino acids may comprise farnesyl, myristoyl, or palmitoyl moieties. Sulfated amino acids may be part of sulfotyrosine or a disulfide bond.

[0204] In some embodiments, the polypeptide and the moiety may belong to two different proteins in the same protein complex. In other embodiments, the moiety may be a portion of a polynucleotide molecule (e.g., a DNA or RNA molecule) that is bound, complexed, or in close proximity to a polypeptide in a sample.

[0205] Any suitable technique or procedure can be used to evaluate the at least partial sequence of a polypeptide tag, a partial tag, a polypeptide, and / or at least partial identity of the portion. For example, if the polypeptide tag, the portion, and / or the partial tag comprises a polypeptide and / or a polynucleotide, any suitable technique or procedure for evaluating the identity or sequence of the polypeptide and / or polynucleotide can be used. Similarly, any suitable technique or procedure for evaluating a polypeptide can be used to evaluate at least a partial sequence of a polypeptide.

[0206] In some embodiments, the polypeptide tag and / or partial tag comprises a polypeptide, and the polypeptide tag and / or partial tag can be evaluated using a binding assay (e.g., an immunoassay). Exemplary immunoassays include enzyme-linked immunosorbent assay (ELISA), immunoblotting, immunoprecipitation, radioimmunoassay (RIA), immunostaining, latex agglutination, indirect hemagglutination assay (IHA), complement fixation, indirect immunofluorescence assay (IFA), turbidimetry, flow cytometry, surface plasmon resonance (SPR), chemiluminescence assay, lateral flow immunoassay, u-capture assay, inhibition assay, and affinity assay.

[0207] In some embodiments, the polypeptide tag and / or partial tag comprises a polynucleotide, such as DNA or RNA. Before or simultaneously with the evaluation, the polynucleotide can be amplified. The polynucleotides in the polypeptide tag and / or partial tag can be amplified using any suitable technique or procedure. For example, the polynucleotide can be amplified using polymerase chain reaction (PCR), strand displacement amplification (SDA), transcription-mediated amplification (TMA), ligase chain reaction (LCR), nucleic acid sequence-based amplification (NASBA), primer extension, rolling circle amplification (RCA), autonomous sequence replication system (3SR), or loop-mediated isothermal amplification (LAMP) procedures.

[0208] Any suitable technique or procedure may be used to assess at least a portion of the sequence of the polypeptide or at least a portion of the identity of the portion. If the portion comprises a polypeptide, at least a portion of the sequence of both the polypeptide and the portion may be assessed by any suitable polypeptide sequencing technique or procedure. For example, at least a portion of the sequence of both the polypeptide and the portion may be assessed by N-terminal amino acid analysis, C-terminal amino acid analysis, Edman degradation, and mass spectrometry. In another example, at least a portion of the sequence of both the polypeptide and the portion can be evaluated by techniques disclosed and / or claimed in U.S. Provisional Patent Application Nos. 62 / 330,841, 62 / 339,071, 62 / 376,886, 62 / 579,844, 62 / 582,312, 62 / 583,448, 62 / 579,870, 62 / 579,840, and 62 / 582,916, and International Patent Application No. PCT / US2017 / 030702 (published as WO 2017 / 192633 A1). For example, any technique or procedure for evaluating a macromolecule (e.g., a polypeptide) provided herein, such as a polypeptide described in Section I, can be used to evaluate at least a portion of the sequence of a polypeptide or at least a portion of the identity.

[0209] In some embodiments, at least a partial sequence of a polypeptide is evaluated using a procedure comprising the following steps: a1) providing a polypeptide and an associated polypeptide tag as a record tag; b1) contacting the polypeptide with a first binding agent capable of binding to the polypeptide, wherein the first binding agent comprises a first coding tag having identification information about the first binding agent; c1) transferring the information of the first coding tag to the record tag to generate a first order extended record tag; d1) analyzing the first order extended record tag. This step a1) may further comprise providing a polypeptide bound to a solid support and an associated polypeptide tag. The method may further comprise contacting the polypeptide with a second (or higher order) binding agent, the second (or higher order) binding agent comprising a second (or higher order) binding moiety capable of binding to the polypeptide and a coding tag having identification information about the second (or higher order) binding agent, transferring the information of the second (or higher order) coding tag to the first order extended record tag to generate a second order (or higher order) extended record tag, and analyzing the second order (or higher order) extended record tag.

[0210] In some embodiments, at least a partial sequence of a polypeptide is evaluated using a procedure comprising the following steps: a1) providing a polypeptide and an associated polypeptide tag as a record tag; b1) contacting the polypeptide with a first binding agent capable of binding to the N-terminal amino acid (NTAA) of the polypeptide, wherein the first binding agent comprises a first coding tag having identification information about the first binding agent; c1) transferring the information of the first coding tag to the record tag to generate a first order extended record tag; d1) analyzing the extended record tag. The method may further comprise providing a polypeptide and an associated polypeptide tag bound to a solid support. The method also comprises contacting the target polypeptide with a second (or higher order) binding agent comprising a second (or higher order) coding tag, the second (or higher order) coding tag having identification information about the second (or higher order) binding agent, wherein the second (or higher order) binding agent is capable of binding to an NTAA other than the NTAA of the polypeptide. The contact between the polypeptide and the second (or higher order) binding agent may be performed in any suitable manner. For example, after the polypeptide is contacted with the first binding agent, the polypeptide may then be contacted with the second (or higher order) binding agent. In another example, contacting the polypeptide with a second (or higher order) binding agent can occur simultaneously with contacting the polypeptide with a first binding agent.

[0211] In some embodiments, at least a portion of the sequence of a polypeptide is evaluated using a procedure comprising the following steps: a1) providing a polypeptide and an associated polypeptide tag as a record tag; b1) contacting the polypeptide with a first binding agent capable of binding to the N-terminal amino acid (NTAA) of the polypeptide, wherein the first binding agent comprises a first encoding tag having identification information about the first binding agent; c1) transferring the information of the first encoding tag to the record tag to generate a first order extended record tag; d1) removing the NTAA to expose a new NTAA of the target polypeptide; e1) contacting the polypeptide with a second (or higher order binding agent); The method comprises contacting a first extended recording tag with a second (or higher order) binding agent, wherein the second (or higher order) binding agent comprises a second (or higher order) coding tag having identification information about the second (or higher order) binding agent, wherein the second (or higher order) binding agent is capable of binding to the new NTAA, wherein the second (or higher order) binding agent comprises a second coding tag having identification information about the second (or higher order) binding agent; f1) transferring the information of the second (or higher order) coding tag to the first extended recording tag to generate a second (or higher order) extended recording tag; g1) analyzing the second (or higher order) extended recording tag. Steps d1)-g1) can be repeated one or more times. The method can further include providing a polypeptide and an associated polypeptide tag bound to a solid support.

[0212] In some embodiments, at least a partial sequence of a polypeptide is evaluated using a procedure comprising the following steps: a1) providing a polypeptide and an associated polypeptide tag as a record tag; b1) modifying the N-terminal amino acid (NTAA) of the polypeptide, for example, using a chemical agent; c1) contacting the polypeptide with a first binding agent capable of binding to the modified NTAA, wherein the first binding agent comprises a first coding tag having identification information about the first binding agent; d1) transferring the information of the first coding tag to the record tag to generate a first order extended record tag; e1) analyzing the first order extended record tag. This step a1) may further comprise providing a polypeptide bound to a solid support and an associated polypeptide tag. The method also comprises contacting the polypeptide with a second (or higher order) binding agent comprising a second (or higher order) coding tag, the second (or higher order) coding tag having identification information about the second (or higher order) binding agent, wherein the second (or higher order) binding agent is capable of binding to a modified NTAA other than the modified NTAA of step b1). The contact between the polypeptide and the second (or higher order) binding agent may be performed in any suitable manner. For example, after the target polypeptide is contacted with the first binding agent, the polypeptide can then be contacted with the second (or higher order) binding agent. In another example, the contact of the polypeptide with the second (or higher order) binding agent can occur simultaneously with the contact of the polypeptide with the first binding agent.

[0213] In some embodiments, analysis of the first order and / or second order (or higher order) extension record tags also evaluates the polypeptide tags.

[0214] In some embodiments, the portion comprises a portion of a polypeptide, and at least partial identity or sequence of the portion is evaluated using a procedure comprising the following steps: a2) providing a portion of a polypeptide and an associated portion tag as a record tag; b2) contacting the portion of the polypeptide with a first binding agent capable of binding to the portion of the polypeptide, wherein the first binding agent comprises a first coding tag having identification information about the first binding agent; c2) transferring information of the first coding tag to the record tag to generate a first order extended record tag; d2) analyzing the first order extended record tag. The method may further comprise contacting the portion of the polypeptide with a second (or higher order) binding agent, the second (or higher order) binding agent comprising a second (or higher order) binding moiety capable of binding to the portion of the polypeptide and a coding tag having identification information about the second (or higher order) binding agent, transferring information of the second (or higher order) coding tag to the first order extended record tag to generate a second order (or higher order) extended record tag, and analyzing the second order (or higher order) extended record tag.

[0215] In some embodiments, at least partial identity or sequence of a portion is evaluated using a procedure comprising the following steps: a2) providing a partial polypeptide and an associated partial tag as a record tag; b2) contacting the partial polypeptide with a first binding agent capable of binding to the N-terminal amino acid (NTAA) of the partial polypeptide, wherein the first binding agent comprises a first coding tag having identification information about the first binding agent; c2) transferring the information of the first coding tag to the record tag to generate a first order extended record tag; d2) analyzing the extended record tag. The method may further comprise providing a partial polypeptide and an associated polypeptide tag bound to a solid support. The method also comprises contacting the partial polypeptide with a second (or higher order) binding agent comprising a second (or higher order) coding tag, the second (or higher order) coding tag having identification information about the second (or higher order) binding agent, wherein the second (or higher order) binding agent is capable of binding to an NTAA other than the NTAA of the polypeptide. The contact between the partial polypeptide and the second (or higher order) binding agent may be performed in any suitable manner. For example, after the partial polypeptide is contacted with the first binding agent, the partial polypeptide can then be contacted with the second (or higher order) binding agent. In another example, the partial polypeptide contacting with the second (or higher order) binding agent can occur simultaneously with the partial polypeptide contacting with the first binding agent.

[0216] In some embodiments, at least a portion of the sequence of a partial polypeptide is evaluated using a procedure comprising the following steps: a2) providing a partial polypeptide and an associated partial tag as a record tag; b2) contacting the partial polypeptide with a first binding agent capable of binding to the N-terminal amino acid (NTAA) of the partial polypeptide, wherein the first binding agent comprises a first coding tag having identification information about the first binding agent; c2) transferring the information of the first coding tag to the record tag to generate a first sequence extension record tag; d2) removing the NTAA to expose a new NTAA of the partial polypeptide; e2) contacting the partial polypeptide with a second (or more The method further comprises contacting a first (or higher order) binding agent with a second (or higher order) binding agent, the second (or higher order) binding agent comprising a second (or higher order) coding tag having identification information about the second (or higher order) binding agent, wherein the second (or higher order) binding agent is capable of binding to the new NTAA, wherein the second (or higher order) binding agent comprises a second coding tag having identification information about the second (or higher order) binding agent; f2) transferring the information of the second (or higher order) coding tag to the first extended recording tag to generate a second (or higher order) extended recording tag; g2) analyzing the second (or higher order) extended recording tag. Steps d2)-g2) can be repeated one or more times. The method can further comprise providing a polypeptide and an associated polypeptide tag bound to a solid support.

[0217] In some embodiments, at least a partial sequence of a partial polypeptide is evaluated using a procedure comprising the following steps: a2) providing a partial polypeptide and an associated partial tag as a record tag; b2) modifying the N-terminal amino acid (NTAA) of the partial polypeptide, for example, using a chemical agent for modification; c2) contacting the partial polypeptide with a first binding agent capable of binding to the modified NTAA, wherein the first binding agent comprises a first coding tag having identification information about the first binding agent; d2) transferring the information of the first coding tag to the record tag to generate a first order extended record tag; e2) analyzing the first order extended record tag. This step a2) may further comprise providing a polypeptide attached to a solid support and an associated partial tag. The method also comprises contacting the polypeptide with a second (or higher order) binding agent comprising a second (or higher order) coding tag, the second (or higher order) coding tag having identification information about the second (or higher order) binding agent, wherein the second (or higher order) binding agent is capable of binding to a modified NTAA other than the modified NTAA of step b1). The contact between the partial polypeptide and the second (or higher order) binding agent may be performed in any suitable manner. For example, after the partial polypeptide is contacted with the first binding agent, the partial polypeptide can then be contacted with the second (or higher order) binding agent. In another example, the partial polypeptide contacting with the second (or higher order) binding agent can occur simultaneously with the partial polypeptide contacting with the first binding agent.

[0218] In some embodiments, analyzing first order and / or second order (or higher order) extended record tags also evaluates partial tags.

[0219] In some embodiments, the first order and / or the second order (or higher order) extension record tag comprises a polynucleotide, such as DNA or RNA, and the first order and / or the second order (or higher order) extension record tag is evaluated to evaluate at least a partial sequence of the polynucleotide and / or a portion of the polypeptide, and / or to evaluate the polypeptide tag and / or a partial tag. Any suitable technique or program can be used to evaluate the polynucleotide sequence. For example, the polynucleotide sequence can be evaluated using Maxam-Gilbert sequencing, chain termination, shotgun sequencing, bridge PCR, single molecule real-time sequencing, ion semiconductor (ion flow sequencing), synthetic sequencing, connection sequencing (SOLiD sequencing), chain termination (Sanger sequencing), massively parallel tag sequencing (MPSS), polony sequencing, 454 pyrophosphate sequencing, Illumina (Solexa) sequencing, DNA nanoball sequencing, heliscope single molecule sequencing, single molecule real-time (SMRT) sequencing, nanopore DNA sequencing, tunnel current DNA sequencing, hybridization sequencing, mass spectrometry sequencing, microfluidic Sanger sequencing, microscopy-based technology, RNAP sequencing or in vitro virus high-throughput sequencing.

[0220] The method of the present invention can be used to assess the spatial proximity of any suitable type between polypeptide and part in a sample. In certain embodiments, polypeptide and part are all parts of a larger polypeptide. In some examples, a larger polypeptide has a primary protein structure, and polypeptide and part are spatially adjacent to the primary protein structure. In some instances, a larger polypeptide has a secondary, tertiary and / or quaternary protein structure, and polypeptide and part are spatially adjacent to the secondary, tertiary and / or quaternary protein structure. In other embodiments, polypeptide and part belong to two different molecules. For example, polypeptide and part can belong to two different proteins in the same protein complex. In other examples, the part can be a part of a polynucleotide molecule (e.g., DNA or RNA molecule) that is combined, compounded or next to the polypeptide in the sample. In these embodiments, the method of the present invention can be used to assess the spatial proximity of any suitable type between or among different molecules, such as spatial proximity between or among different subunits in a protein complex, a protein-DNA complex or a protein-RNA complex.

[0221] III. Uses of the Method

[0222] The present method can be used for any suitable purpose. In certain embodiments, the method of the present invention can be used for assessing the spatial relationship between a single polypeptide and a single part in a sample. In other embodiments, the method of the present invention can be used for assessing the spatial relationship between a single polypeptide and multiple parts in a sample. In other embodiments, the method of the present invention can be used for assessing the spatial relationship between multiple polypeptides and multiple parts in a sample.

[0223] In some embodiments, polypeptide and part all belong to the same molecule, and this method is used to identify and / or evaluate the interaction between polypeptide and part in the same molecule.For example, part can be partial amino acid or partial polypeptide in the same protein of polypeptide, and this method is used to identify and / or evaluate the interaction between partial amino acid or partial polypeptide in polypeptide and protein.In another example, method of the present invention is used to identify and / or evaluate the interaction region or domain in the same protein.In another example, part is partial amino acid of modification or partial polypeptide of modification, and this method is used to identify and / or evaluate the interaction between polypeptide and partial amino acid of modification or partial polypeptide of modification in protein.In some embodiments, polypeptide and part are all part of larger polypeptide, and polypeptide and part are spatially adjacent to secondary, tertiary and / or quaternary protein structure.

[0224] In some embodiments, the methods of the present invention may further comprise preserving the structure of the target molecule prior to analysis, such as by cross-linking. For example, the target molecule may be a target protein, and the methods of the present invention may further comprise preserving the structure of the target protein prior to analysis, such as by cross-linking. In such examples, the methods of the present invention may be used to identify and / or assess disulfide bonds in the target protein.

[0225] In some embodiments, the portion belongs to a molecule that is bound, compounded, or immediately adjacent to a target protein comprising a target polypeptide, and the method is used to identify and / or evaluate the interaction between a target protein and a molecule that is bound, compounded, or immediately adjacent to the target protein in a sample. For example, a portion can be a portion of an amino acid or a portion of a polypeptide in a portion of a protein, which is bound, compounded, or immediately adjacent to a target protein comprising a target polypeptide, and the method is used to identify and / or evaluate the interaction between a target protein and a portion of a protein in a sample. In another example, the method is used to identify and / or evaluate the target protein and an interaction region or domain in a portion of a protein that is bound, compounded, or immediately adjacent to the target protein, for example, to identify and / or evaluate the interaction region or domain involved in protein subunit binding or compounding, or protein-ligand binding or compounding. In yet another example, the method is used to evaluate the probability that two or more polypeptide regions or domains belong to the same protein, the same protein binding pair, or the same protein complex.

[0226] In some embodiments, the at least partial sequence of the polypeptide and the at least partial identity of the part are evaluated separately from the connection structure between the polypeptide and the part. For example, after the connection structure is formed between the polypeptide and the part and after the information is transferred between the polypeptide tag and the part tag to form a shared unique molecular identifier and / or barcode, the at least partial sequence of the polypeptide and the at least partial identity of the part are evaluated. In some examples, the at least partial sequence of the polypeptide and the at least partial identity of the part are evaluated after the polypeptide and the part are dissociated. In some aspects, after the polypeptide (with the associated polypeptide tag) is fixed on the support, and after the part (with the associated part tag) is fixed on the solid support, the at least partial sequence of the polypeptide and the at least partial identity of the part are evaluated. In some of any such embodiments, the at least partial sequence of the polypeptide and the at least partial identity of the part are evaluated, including contacting the polypeptide and the part with one or more binding agents. In some examples, contacting the polypeptide and moiety with one or more binding agents is performed as follows: after formation of a linkage structure between the polypeptide and the moiety and after transfer of information between the polypeptide tag and the moiety tag to form a shared unique molecular identifier and / or barcode; after dissociation of the polypeptide from the moiety; after immobilization of the polypeptide (with associated polypeptide tag) on ​​a support and after immobilization of the moiety (with associated moiety tag) on ​​a solid support.

[0227] In some embodiments, the method further comprises a physical partitioning step, for example, partitioning by emulsion or other physical partitioning techniques. In some embodiments, the method does not comprise a physical partitioning step.

[0228] In certain embodiments, the method of the present invention is also included in limiting the number of proteins in the analysis, for example limiting the average number of proteins. The number of proteins in the analysis can be limited by any suitable technology or procedure. For example, the number of proteins can be limited by dilution. In another example, the number of proteins can be limited by binding the protein to a solid support such as beads. In certain embodiments, paired or interactive polypeptides and parts are immobilized on a solid support to achieve desired sampling. In some cases, polypeptides and parts are fixed to increase the possibility that both polypeptides and parts are fixed on the same solid support. In some instances, polypeptides or parts (and their associated labels) are fixed on a solid support, then the polypeptides are dissociated from the parts, and the other of the polypeptides or parts is fixed on the same solid support (for example, the same beads).

[0229] In some embodiments, the methods of the invention can be used to analyze proteins in their native conformation. In some embodiments, the formation of the junction structure between the polypeptide and the moiety is performed on the polypeptide and the moiety in a sample that is interactive or spatially close while each retaining its secondary, tertiary and / or quaternary protein structure. In other embodiments, the methods of the invention can be used to analyze denatured or renatured proteins.

[0230] In some embodiments, the methods of the invention can be used to analyze a proteome, such as an entire proteome. The proteome can be a viral proteome, a viral fraction, a cell fraction, an organelle, a cell, a tissue, an organ, an organism, or a biological sample.

[0231] The methods of the present invention can be used to assess the spatial relationship between a polypeptide and a moiety in any suitable sample. In some embodiments, the methods of the present invention can be used to assess the spatial relationship between a target polypeptide and a moiety in a biological sample (e.g., blood, plasma, serum or urine sample).

[0232] In some embodiments, the methods of the invention can be performed homogeneously, for example in solution. In some embodiments, the methods of the invention can be performed heterogeneously, for example in suspension.

[0233] IV. Kits and Articles for Assessing Spatial Relationships

[0234] Provided herein are kits for assessing the spatial relationship between one or more polypeptides and one or more moieties in a sample, comprising using any of the methods provided herein. In one aspect, the kit further comprises instructions describing a method for assessing a sample using the methods provided herein. In some embodiments, provided herein are kits and components for a method of analyzing a macromolecule, the method comprising: a) forming a junction structure between a site of a polypeptide in the sample and a site of a moiety in the sample, the junction structure comprising a polypeptide tag associated with the site of the polypeptide and a moiety tag associated with the site of the moiety, wherein the polypeptide tag and the moiety tag are associated; b) transferring information between the associated polypeptide tag and the moiety tag or connecting the associated polypeptide tag and the moiety tag to form a shared unique molecular identifier (UMI) and / or barcode; c) destroying the junction structure by dissociating the polypeptide from the moiety and dissociating the polypeptide tag from the moiety tag while maintaining the association between the polypeptide and the polypeptide tag and maintaining the association between the moiety and the moiety tag; and d) evaluating at least a partial sequence of the polypeptide tag and the polypeptide, and evaluating at least a partial identity of the moiety tag and the moiety; wherein the polypeptide tag and the evaluated portions of the moiety tag comprise a shared unique molecular identifier (UMI) and / or barcode that indicates the spatial proximity of the site of the polypeptide and the site of the moiety in the sample.

[0235] In some embodiments, provided herein are kits and components in a method for assessing the identity and spatial relationship between a polypeptide and a moiety, the method comprising: a) forming a junction structure between a site of a polypeptide in the sample and a site of a moiety in the sample, the junction structure comprising a polypeptide tag associated with the site of the polypeptide and a moiety tag associated with the site of the moiety, wherein the polypeptide tag and the moiety tag are associated; b) transferring information between the associated polypeptide tag and the moiety tag to form a shared unique molecular identifier (UMI) and / or barcode, wherein the shared UMI and / or barcode is formed as a separate record polynucleotide; c) disrupting the junction structure by dissociating the polypeptide from the moiety and dissociating the polypeptide tag from the moiety tag while maintaining the association between the polypeptide and the polypeptide tag and maintaining the association between the moiety and the moiety tag; d) assessing at least a partial sequence of the polypeptide tag and the polypeptide, and assessing at least a partial identity of the moiety tag and the moiety; and e) assessing the separate record polynucleotide to establish a spatial relationship between the site of the polypeptide and the site of the moiety.

[0236] In some embodiments, provided herein are kits and components for methods for a) providing a preassembled structure comprising a shared unique molecular identifier (UMI) and / or barcode in a middle portion, the UMI and / or barcode flanked on one side by a polypeptide tag and on another side by a moiety tag; b) forming a junction structure between a site of a polypeptide in a sample and a site of a moiety in the sample by associating the polypeptide tag of the preassembled structure with a site of the polypeptide and associating the moiety tag of the preassembled structure with a site of the moiety; c) disrupting the junction structure by dissociating the polypeptide from the moiety and the polypeptide tag from the moiety tag while maintaining an association between the polypeptide and the polypeptide tag and maintaining an association between the moiety and the moiety tag; and d) evaluating at least a partial sequence of the polypeptide tag and the polypeptide, and evaluating at least a partial identity of the moiety tag and the moiety; wherein the polypeptide tag and the evaluation portion of the moiety tag comprise a shared unique molecular identifier (UMI) and / or barcode that indicates the spatial proximity of the site of the polypeptide and the site of the moiety in the sample.

[0237] In some embodiments, kits provided herein include components for performing methods of assessing spatial interactions and / or relationships, reaction mixture compositions comprising the components, and kits for constructing reaction mixtures.

[0238] In some embodiments, the kit comprises one or more polypeptide tags and one or more part tags; a reagent for forming a connection structure between a polypeptide and a part in a sample; and a reagent for evaluating the identity of a part to at least a portion of the sequence of a polypeptide. In some embodiments, the kit also comprises instructions for evaluating the identity and spatial relationship between polypeptides. In some embodiments, the kit comprises instructions for preparing a sample. In some embodiments, the kit comprises components, such as the polypeptides and polynucleotides described in Sections I and II.

[0239] In some embodiments, the kit comprises one or more polypeptide tags and one or more moiety tags; a reagent for forming a connection structure between the polypeptide and the moiety in a sample, wherein the connection structure is formed as an isolated record polynucleotide; and a reagent for evaluating the identity of the moiety to at least a portion of the sequence of the polypeptide. In some of any of the embodiments provided, the kit further comprises a reagent for analyzing the isolated record polynucleotide.

[0240] In some of any of the embodiments provided, the kit further comprises one or more reagents for ligation (e.g., enzymatic or chemical ligation, splint ligation, sticky end ligation, single-stranded (ss) ligation such as ssDNA ligation, or any combination thereof), polymerase-mediated reaction (e.g., primer extension of single-stranded nucleic acid or double-stranded nucleic acid), or any combination thereof. In some embodiments, the ligation reagent is (i) a chemical ligation reagent or a biological ligation reagent, for example, a ligase, such as a DNA ligase or RNA ligase for ligating single-stranded nucleic acid or double-stranded nucleic acid, or (ii) a reagent for primer extension of single-stranded nucleic acid or double-stranded nucleic acid, optionally, wherein the kit further comprises a ligation reagent, the ligation reagent comprises at least two ligases or variants thereof (e.g., at least two DNA ligases, or at least two RNA ligases, or at least one DNA ligase and at least one RNA ligase), wherein the at least two ligases or variants thereof comprise an adenylating ligase and a constitutively non-adenylated ligase, or optionally wherein the kit further comprises a ligation reagent, the ligation reagent comprises a DNA or RNA ligase and a DNA / RNA deadenylase.

[0241] In some embodiments, the kit comprises reagents for assessing the identity of a portion and at least a partial sequence of a polypeptide. In some cases, the kit comprises a library of binding agents, wherein each binding agent comprises a binding moiety and an encoded polymer, the encoded polymer comprising identification information about the binding moiety, in some embodiments, the binding moiety is capable of binding to one or more N-terminal, internal, or C-terminal amino acids of the fragment, or is capable of binding to one or more N-terminal, internal, or C-terminal amino acids modified by a functionalizing agent;

[0242] In some embodiments, the kit comprises a reagent for providing a polypeptide directly or indirectly associated with a polypeptide tag and a reagent for providing a moiety directly or indirectly associated with a moiety tag; a reagent for functionalizing the N-terminal amino acid (NTAA) of the polypeptide; a first binding agent or a first detectable label, the first binding agent comprising a first binding moiety capable of binding to functionalized NTAA and a first coding tag having identification information about the first binding agent; and a reagent for transferring the information of the first coding tag to a recording tag to generate an extended recording tag. In some embodiments, the kit further comprises a reagent for analyzing the extended recording tag or a reagent for detecting the first detectable tag.

[0243] In some embodiments, the kit further comprises a reagent for removing the functionalized NTAA to expose the new NTAA. Any suitable removal reagent can be used. In some embodiments, the amino acid removed is an amino acid modified using any method or reagent provided herein. For example, the reagent may comprise an enzyme or chemical reagent to remove one or more terminal amino acids. For example, in some cases, the reagent for removing the functionalized NTAA is a carboxypeptidase, an aminopeptidase, or a dipeptidyl peptidase, a dipeptidyl aminopeptidase, or a variant, mutant, or modified protein thereof; a hydrolase or a variant, mutant, or modified protein thereof; a mild Edman degradation reagent; an Edmanase enzyme; TFA; a base; or any combination thereof. In some cases, the removal reagent comprises trifluoroacetic acid or hydrochloric acid. In some examples, the removal reagent comprises an acyl peptide hydrolase (APH). In some embodiments, the removal reagent comprises a carboxypeptidase or an aminopeptidase or a variant, mutant, or modified protein thereof; a hydrolase or a variant, mutant, or modified protein thereof; a mild Edman degradation reagent; an Edmanase enzyme; anhydrous TFA, a base; or any combination thereof. In some embodiments, the mild Edman degradation reagent uses dichloro acid or monochloro acid; the mild Edman degradation reagent uses TFA, TCA or DCA; or the mild Edman degradation reagent uses triethylamine, triethanolamine or triethylammonium acetate (Et3NHOAc).

[0244] In some cases, the reagent for removing the amino acid comprises a base. In some embodiments, the base is a hydroxide, an alkylated amine, a cyclic amine, a carbonate buffer, or a metal salt. In some embodiments, the hydroxide is sodium hydroxide; the alkylated amine is selected from methylamine, ethylamine, propylamine, dimethylamine, diethylamine, dipropylamine, trimethylamine, triethylamine, tripropylamine, cyclohexylamine, benzylamine, aniline, diphenylamine, N,N-diisopropylethylamine (DIPEA) and lithium diisopropylaminoamide (LDA); the cyclic amine is selected from pyridine, pyrimidine, imidazole, pyrrole, indole, piperidine, pyrrolidine (prolidine), 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), 1,5-diazabicyclo[4.3.0]non-5-ene (DBN); the carbonate buffer comprises sodium carbonate, potassium carbonate, calcium carbonate, sodium bicarbonate, potassium bicarbonate or calcium bicarbonate; the metal salt comprises silver; or the metal salt is AgClO4.

[0245] In some embodiments, the method further comprises contacting the polypeptide with a peptide coupling agent. In some embodiments, the peptide coupling agent is a carbodiimide compound. In some examples, the carbodiimide compound is diisopropylcarbodiimide (DIC) or 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC).

[0246] In one aspect, the kit also includes a buffer for use with the provided method. In some instances, the kit also includes a detergent or a surfactant. In certain embodiments, the provided kit includes a buffer for information transfer between a polypeptide tag and a partial tag, for extension of polynucleotides, for primer extension reactions and / or for ligation reactions. In one aspect, the kit also includes one or more solutions or buffers (e.g., Tris, MOPS, etc.) for implementing the method according to any method of the present invention.

[0247] In any of the foregoing embodiments, the kit can comprise a support or substrate, such as a rigid solid support, a flexible solid support, or a soft solid support, and including a porous support or a non-porous support.

[0248] In any of the foregoing embodiments, the kit can include a support comprising beads, porous beads, porous matrices, arrays, surfaces, glass surfaces, silicon surfaces, plastic surfaces, slides, filters, nylon, chips, silicon crystal chips, flow-through chips, biochips comprising signal transduction electronic devices, wells, microtiter wells, plates, ELISA plates, disks, rotating interferometry disks, membranes, nitrocellulose membranes, nitrocellulose-based polymer surfaces, nanoparticles (e.g., comprising metals such as magnetic nanoparticles (Fe3O4), gold nanoparticles, and / or silver nanoparticles), quantum dots, nanoshells, nanomicrospheres, or any combination thereof. In one embodiment, the support comprises polystyrene beads, polymer beads, agarose beads, acrylamide beads, solid core beads, porous beads, paramagnetic beads, glass beads, or controlled pore beads, or any combination thereof. In some embodiments, the support or substrate comprises a plurality of spatially resolved attachment sites.

[0249] In any provided embodiment, the kit can include a support and / or can be used to analyze multiple analytes (e.g., polypeptides) in a sequential reaction, a parallel reaction, or a combination of sequential and parallel reactions. In one embodiment, the analytes are spaced on the support at an average distance of equal to or greater than about 10 nm, equal to or greater than about 15 nm, equal to or greater than about 20 nm, equal to or greater than about 50 nm, equal to or greater than about 100 nm, equal to or greater than about 150 nm, equal to or greater than about 200 nm, equal to or greater than about 250 nm, equal to or greater than about 300 nm, equal to or greater than about 350 nm, equal to or greater than about 400 nm, equal to or greater than about 450 nm, or equal to or greater than about 500 nm.

[0250] In some embodiments, the kit further comprises one or more containers or vessels, such as tubular containers (eg, test tubes, capillaries, Eppendorf tubes) for implementing the methods of use. In some examples, each component is provided in a separate vessel.

[0251] In one aspect, the kit further comprises one or more oligonucleotides, and in one aspect (optionally) free nucleotides, and in one aspect (optionally) sufficient free nucleotides to perform a PCR reaction, rolling circle replication, ligase chain reaction, reverse transcription, nucleic acid labeling or labeling reaction or a derivative method thereof.

[0252] In one aspect, the kit further comprises at least one enzyme, wherein in one aspect (optionally), the enzyme is a polymerase. In one aspect, the kit further comprises one or more oligonucleotides, free nucleotides and at least one polymerase or enzyme capable of amplifying nucleic acids in PCR reactions, rolling circle replication, ligase chain reaction, reverse transcription or its derivative methods. One or more oligonucleotides can be specifically hybridized with nucleic acid from a sample of an object (e.g., from an animal, plant, insect, yeast, virus, bacteriophage, nematode, bacterium or fungus).

[0253] In some embodiments, the kit also includes reagents and components for purifying, separating and / or collecting polypeptides, parts, tags and / or polynucleotides (e.g., separated record polynucleotides). In some embodiments, the kit also includes reagents for linking and collecting polypeptides, parts, tags and / or polynucleotides (e.g., separated record polynucleotides). In some embodiments, the kit also includes instructions for preparing samples. In some cases, the kit includes reagents and components for nucleic acid (e.g., DNA) separation, precipitation and / or collection.

[0254] Exemplary Embodiments

[0255] Examples provided include:

[0256] 1. A method for assessing the identity and spatial relationship between a polypeptide and a moiety in a sample, the method comprising:

[0257] a) forming a connection structure between a site of a polypeptide in a sample and a site of a moiety in the sample, the connection structure comprising a polypeptide tag associated with the site of the polypeptide and a moiety tag associated with the site of the moiety, wherein the polypeptide tag and the moiety tag are associated;

[0258] b) transferring information between the associated polypeptide tag and the partial tag or linking the associated polypeptide tag and the partial tag to form a shared unique molecular identifier (UMI) and / or barcode;

[0259] c) disrupting the linking structure by dissociating the polypeptide from the moiety and the polypeptide tag from the moiety tag while maintaining the association between the polypeptide and the polypeptide tag and maintaining the association between the moiety and the moiety tag; and

[0260] d) evaluating the sequence of said polypeptide tag and at least a portion of said polypeptide, and evaluating at least a partial identity of said portion of the tag and said portion,

[0261] Wherein the polypeptide tag and the evaluation portion of the portion tag comprise the shared unique molecular identifier (UMI) and / or barcode, which indicates the spatial proximity of the site of the polypeptide and the site of the portion in the sample.

[0262] 2. The method of embodiment 1, wherein the portion comprises a polypeptide.

[0263] 3. The method of embodiment 1, wherein the portion comprises a polynucleotide.

[0264] 4. The method of any one of embodiments 1-3, wherein the polypeptide tag comprises a polynucleotide.

[0265] 5. The method of any one of embodiments 1-4, wherein the partial tag comprises a polynucleotide.

[0266] 6. The method according to embodiment 5, wherein the polypeptide tag comprises a first polynucleotide and the partial tag comprises a second polynucleotide, the first and second polynucleotides comprise complementary sequences, and the polypeptide tag and the partial tag are associated via the complementary sequence.

[0267] 7. The method of embodiment 6, wherein transferring information between the associated polypeptide tag and the partial tag comprises extending both the first polynucleotide of the polypeptide tag and the second polynucleotide of the partial tag to form the shared UMI and / or barcode.

[0268] 8. The method of embodiment 6, wherein transferring information between the associated polypeptide tag and the partial tag comprises extending one of the first polynucleotide of the polypeptide tag and the second polynucleotide of the partial tag to form the shared UMI and / or barcode.

[0269] 9. The method of embodiment 5, wherein the polypeptide tag comprises a double-stranded polynucleotide, and the partial tag comprises a double-stranded polynucleotide, and transferring information between the associated polypeptide tag and the partial tag comprises linking the double-stranded polynucleotides to form the shared UMI and / or barcode.

[0270] 10. The method of embodiment 9, wherein the shared UMI and / or barcode comprises a sequence of the double-stranded polynucleotide.

[0271] 11. The method of embodiment 9, wherein the shared UMI and / or barcode comprises a sequence of one of the double-stranded polynucleotides.

[0272] 12. The method according to any one of embodiments 1-11, wherein, in the connection structure, the polypeptide tag and the partial tag are stably associated.

[0273] 13. The method of any one of embodiments 1-11, wherein in the connection structure, the polypeptide tag and the partial tag are transiently associated.

[0274] 14. The method according to any one of embodiments 1-13, wherein, in the connection structure, the polypeptide tag and the partial tag are directly associated.

[0275] 15. The method of any one of embodiments 1-13, wherein in the connection structure, the polypeptide tag and the partial tag are indirectly associated, for example, via a linker or a UMI between the polypeptide tag and the partial tag.

[0276] 16. A method for assessing the identity and spatial relationship between a polypeptide and a moiety in a sample, the method comprising:

[0277] a) forming a connection structure between a site of a polypeptide in a sample and a site of a moiety in the sample, the connection structure comprising a polypeptide tag associated with the site of the polypeptide and a moiety tag associated with the site of the moiety, wherein the polypeptide tag and the moiety tag are associated;

[0278] b) transferring information between the associated polypeptide tag and the moiety tag to form a shared unique molecular identifier (UMI) and / or barcode, wherein the shared UMI and / or barcode is formed as a separate record polynucleotide;

[0279] c) destroying the linking structure by dissociating the polypeptide from the moiety and the polypeptide tag from the moiety tag while maintaining the association between the polypeptide and the polypeptide tag and maintaining the association between the moiety and the moiety tag;

[0280] d) evaluating the sequence of said polypeptide tag and at least a portion of said polypeptide, and evaluating at least a partial identity of said portion tag and said portion; and

[0281] e) evaluating said isolated record polynucleotides to establish a spatial relationship between the site of said polypeptide and the site of said moiety.

[0282] 17. The method of embodiment 16, wherein the polypeptide tag and the partial tag comprise polynucleotides.

[0283] 18. The method according to embodiment 16 or embodiment 17, wherein the connection structure is formed between the polypeptide tag and the partial tag via the separated recording polynucleotide.

[0284] 19. The method of any one of embodiments 16-18, wherein the method forms a plurality of separate recorded polypeptides between the polypeptide tag and more than one site or more than one portion of the portion.

[0285] 20. The method of any one of embodiments 16-19, wherein step e) establishes the spatial relationship between the site of the polypeptide and two or more sites of the moiety or two or more moieties.

[0286] 21. The method of any one of embodiments 16-20, wherein the polypeptide tag and the isolated recording polynucleotide are transiently associated in the connection structure.

[0287] 22. The method of any one of embodiments 16-21, wherein the polypeptide tag and the separated recording polynucleotide are directly associated in the connection structure.

[0288] 23. The method of any one of embodiments 16-22, wherein the partial tag and the separated recording polynucleotide are transiently associated in the connection structure.

[0289] 24. The method of any one of embodiments 16-23, wherein the partial tag and the separated recording polynucleotide are directly associated in the connection structure.

[0290] 25. A method according to any one of embodiments 16-24, wherein the separated recording polynucleotide is formed by extension, such as primer extension.

[0291] 26. A method according to any one of embodiments 16-24, wherein the isolated recording polynucleotide is formed by ligation.

[0292] 27. The method of any one of embodiments 16-26, wherein the isolated recording polynucleotide is released from the polypeptide tag and the partial tag.

[0293] 28. The method of any one of embodiments 16-27, further comprising collecting the isolated record polynucleotide prior to evaluating the isolated record polynucleotide.

[0294] 29. The method of embodiment 28, wherein evaluating the isolated record polynucleotides comprises sequencing the collected shared unique molecular identifiers (UMIs) and / or barcodes to generate sequencing data.

[0295] 30. The method of any one of embodiments 16-29, further comprising conjugating the collected isolated transcript polynucleotides prior to evaluating the isolated transcript polynucleotides.

[0296] 31. The method of embodiment 30, wherein evaluating the isolated record polynucleotide comprises sequencing the concatenated isolated record polynucleotide.

[0297] 32. The method of any one of embodiments 1-31, wherein, in forming the connection structure, a single polypeptide tag is associated with a single site of the polypeptide, a single polypeptide tag is associated with multiple sites of the polypeptide, or multiple polypeptide tags are associated with multiple sites of the polypeptide.

[0298] 33. A method according to any one of embodiments 1-32, wherein, in forming the connection structure, a single part tag is associated with a single site of the part, a single part tag is associated with multiple sites of the part, or multiple part tags are associated with multiple sites of the part.

[0299] 34. The method of any one of embodiments 1-33, wherein information is transferred between the associated polypeptide tag and the partial tag or the associated polypeptide tag and the partial tag are linked to form a single shared unique molecular identifier (UMI) and / or barcode.

[0300] 35. The method of embodiment 34, wherein the single shared unique molecular identifier (UMI) and / or barcode is formed by combining multiple sequences from the polypeptide tag and / or the partial tag, e.g., multiple UMIs and / or barcodes.

[0301] 36. The method of any one of embodiments 1-33, wherein information is transferred between the associated polypeptide tags and the partial tags or the associated polypeptide tags and the partial tags are linked to form a plurality of shared unique molecular identifiers (UMIs) and / or barcodes.

[0302] 37. The method of any one of embodiments 1-36, wherein in the ligated structure, the shared UMI and / or barcode comprises a complementary polynucleotide hybrid, and dissociating the polypeptide tag from the partial tag comprises denaturing the complementary polynucleotide hybrid.

[0303] 38. The method of any one of embodiments 1-37, wherein the polypeptide and the portion are both parts of a larger polypeptide, and dissociating the polypeptide from the portion comprises fragmenting the larger polypeptide into peptide fragments.

[0304] 39. The method of embodiment 38, wherein the larger polypeptide is fragmented into peptide fragments by protease digestion.

[0305] 40. The method of any one of embodiments 1-39, wherein the moiety is a portion of a molecule that is bound to, complexed with, or in close proximity to a polypeptide in the sample.

[0306] 41. The method of embodiment 40, wherein the polypeptide and the portion belong to two different proteins in the same protein complex.

[0307] 42. The method of embodiment 40, wherein the portion is a portion of a polynucleotide molecule that is bound to, complexed with, or in close proximity to a polypeptide in the sample.

[0308] 43. The method of any one of embodiments 1-42, wherein at least a portion of the sequence of the polypeptide is evaluated using a program comprising the steps of:

[0309] a1) providing the polypeptide and the associated polypeptide tag used as a record tag;

[0310] b1) contacting the polypeptide with a first binding agent capable of binding to the polypeptide, wherein the first binding agent comprises a first coding tag having identification information about the first binding agent;

[0311] c1) transferring the information of the first coding label to the recording label to generate a first sequence extended recording label; and

[0312] d1) Analyzing the first sequence extension record label.

[0313] 44. The method of embodiment 43, wherein analyzing the first sequence extension record signature also evaluates the polypeptide signature.

[0314] 45. The method of any one of embodiments 1-44, wherein the portion comprises a portion of a polypeptide, and the at least partial identity of the portion is assessed using a procedure comprising the following steps:

[0315] a2) providing the partial polypeptide and the associated partial tag used as a record tag;

[0316] b2) contacting the partial polypeptide with a first binding agent capable of binding to the partial polypeptide, wherein the first binding agent comprises a first coding tag having identification information about the first binding agent;

[0317] c2) transferring the information of the first encoding label to the recording label to generate a first sequence extended recording label; and

[0318] d2) Analyzing the first sequence extension record label.

[0319] 46. ​​The method of embodiment 45, wherein analyzing the first sequence extended record signature also evaluates the partial signature.

[0320] 47. A method for assessing the identity and spatial relationship between a polypeptide and a moiety in a sample, the method comprising:

[0321] a) providing a preassembled structure comprising a shared unique molecular identifier (UMI) and / or barcode in the middle portion, the UMI and / or barcode flanked by a polypeptide tag on one side and by a moiety tag on the other side;

[0322] b) forming a junction structure between the site of the polypeptide in the sample and the site of the moiety in the sample by associating the polypeptide tag of the preassembled structure with the site of the polypeptide and associating the moiety tag of the preassembled structure with the site of the moiety;

[0323] c) disrupting the linking structure by dissociating the polypeptide from the moiety and the polypeptide tag from the moiety tag while maintaining the association between the polypeptide and the polypeptide tag and maintaining the association between the moiety and the moiety tag; and

[0324] d) evaluating the sequence of said polypeptide tag and at least a portion of said polypeptide, and evaluating at least a partial identity of said portion of the tag and said portion,

[0325] wherein the polypeptide tag and the evaluation portion of the portion tag comprise the shared unique molecular identifier (UMI) and / or barcode indicating the spatial proximity of the site of the polypeptide and the site of the portion in the sample.

[0326] 48. The method of embodiment 47, wherein the portion comprises a polypeptide.

[0327] 49. A method according to embodiment 47, wherein the portion comprises a polynucleotide.

[0328] 50. The method of any one of embodiments 47-49, wherein the polypeptide tag comprises a polynucleotide.

[0329] 51. The method of any one of embodiments 47-50, wherein the partial tag comprises a polynucleotide.

[0330] 52. The method of any one of embodiments 47-51, wherein the polypeptide tag and the partial tag are stably associated in the connection structure.

[0331] 53. A method according to any one of embodiments 47-51, wherein, in the connection structure, the polypeptide tag and the partial tag are transiently associated.

[0332] 54. The method of any one of embodiments 47-53, wherein, in the connection structure, the polypeptide tag and the partial tag are directly associated.

[0333] 55. The method of any one of embodiments 47-53, wherein in the connection structure, the polypeptide tag and the partial tag are indirectly associated, for example, via a linker or a UMI between the polypeptide tag and the partial tag.

[0334] 56. A method according to any one of embodiments 47-55, wherein, in forming the connection structure, a single polypeptide tag is associated with a single site of the polypeptide, a single polypeptide tag is associated with multiple sites of the polypeptide, or multiple polypeptide tags are associated with multiple sites of the polypeptide.

[0335] 57. A method according to any one of embodiments 47-56, wherein, in forming the connection structure, a single part tag is associated with a single site of the part, a single part tag is associated with multiple sites of the part, or multiple part tags are associated with multiple sites of the part.

[0336] 58. The method of any one of embodiments 47-57, wherein the formed ligation structure comprises a single shared unique molecular identifier (UMI) / barcode.

[0337] 59. The method of any one of embodiments 47-57, wherein the formed ligation structure comprises a plurality of shared unique molecular identifiers (UMIs) / barcodes.

[0338] 60. The method of any one of embodiments 47-57, wherein the polypeptide tag comprises a first polynucleotide and the partial tag comprises a second polynucleotide.

[0339] 61. The method of any one of embodiments 47-60, wherein in the ligated structure, the shared UMI and / or barcode comprises a complementary polynucleotide hybrid, and dissociating the polypeptide tag from the partial tag comprises denaturing the complementary polynucleotide hybrid.

[0340] 62. The method of any one of embodiments 47-61, wherein the polypeptide and the portion are both parts of a larger polypeptide, and dissociating the polypeptide from the portion comprises fragmenting the larger polypeptide into peptide fragments.

[0341] 63. The method of embodiment 62, wherein the larger polypeptide is fragmented into peptide fragments by protease digestion.

[0342] 64. The method of any one of embodiments 47-63, wherein the moiety is a portion of a molecule that is bound to, complexed with, or in close proximity to a polypeptide in the sample.

[0343] 65. The method of embodiment 64, wherein said polypeptide and said portion belong to two different proteins in the same protein complex.

[0344] 66. A method according to embodiment 64, wherein the portion is a portion of a polynucleotide molecule that is bound to, complexed with, or adjacent to a polypeptide in the sample.

[0345] 67. The method of any one of embodiments 47-66, wherein at least a portion of the sequence of the polypeptide is evaluated using a program comprising the steps of:

[0346] a3) providing the polypeptide and the associated polypeptide tag for use as a record tag;

[0347] b3) contacting the polypeptide with a first binding agent capable of binding to the polypeptide, wherein the first binding agent comprises a first coding tag having identification information about the first binding agent;

[0348] c3) transferring the information of the first encoding label to the recording label to generate a first sequence extended recording label; and

[0349] d3) Analyzing the first sequence extension record label.

[0350] 68. The method of embodiment 67, wherein analyzing the first sequence extension record tag also evaluates the polypeptide tag.

[0351] 69. The method of any one of embodiments 47-68, wherein the portion comprises a portion of a polypeptide, and the at least partial identity of the portion is assessed using a procedure comprising the following steps:

[0352] a4) providing the partial polypeptide and the associated partial tag for use as a record tag;

[0353] b4) contacting the partial polypeptide with a first binding agent capable of binding to the partial multi-polypeptide, wherein the first binding agent comprises a first coding tag having identification information about the first binding agent;

[0354] c4) transferring the information of the first encoding label to the recording label to generate a first sequence extended recording label; and

[0355] d4) Analyzing the first sequence extension record label.

[0356] 70. The method of embodiment 69, wherein analyzing the first sequence extended record signature also evaluates the partial signature.

[0357] 71. A method according to any one of embodiments 1-70, wherein the evaluation of at least partial sequence identity of the polypeptide and the portion is performed after forming a connecting structure between the site of the polypeptide and the site of the portion.

[0358] 72. The method of any one of embodiments 1-71, wherein the evaluation of at least a partial sequence of the polypeptide and at least a partial identity of the portion is performed after dissociation of the polypeptide from the portion.

[0359] 73. The method of any one of embodiments 43-46 and 67-70, wherein contacting the polypeptide and the moiety with one or more binding agents is performed after forming a linkage structure between the polypeptide and the moiety.

[0360] 74. The method of any one of embodiments 43-46, 67-70 and 73, wherein contacting the polypeptide and the moiety with one or more binding agents is performed after dissociation of the polypeptide from the moiety.

[0361] 75. A kit for assessing the identity and spatial relationship between polypeptides and moieties in a sample, comprising:

[0362] (a) one or more polypeptide tags and one or more partial tags;

[0363] (b) a reagent for forming a linking structure between the polypeptide and the moiety in the sample; and

[0364] (c) reagents for assessing the identity of said portion and at least a partial sequence of said polypeptide.

[0365] 76. A kit for assessing the identity and spatial relationship between polypeptides and moieties in a sample, comprising:

[0366] (a) one or more polypeptide tags and one or more partial tags;

[0367] (b) a reagent for forming a linking structure between the polypeptide and the moiety in a sample, wherein the linking structure is formed as a separate record polynucleotide; and

[0368] (c) reagents for assessing the identity of said portion and at least a partial sequence of said polypeptide.

[0369] 77. The kit of embodiment 76 further comprises one or more reagents for analyzing the isolated recorded polynucleotide.

[0370] 78. A kit according to any one of embodiments 75-77, wherein the reagents for evaluating the identity of the portion and at least a partial sequence of the polypeptide comprise a library of binders, wherein each binder comprises a binding portion and an encoded polymer, wherein the encoded polymer comprises identification information about the binding portion, wherein the binding portion is capable of binding to one or more N-terminal, internal or C-terminal amino acids of the fragment, or is capable of binding to one or more N-terminal, internal or C-terminal amino acids modified by a functionalizing agent.

[0371] 79. A kit for evaluating spatial relationships, comprising:

[0372] (a) reagents for providing a polypeptide that is directly or indirectly associated with a polypeptide tag and for providing a moiety that is directly or indirectly associated with a moiety tag;

[0373] (b) a reagent for functionalizing the N-terminal amino acid (NTAA) of the polypeptide;

[0374] (c) a first binding agent comprising a first binding moiety capable of binding to the functionalized NTAA and (c1) a first coded tag having identification information about the first binding agent, or (c2) a first detectable tag; and

[0375] (d) a reagent that transfers information of the first encoding tag to the recording tag to produce an extended recording tag; and

[0376] (e) A reagent for analyzing the extended recording label or a reagent for detecting the first detectable label.

[0377] 80. The kit of embodiment 79, wherein the kit further comprises reagents for eliminating functionalized NTAA to expose new NTAA.

[0378] 81. A kit according to embodiment 80, wherein the reagent for eliminating functionalized NTAA is a carboxypeptidase or an aminopeptidase or a variant, mutant or modified protein thereof; a hydrolase or a variant, mutant or modified protein thereof; a mild Edman degradation reagent; an Edmanase enzyme; TFA; a base; or any combination thereof.

[0379] 82. The kit of any one of embodiments 75-79, further comprising a support or substrate.

[0380] 83. The kit of embodiment 82, wherein the support or substrate is a bead, a porous bead, a porous matrix, an array, a glass surface, a silicon surface, a plastic surface, a filter, a membrane, nylon, a silicon crystal chip, a flow chip, a biochip including a signal transduction electronic device, a microtiter well, an ELISA plate, a rotating interferometry disk, a nitrocellulose membrane, a nitrocellulose-based polymer surface, a nanoparticle or a microsphere.

[0381] 84. The kit of Embodiment 82 or Embodiment 83, wherein the support or substrate comprises a plurality of spatially resolved attachment points.

[0382] Example

[0383] The following examples are provided to illustrate but not to limit the methods, compositions, and uses provided herein.

[0384] Example 1: Paired Association

[0385] In this example, peptide 1 (Pep 1) and peptide 2 (Pep 2) are subsequences of protein 1. DNA tags containing UMIs are covalently attached to sites in the protein sample. These sites should be evenly and appropriately spaced to optimize the yield of useful information for each assay design.

[0386] In a protein sample, a DNA tag with UMI 1 is linked to Pep 1 and a DNA tag with UMI 2 is linked to Pep 2. The DNA tags are designed so that the UMI sequence can be copied from one tag to another by a DNA polymerase, for example using a universal complementary 3' end as a primer. A reaction is performed to copy the tag information, for example, using a DNA polymerase to perform a cycle of annealing + extension. (See, e.g., Assarsson, Lundberg et al., 2014). By proximity, UMI 1 and UMI 2 write to each other. In some examples, only a single extension cycle is performed so that a unique tag pair is formed. Other variations are also possible in which sequences are spread between multiple tags. Such a system should be designed not to produce unwanted tag multimers or at least to minimize them.

[0387] Next, protein 1 was cleaved and the peptide-UMI-tag pairs were processed to generate NGPS data. The DNA tags bound to the UMIs were used as record tags (or written to record tags) in the NGPS assay. Following NGS sequencing and sequence analysis, the following sequence constructs were extracted:

[0388] {Pep1, UMI1-UMI2}

[0389] {Pep2, UMI2-UMI1}

[0390] Assuming that UMI 1 and UMI 2 are, to a first approximation, "unique" (i.e., have a suitably low probability of occurring more than once by chance in a sample), we can use this information to infer with high confidence that Pep 1 and Pep 2 are in close proximity in a protein sample. In particular, if we empirically tune and calibrate the system so that peptides linked using partitioning by association (PBA) are likely to be part of the same protein, we can infer that Pep 1 and Pep 2 are likely subsequences of a single protein. This additional information is not obtained from NGPS alone. When combined with peptide sequence data, it allows us to identify protein sequences with much higher confidence because we can look for consistent pairs (or more) of peptide sequence matches.

[0391] Example 2: Network Reconfiguration

[0392] It is not required that the peptide pairs are from the same protein. In some examples, the PBA method is applied to complex protein samples. The samples are labeled with DNA tags and UMI pairs are formed as described in Example 1. In some cases, the UMI pairs will associate subsequences of proteins (cis-protein association or CPA). In other cases, UMI pairs will be formed between proteins (trans-protein association or TPA). In complex protein samples, a mixture of CPA and TPA may be present.

[0393] Even with only one CPA per protein, PBA significantly increases the ability to uniquely identify proteins. However, additional performance is gained by reconstructing the network of pairs. For example, if Pep 3 and Pep 4 are subsequences of protein 2. Let us assume that PBA enables the following associations:

[0394] Pep 1 from protein 1 and Pep 3 from protein 2.

[0395] Pep 2 from protein 1 and Pep 4 from protein 2.

[0396] Suppose we can map Pep 1 and Pep 2 to protein 1, but we cannot map Pep 3 and Pep 4 to protein 1. However, we can infer that Pep 3 and Pep 4 have a reasonable likelihood of belonging to the same protein (or a small subgroup of proteins neighboring protein 1). We can therefore use this "partitioning" information to identify high-likelihood matches and together initiate a network of pairwise relationships that allows us to identify proteins using PBA using shorter and less accurate sequences than would be required without PBA.

[0397] PBA can be used with physical partitioning. However, due to this "network" effect, physical partitioning is usually not required. PBA can be performed in bulk without the need for emulsions, or other complex partitioning techniques. Instead, partitioning based on "virtual" proximity is established at the molecular level and reconstructed in an informative manner.

[0398] In some cases, it is preferred to limit the number of encoded proteins that are in close enough proximity to produce pairs, preferably, the PBA will produce many relatively discrete "networks", rather than one large, diffuse network that could in principle contain the entire protein sample. Simple methods to limit the average number of proteins that associate together include dilution and physical separation, for example by adsorption or other attachment to a solid support such as beads.

[0399] Example 3: Labeling proteins and protein complexes using DNA tags

[0400] DNA tags consisting of a common primer sequence flanking the UMI / barcode and a 5' coupling portion (for direct or indirect coupling to a polypeptide) can be coupled to native proteins or protein complexes. Many standard bioconjugation methods (e.g., Hermanson 2013) can be used to directly couple DNA tags to reactive amino acid residues (e.g., Lys, Cys, tyrosine, etc., see Ref), or indirectly coupled via heterobifunctional linkers. For example, heterobifunctional linkers, such as NHS-PEGl 1-mTet, can be used to chemically label lysine residues in buffers such as 50mM sodium borate or HEPES (pH 8.5), and to generate orthogonal chemical "click" groups for subsequent coupling to DNA tags with 5' trans-cyclooctane (TCO) groups. After lysine labeling with NHS-PEG11-mTet, excess NHS-PEG11-mTet linkers are removed using a 10k MWCO filter or reverse phase purification resin (RP-S).

[0401] The 5'TCO-labeled DNA tag is coupled to the mTet-labeled protein in 1X PBS buffer (pH 7.5). Excess DNA tags can be removed by cleaning on mTet scavenger resin. After removing excess DNA tags, information is transferred between adjacent DNA tags using a proximity-based primer extension step. Specifically, adjacent DNA tags are allowed to anneal at room temperature for 5 minutes in extension buffer (50mM Tris-Cl (pH 7.5), 2mM MgSO4, 125μM dNTPs, 50mM sodium chloride, 1mM dithiothreitol, 0.1% Tween-20 and 0.1mg / mL BSA), and after a short 2 minutes, a heating step is performed to 45°C. After annealing, Klenow exo-DNA polymerase (NEB, 5U / μL) is added to the beads to reach a final concentration of 0.125U / μl, and incubated at 23°C for 5 minutes. After primer extension, the reaction was quenched by adding urea to 8 M to denature proteins and protein complexes.

[0402] Example 4: Processing of peptides adjacent to DNA markers

[0403] After primer extension and protein denaturation, the denatured polypeptide is acylated at remaining unreacted cysteine ​​or lysine residues and then protease digested with endopeptidases such as trypsin, Lysc, Argc, etc. The DNA tag proximal to the extension on the labeled peptide acts as a record tag in our NGPS protein encoding assay as described in PCT / US2017 / 030702. The DNA-labeled peptide is immobilized to a sequencing substrate (e.g., beads) by direct chemical conjugation or by hybridization capture and ligation with a DNA capture probe directly attached to the sequencing substrate (see, e.g., Figure 6 ).

[0404] After the DNA-peptide construct is attached to the sequencing substrate, at least two DNA tags are present (see, e.g., Figure 5 C), one DNA tag type includes a 3'Spl' sequence and the other DNA tag type includes a 3'Sp2' sequence. These two sequence types are converted to universal Sp spacer sequences by annealing conversion primers (Sp2-Sp' and Spl-Spl). Extension on these primer sequences generates the final recorded tag for ProteoCode sequencing.

[0405] Example 5: Connectivity-based proximity loop

[0406] This example describes a method for evaluating proximity interactions between a polypeptide and one or more moieties using proximity cycles based on ligation. The polypeptide and moiety are each labeled with a DNA tag. The DNA tags are designed to interact through cycles of extension, ligation, and denaturation.

[0407] In the first step of a given cycle, the common primer anneals to the F' site on the 3' end of the DNA tag. The DNA tag on the polypeptide is oriented with its 3' end away from the polypeptide and the extra T base, and the DNA tag on the moiety is oriented so that its 3' end is attached to the moiety and the 5' end is free ( Fig. 8A In some embodiments, the reverse design can be used. After the F primer anneals with the DNA tag (polypeptide tag and partial tag), the primer is extended to produce a double-stranded DNA tag product, and the A extension enzyme activity of the polymerase generates an A overhang on the double-stranded DNA tag product annealed with the partial DNA tag ( Figure 8B The A overhang on the partial tag and the T overhang on the peptide tag enable ligation ( Figure 8C ). The 5' end of some DNA tags is non-phosphorylated and non-ligatable, while the 5' end of the F primer is phosphorylated and ligatable. Fig.8D As shown, ligation produces a separate transcript of P-M1 polynucleotides. In some cases, the polypeptides are spatially adjacent to more than one moiety (e.g., M1, M2, etc.). Cyclic annealing, extension, and ligation produce multiple linear transcripts (e.g., separate transcript polynucleotides) of P-M1, P-M2, etc. ( Figures 9A-9B Indirect or overlapping information from multiple separate recorded polynucleotides further indicates spatial proximity information of a polypeptide having two or more parts ( Fig. 9C ).

[0408] Cyclic annealing, extension, and ligation were performed as follows: a 50 μl reaction included 100 ng of DNA-labeled protein complex, 200 nM F primer, 0.5 U Taq polymerase (NEB), and 2 U Pfu DNA ligase (D540K mutant) (U.S. Pat. No. 5,427,930; Tanabe et al., Archaea (2015) 2015:267570) in 1X Ext-Lig buffer (20 mM Tris-HCl, pH 8.0, 25 mM potassium acetate, 2 mM magnesium acetate, 1 mM NAD, 200 μM dNTPs, in addition to 500 μM dATP, 10 mM DTT, 0.1% Triton X-100). The reaction was cycled for 30 cycles under the following conditions: 94° C. for 2 minutes, then 60° C. for 1 minute, 40° C. for 5 minutes, and 94° C. for 30 seconds for 30 cycles. After extension ligation thermocycling in the presence of the F primer, the resulting transcript was PCR amplified using the F and R primers using standard PCR conditions.

[0409] The provided methods can be used to determine the proximity of P to adjacent M1, M2, etc. The sequence or identity of P and portions of M1, M2 is further determined using ProteoCode sequencing (e.g., see International Patent Application Publication No. WO 2017 / 192633).

[0410] Example 6: Concatenating DNA libraries for nanopore sequencing

[0411] The DNA library was PCR amplified (20 cycles) using VeraSeq 2.0Ultra DNA polymerase with a 5' phosphorylated primer to produce library amplicons suitable for blunt-end ligation (about 20ng / μL PCR yield). To connect the PCR products, 20μL of PCR reaction solution was mixed with 20μL 2X rapid ligase buffer and 1μL rapid ligase (NEB) and incubated at room temperature for about 16 hours. The resulting ligation product was purified using a Zymo purification column, with a length of about 0.5-2kb (possibly also a mixture of some circular products) and eluted into 20μL water. The resulting cascaded products were prepared for nanopore sequencing using a rapid sequencing preparation kit (SQK-RAD002) based on transposase adapter addition and analyzed on a MinION Mk 1B (R9.4) device. Other methods for concatenating DNA libraries include using Gibson assembly, the method described by Schlecht et al., and can also be used to concatenate DNA libraries as described above and used for nanopore sequencing (Schlecht et al., (2017) Sci Rep 7(1):5252).

[0412] Example 7: Peptide labeling and information transfer between neighboring molecules

[0413] This example describes the transfer of information between two parts of a polypeptide in a proximity model system: a biotin-containing part of the peptide (part) and a phenylalanine (F)-containing part of the peptide (part).

[0414] The polypeptide tag (DNA 1) was prepared by extension and ligation of synthetic oligonucleotides, which contained Fig. 10A The complementary spacers (sp' and sp), PEG linkers and complementary UMI sequences (UMI 1 and UMI 1') are shown. The 3' end of DNA1 contains an overlapping region (OL') that is complementary to the OL region on DNA2.

[0415] The partial tag (DNA 1) and the peptide tag (DNA 2) were linked to a model polypeptide (K(biotin)GSGSK(N3)GSGSRFAGVAMPGAEDDVVGSGS-K(N3)-NH2) according to SEQ ID NO:1), which contains biotin at the N-terminus and an internal phenylalanine. The DNA1 and DNA 2 tags were linked to the peptide using a DBCO click reaction, where DNA 1 (5 μM), DNA 2 (5 μM) and peptide (1 μM) were mixed in 100 mM HEPES (pH 7.5) and 150 mM sodium chloride buffer and heated at 60°C overnight. Because each peptide has two DNA attachment sites, three different products were produced: peptides with two DNA1 attachments, peptides with two DNA2 attachments, or peptides with DNA 1 and DNA 2 attachments. Peptides attached only to DNA 1 and DNA2 contain the necessary hybridization regions for information transfer. To remove free excess DNA, streptavidin beads (MyOne Streptavidin T1, Thermo Fisher, USA) were used to separate the complex of polypeptide and DNA by binding to biotin. Twenty (20) μL of the reaction mixture was incubated with streptavidin beads (10 μL) at 25°C for 40 minutes. After removing the supernatant and rinsing twice with PBS+0.1% Tween 20, the sample was eluted in 20 μL of 95% formamide at 60°C for 5 minutes. As a control, DNA 3 oligonucleotides were incubated with the same peptide as SEQ ID NO:1, except that it contained only 1 azide group. DNA 3-peptide complexes were prepared by incubation at 60°C overnight to produce control complexes and purified as described above. The attachment of DNA to polypeptides before and after purification was confirmed by mobility shift on a 15% denaturing polyacrylamide (TBU) gel.

[0416] The purified DNA 1-DNA 2-peptide complex was captured on magnetic agarose beads by hybridization of DNA 1 and ligation of DNA 1 to the bead-attached DNA 1 capture DNA ( Fig. 10A). By design, the beads contain two types of capture DNA, one with a region complementary to DNA 1 and the other with a region complementary to DNA 2. However, the hybridization site of DNA 2 is pre-blocked with complementary single-stranded DNA to enable capture by DNA 1. Equal concentrations of purified DBCO click reaction mixture containing DNA 1-DNA 2-peptide and DNA 3-peptide (total concentration: 0.1 nM) were mixed with magnetic agarose beads and hybridized to agarose beads in buffer (containing 5X SSC, 0.02% SDS and 15% formamide), followed by washing with PBS + 0.1% Tween20 and ligation. After ligation, the unligated substrate and capture DNA blocker for DNA 2 were rinsed with 0.1 M sodium hydroxide + 0.1% Tween 20.

[0417] For information transfer between DNA1 and DNA2, 0.125 U / μL Klenow fragment (3'->5'exo-) (KF) was used in the presence of dNTP mixture (125 μM each), 50 mM Tris-HCl (pH 7.5), 2 mM MgSO4, 50 mM NaCl, 1 mM DTT, 0.1% Tween 20 and 0.1 mg / mL BSA. - ). The reaction was incubated at 37°C for 5 minutes to perform intramolecular extension of DNA 2 using DNA 1 as a template.

[0418] After information transfer, the linker between DNA 1 and DNA 2 (the polypeptide and part of the tag) is broken by cleavage at the presence of a single uracil (U). Fig. 10A ). The cleavage reaction contained 0.05 U / μL USER Enzyme, 0.2 U / μL T4 PNK, 1 mM ATP, 5 mM DTT in the presence of 1X CutSmart buffer from NEB and was incubated at 37°C for 60 minutes. Next, trypsin digestion was performed to separate the peptides from the moieties (in this example, the F-containing moiety of the model polypeptide and the biotin-containing moiety of the model polypeptide, respectively), as Fig. 10B As shown. Digestion was performed at 37°C for 2 hours with 0.02 mg / mL trypsin, 0.1% Tween 20, 500 mM sodium chloride and 50 mM HEPE (pH 8.0). During the trypsin cleavage reaction, the separated part-DNA 2 was recaptured by hybridization with the bead-attached DNA 2 capture DNA. After washing with PBS + 0.1% Tween, the sample was incubated in a rapid ligase mixture at 25°C for 30 minutes as described above for the first connection to covalently link part-DNA 2 to the bead-attached DNA 2 capture DNA.

[0419] The oligonucleotides (R1′-sp′) were added to the KF beads as described previously in the presence of dNTPs (125 μM each). - The final capping step is performed in the reaction mixture to produce a final product having the terminal sequence (R1) for both DNA 1 and DNA 2 at the 3' end, such as Fig. 10B As shown. R1 and another DNA region (at the 5' end of DNA 1 and DNA 2) were used as annealing sites for adapter PCR for NGS. After amplification by adapter and index PCR and introduction of binding sites and index sequences, the samples were sequenced using MiSeq kit v3 (Illumina, USA). Amplicons were sequenced and counted using MiSeq.

[0420] The results demonstrating information transfer are shown in Table 1. An average of 491 information transfer events were detected in replicate experiments (replicate 1 = 617, replicate 2 = 365). Events were detected by identifying unique UMI-1 matches between DNA 1 and DNA 2, corresponding to unique pairings between single peptide-DNA 1 and moiety-DNA 2 constructs.

[0421] Table 1. Information transfer results

[0422]

[0423] To detect the background of this experiment, a control sample DNA 3-peptide was mixed with DNA1-DNA 2-peptide in equal proportions during the first hybridization / ligation step. The NGS output ratio of DNA 3 to DNA 2 was equal to or less than 0.0066, indicating that almost all information transfer events occurred in Fig. 10B within the same molecule.

[0424] In summary, this example demonstrates that information transfer between a peptide and moieties (biotin- and F-containing portions of the peptide) in a model polypeptide is efficient with low background.

[0425] In some cases, the polypeptide and the portion are evaluated for at least partial sequence of the polypeptide and at least partial identity of the portion prior to the final capping step described above. Fig. 10B ). The encoding step is performed to evaluate at least a portion of the sequence of the peptide. The binding agent with the coding tag oligonucleotide containing information about the binding agent can recognize the N-terminal amino acid or recognize a portion of the polypeptide or part. After the binding agent binds to its corresponding target, the 3'-spacer region of the coding tag hybridizes with the 3'-spacer region of the DNA oligonucleotide linked to the same peptide. The DNA linked to the peptide can be obtained by using KF - Extension is performed to replicate the coding tag and, as a result, the information from the coding tag is transferred to the DNA sequences (DNA 1 and DNA 2) linked to the peptide for analysis.

[0426] This encoding step is then followed by a final capping step as described above, in which an oligonucleotide containing a universal priming sequence (R1'-sp') is added to the KF with these peptides (associated with DNA 1 and DNA 2) in the presence of dNTPs (e.g., 125 μM each). - The reaction mixture is then added to produce the final product for NGS readout.

[0427] Example 8: Evaluation of encoding function using a mixture of binders

[0428] This example describes an exemplary encoding assay using a binding agent that recognizes a portion of a peptide (eg, the N-terminal amino acid).

[0429] In an exemplary model system for evaluating at least a portion of polypeptides and moieties, a peptide comprising phenylalanine attached to a DNA record tag (F-peptide) and biotin attached to a DNA record tag was evaluated in a coding assay. A binder that does not bind to biotin or N-terminal phenylalanine (F) on the peptide is also included as a negative control. Two hundred (200) nM of an exemplary binder that binds to phenylalanine when phenylalanine is the N-terminal amino acid residue (F-binder), 44 nM of a single-chain streptavidin binder (mSA-binder) that recognizes biotin, and 200 nM of a negative control binder were incubated with biotin attached to a record tag and an F-peptide (F at the N-terminus) attached to a record tag. Each binder attached to a corresponding coding tag that recognizes the binder was incubated with beads conjugated with a biotin-record tag conjugate and an F-peptide-record tag conjugate. After binding and washing, the transfer of the encoded tag information to the recording tag by extension was achieved by incubating the beads in a solution containing 0.125 units / μL Klenow fragment (3'->5'exo-) (MCLAB, USA), dNTP mixture (125 μM each), 50 mM Tris-HCl (pH 7.5), 2 mM MgSO4, 50 mM NaCl, 1 mM DTT, 0.1% Tween 20, and 0.1 mg / mL BSA. The reaction was incubated at 37°C for 5 minutes. The beads were washed after encoding. The extended recording tags of this method were PCR amplified and analyzed by next-generation sequencing technology (NGS).

[0430] As shown in the NGS results in Table 2, mSA and F-binders were able to bind and encode their corresponding targets, and the tested binders showed low encoding signals for peptides that were not the binder targets.

[0431] Table 2. Coding yields for mSA binders and F binders

[0432]

[0433] Exemplary Advantages

[0434] Each peptide derived from a single protein (or physical partition) need not have the same barcode as other peptides (or physical partitions) from the protein. Each site (even within the same protein) can have a different sequence identifier, e.g., often called a UMI. Proteins can be processed in batches, without the need for beads, etc. For convenience and / or to help facilitate, a solid support can be used, but in principle the method can be performed in solution on samples of arbitrarily complexity. For example, an entire proteome sample can be partitioned in batches. In contrast, the heavy lifting is computationally laborious.

[0435] When performed on native proteins in complex, PBA can be used to reconstitute protein complexes. When performed on renatured proteins, PBA can be used to identify proteins with a propensity to associate.

[0436] PBAs can be used to bind other types of molecules, such as DNA-protein complexes. PBAs can be used with sample barcoding so that multiple samples can be pooled and analyzed together.

[0437] The present disclosure is not intended to limit the scope to the specific disclosed embodiments provided, for example, those embodiments used to illustrate various aspects of the present invention. According to the description and teachings herein, various modifications to the compositions and methods will become readily understood. These changes can be practiced without departing from the true scope and spirit of the present disclosure, and these changes are intended to fall within the scope of the present disclosure. These and other changes can be made to the embodiments described in detail above. Generally, in the following claims, the terms used should not be interpreted as limiting the claims to the specific embodiments disclosed in the specification and claims, but should be interpreted that the claims are entitled to include all possible embodiments and all equivalent scopes. Therefore, the claims are not limited by the present disclosure.

[0438] References cited:

[0439] US 2015 / 0224466 A1;

[0440] US 2010 / 0136544 A1;

[0441] U.S. Patent No. 9,029,085 B2;

[0442] U.S. Patent No. 9,085,798 B2;

[0443] U.S. Patent No. 6,511,809 B2;

[0444] WO 2017 / 192633 A1;

[0445] WO 2016 / 123419 A1;

[0446] WO 2015 / 070037 A2;

[0447] WO 2016 / 130704 A2;

[0448] WO 2017 / 075265 A1;

[0449] WO 2016 / 061517 A2;

[0450] WO 2015 / 042506 A1;

[0451] WO 2016 / 0138086 A1;

[0452] Abe, H., Y. Kondo, H. Jinmei, N. Abe, K. Furukawa, A. Uchiyama, S. Tsuneda, K. Aikawa, I. Matsumoto and Y. Ito (2008). "Rapid DNA chemical ligation for amplification of RNA and DNA signal". Bioconjug Chem 19(1):327-333;

[0453] Assarsson, E., M. Lundberg, G. Holmquist, J. Bjorkesten, SB Thorsen, D. Ekman, A. Eriksson, E. Rennel Dickens, S. Ohlsson, G. Edfeldt, A. C Andersson, P. Lindstedt, J. Stenvang, M. Gullberg, and S. Fredriksson (2014). “Homogenous 96-plex PEA immunoassay exhibiting high sensitivity, specificity, and excellent scalability”. PLoS One 9(4):e95192;

[0454] El-Sagheer, AH, VV Cheong and T. Brown (2011). "Rapid chemical ligation of oligonucleotides by the Diels-Alder reaction". Org Biomol Chem 9(1):232-235;

[0455] El-Sagheer, A.H., A.P. Sanzone, R. Gao, A. Tavassoli, and T. Brown (2011). "Biocompatible artificial DNA linker that is read through by DNA polymerases and is functional in Escherichia coli". Proc Natl Acad Sci USA 108(28):11338-11343;

[0456] Hermanson, G. (2013). Bioconjugation Techniques , Academic Press;

[0457] Holding, AN (2015). “XL-MS: Protein cross-linking coupled with mass spectrometry” Methods 89:54-63;

[0458] Kilpatrick, LE and EL Kilpatrick (2017). "Optimizing High-Resolution Mass Spectrometry for the Identification of Low-Abundance Post-Translational Modifications of Intact Proteins". J Proteome Res 16(9):3255-3265;

[0459] Park, J., M. Koh, JY Koo, S. Lee, and SB Park (2016). "Investigation of Specific Binding Proteins to Photoaffinity Linkers for Efficient Deconvolution of Target Protein". ACS Chem Biol 11(1):44-52;

[0460] Schaus, TE, et al. (2017). “A DNAnanoscope via auto-cycling proximity recording”. Nat Commun 8(1):696.

[0461] Schneider, M., A. Belsom and J. Rappsilber (2018). “Protein Tertiary Structure by Crosslinking / Mass Spectrometry”. Trends Biochem Sci 43(3):157-169; and

[0462] Switzar, L., M. Giera, and W. M. Niessen (2013). “Protein digestion: an overview of the available techniques and recent developments” J Proteome Res 12(3):1067-1077. SEQUENCE LISTING <110> Encodia, Inc. Chee, Mark Gunderson, Kevin <120> PROXIMITY INTERACTION ANALYSIS <130> 4614-2000940 <150> US 62 / 726,933 <151> 2018-09-04 <150> US 62 / 726,959 <151> 2018-09-04 <150> US 62 / 812,861 <151> 2019-03-01 <160> 1 <170> PatentIn version 3.5 <210> 1 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide: Biotin and F containing peptide <220> <221> MISC_FEATURE <222> (1)..(1) <223> Biotin <220> <221> MOD_RES <222> (6)..(6) <223> Azide Modification <220> <221> MOD_RES <222> (30)..(30) <223> Azide Modification <220> <221> MISC_FEATURE <222> (30)..(30) <223> Amine <400> 1 Lys Gly Ser Gly Ser Lys Gly Ser Gly Ser Arg Phe Ala Gly Val Ala 1 5 10 15 Met Pro Gly Ala Glu Asp Asp Val Val Gly Ser Gly Ser Lys 20 25 30

Claims

1. A method for assessing the identity and spatial relationship between a polypeptide and a moiety in a sample, wherein the moiety comprises another polypeptide or a polynucleotide, the method comprising: a) forming a connection structure between a site of a polypeptide in a sample and a site of a moiety in the sample, the connection structure comprising a polypeptide tag associated with the site of the polypeptide and a moiety tag associated with the site of the moiety, wherein the polypeptide tag and the moiety tag comprise nucleotides, and the polypeptide tag and the moiety tag are associated; b) transferring information between the associated polypeptide tag and the moiety tag by primer extension or ligation to form a shared unique molecular identifier (UMI) and / or barcode; c) destroying the linking structure by dissociating the polypeptide from the moiety and the polypeptide tag from the moiety tag while maintaining the association between the polypeptide and the polypeptide tag and maintaining the association between the moiety and the moiety tag; as well as d) evaluating the polypeptide tag and at least a partial sequence of the polypeptide, and evaluating the partial tag and at least a partial sequence of the portion, Wherein the polypeptide tag and the evaluation portion of the portion tag comprise the shared unique molecular identifier (UMI) and / or barcode, which indicates the spatial proximity of the site of the polypeptide and the site of the portion in the sample.

2. The method according to claim 1, wherein: The portion comprises a polypeptide.

3. The method according to claim 1, wherein: The portion comprises a polynucleotide.

4. The method according to claim 1, wherein: The polypeptide tag comprises a first polynucleotide, and the partial tag comprises a second polynucleotide, the first and second polynucleotides comprise complementary sequences, and the polypeptide tag and the partial tag are associated via the complementary sequence.

5. The method according to claim 4, wherein: Transferring information between the associated polypeptide tag and partial tag includes extending both the first polynucleotide of the polypeptide tag and the second polynucleotide of the partial tag to form the shared UMI and / or barcode.

6. The method according to claim 4, wherein: Transferring information between the associated polypeptide tag and the partial tag comprises extending one of the first polynucleotide of the polypeptide tag and the second polynucleotide of the partial tag to form the shared UMI and / or barcode.

7. The method according to claim 1, wherein: The polypeptide tag comprises a double-stranded polynucleotide, and the partial tag comprises a double-stranded polynucleotide, and transferring information between the associated polypeptide tag and partial tag comprises linking the double-stranded polynucleotides to form the shared UMI and / or barcode.

8. The method according to claim 7, wherein: The shared UMI and / or barcode comprises the sequence of the double-stranded polynucleotide.

9. The method according to claim 7, wherein: The shared UMI and / or barcode comprises a sequence of one of the double-stranded polynucleotides.

10. The method according to claim 1, wherein: In the linked structure, the polypeptide tag and the moiety tag are stably associated.

11. The method according to claim 1, wherein: In the linked structure, the polypeptide tag and the moiety tag are transiently associated.

12. The method according to claim 1, wherein: In the linked structure, the polypeptide tag and the moiety tag are directly associated.

13. The method according to claim 1, wherein: In the connection structure, the polypeptide tag and the partial tag are associated via a linker or a UMI between the polypeptide tag and the partial tag.

14. A method for assessing the identity and spatial relationship between a polypeptide and a moiety in a sample, wherein the moiety comprises another polypeptide or a polynucleotide, the method comprising: a) forming a connection structure between a site of a polypeptide in a sample and a site of a moiety in the sample, the connection structure comprising a polypeptide tag associated with the site of the polypeptide and a moiety tag associated with the site of the moiety, wherein the polypeptide tag and the moiety tag comprise nucleotides, and the polypeptide tag and the moiety tag are associated; b) transferring information between the associated polypeptide tag and the partial tag by primer extension or ligation to form a shared unique molecular identifier (UMI) and / or barcode, wherein the shared UMI and / or barcode is formed as a separate record polynucleotide; c) destroying the linking structure by dissociating the polypeptide from the moiety and the polypeptide tag from the moiety tag while maintaining the association between the polypeptide and the polypeptide tag and maintaining the association between the moiety and the moiety tag; d) evaluating the polypeptide tag and at least a partial sequence of the polypeptide, and evaluating the partial tag and at least a partial sequence of the portion; as well as e) evaluating said isolated record polynucleotides to establish a spatial relationship between the site of said polypeptide and the site of said moiety.

15. The method according to claim 14, wherein: The connection structure is formed between the polypeptide tag and the partial tag via the separated recording polynucleotide.

16. The method according to claim 14, wherein: In the linked structure, the polypeptide tag and the isolated recorder polynucleotide are transiently associated.

17. The method according to claim 14, wherein: In the linked structure, the polypeptide tag and the isolated record polynucleotide are directly associated.

18. The method according to claim 14, wherein: In the ligated structure, the partial tag and the isolated recorder polynucleotide are transiently associated.

19. The method according to claim 14, wherein: In the linked structure, the partial tag and the isolated recording polynucleotide are directly associated.

20. The method according to claim 14, wherein: The isolated record polynucleotide is formed by primer extension.

21. The method according to claim 14, wherein: The isolated record polynucleotide is formed by ligation.

22. The method according to claim 14, wherein: The separated record polynucleotide is released from the polypeptide tag and the partial tag.

23. The method of claim 14, further comprising collecting the isolated transcript polynucleotide prior to evaluating the isolated transcript polynucleotide.

24. The method according to claim 23, wherein: Evaluating the isolated record polynucleotides comprises sequencing the collected shared unique molecular identifiers (UMIs) and / or barcodes, thereby generating sequencing data.

25. The method of claim 14, further comprising conjugating the collected isolated transcript polynucleotides prior to evaluating the isolated transcript polynucleotides.

26. The method according to claim 25, wherein: Evaluating the isolated transcript polynucleotides comprises sequencing the concatenated isolated transcript polynucleotides.

27. The method of claim 1, wherein: Information is transferred between the associated polypeptide tag and the moiety tag by primer extension or ligation to form a single shared unique molecular identifier (UMI) and / or barcode.

28. The method according to claim 27, wherein: The single shared unique molecular identifier (UMI) and / or barcode is formed by combining multiple UMIs and / or barcodes from the polypeptide tag and / or the partial tag.

29. The method of claim 1, wherein: Information is transferred between the associated polypeptide tags and the partial tags by primer extension or ligation to form multiple shared unique molecular identifiers (UMIs) and / or barcodes.

30. The method of claim 1, wherein: In the ligated structure, the shared UMI and / or barcode comprises a complementary polynucleotide hybrid, and dissociating the polypeptide tag from the partial tag comprises denaturing the complementary polynucleotide hybrid.

31. The method of claim 1, wherein: The polypeptide and the portion are both parts of a larger polypeptide, and dissociating the polypeptide from the portion comprises fragmenting the larger polypeptide into peptide fragments.

32. The method according to claim 31, wherein: The larger polypeptide is fragmented into peptide fragments by protease digestion.

33. The method of claim 1, wherein: The moiety is a portion of a molecule that is bound to, complexed with, or in close proximity to a polypeptide in the sample.

34. The method of claim 33, wherein: The polypeptide and the portion belong to two different proteins in the same protein complex.

35. The method of claim 33, wherein: The moiety is a portion of a polynucleotide molecule that is associated with, complexed with, or in close proximity to a polypeptide in the sample.

36. The method according to any one of claims 1 to 13, wherein: At least a partial sequence of the polypeptide is evaluated using a procedure comprising the following steps: a1) providing the polypeptide and the associated polypeptide tag used as a record tag; b1) contacting the polypeptide with a first binding agent capable of binding to the polypeptide, wherein the first binding agent comprises a first coding tag having identification information about the first binding agent; c1) transferring the information of the first coding label to the recording label by primer extension or ligation to generate a first sequence extension recording label; as well as d1) Analyzing the first sequence extension record label.

37. The method of claim 36, wherein: Analyzing the first sequence extension record signature also evaluates the polypeptide signature.

38. The method of claim 1, wherein: The portion comprises a portion of a polypeptide, and at least a partial sequence of the portion is evaluated using a procedure comprising the following steps: a2) providing the partial polypeptide and the associated partial tag used as a record tag; b2) contacting the partial polypeptide with a first binding agent capable of binding to the partial polypeptide, wherein the first binding agent comprises a first coding tag having identification information about the first binding agent; c2) transferring the information of the first coding label to the recording label by primer extension or ligation to generate a first sequence extension recording label; as well as d2) Analyzing the first sequence extension record label.

39. The method of claim 38, wherein: Analyzing the first order extended record tag also evaluates the partial tag.

40. A method for assessing the identity and spatial relationship between a polypeptide and a moiety in a sample, wherein the moiety comprises another polypeptide or a polynucleotide, the method comprising: a) providing a preassembled structure comprising a shared unique molecular identifier (UMI) and / or barcode in a middle portion, the UMI and / or barcode being flanked on one side by a polypeptide tag and on another side by a moiety tag, wherein the polypeptide tag and the moiety tag comprise nucleotides; b) forming a junction structure between the site of the polypeptide in the sample and the site of the moiety in the sample by associating the polypeptide tag of the preassembled structure with the site of the polypeptide and associating the moiety tag of the preassembled structure with the site of the moiety; c) destroying the linking structure by dissociating the polypeptide from the moiety and the polypeptide tag from the moiety tag while maintaining the association between the polypeptide and the polypeptide tag and maintaining the association between the moiety and the moiety tag; as well as d) evaluating the polypeptide tag and at least a partial sequence of the polypeptide, and evaluating the partial tag and at least a partial sequence of the portion, wherein the polypeptide tag and the evaluation portion of the portion tag comprise the shared unique molecular identifier (UMI) and / or barcode indicating the spatial proximity of the site of the polypeptide and the site of the portion in the sample.

41. The method of claim 40, wherein: The portion comprises a polypeptide.

42. The method of claim 40, wherein: The portion comprises a polynucleotide.

43. The method of claim 40, wherein: In the linked structure, the polypeptide tag and the moiety tag are stably associated.

44. The method of claim 40, wherein: In the linked structure, the polypeptide tag and the moiety tag are transiently associated.

45. The method of claim 40, wherein: In the linked structure, the polypeptide tag and the moiety tag are directly associated.

46. ​​The method of claim 40, wherein: In the connection structure, the polypeptide tag and the partial tag are associated via a linker or a UMI between the polypeptide tag and the partial tag.

47. The method of claim 40, wherein: The formed ligated structure comprises a single shared unique molecular identifier (UMI) / barcode.

48. The method of claim 40, wherein: The formed ligated structure comprises a plurality of shared unique molecular identifiers (UMIs) / barcodes.

49. The method of claim 40, wherein: The polypeptide tag comprises a first polynucleotide and the partial tag comprises a second polynucleotide.

50. The method of claim 40, wherein: In the ligated structure, the shared UMI and / or barcode comprises a complementary polynucleotide hybrid, and dissociating the polypeptide tag from the partial tag comprises denaturing the complementary polynucleotide hybrid.

51. The method of claim 40, wherein: The polypeptide and the portion are both parts of a larger polypeptide, and dissociating the polypeptide from the portion comprises fragmenting the larger polypeptide into peptide fragments.

52. The method of claim 51, wherein: The larger polypeptide is fragmented into peptide fragments by protease digestion.

53. The method of claim 40, wherein: The moiety is a portion of a molecule that is bound to, complexed with, or in close proximity to a polypeptide in the sample.

54. The method of claim 53, wherein: The polypeptide and the portion belong to two different proteins in the same protein complex.

55. The method of claim 53, wherein: The moiety is a portion of a polynucleotide molecule that is associated with, complexed with, or in close proximity to a polypeptide in the sample.

56. The method of claim 40, wherein: At least a partial sequence of the polypeptide is evaluated using a procedure comprising the following steps: a3) providing the polypeptide and the associated polypeptide tag for use as a record tag; b3) contacting the polypeptide with a first binding agent capable of binding to the polypeptide, wherein the first binding agent comprises a first coding tag having identification information about the first binding agent; c3) transferring the information of the first coding label to the recording label by primer extension or ligation to generate a first sequence extension recording label; as well as d3) Analyzing the first sequence extension record label.

57. The method of claim 56, wherein: Analyzing the first sequence extension record signature also evaluates the polypeptide signature.

58. The method of claim 40, wherein: The portion comprises a portion of a polypeptide, and at least a partial sequence of the portion is evaluated using a procedure comprising the following steps: a4) providing the partial polypeptide and the associated partial tag for use as a record tag; b4) contacting the partial polypeptide with a first binding agent capable of binding to the partial multi-polypeptide, wherein the first binding agent comprises a first coding tag having identification information about the first binding agent; c4) transferring the information of the first coding label to the recording label by primer extension or ligation to generate a first sequence extension recording label; as well as d4) Analyzing the first sequence extension record label.

59. The method of claim 58, wherein: Analyzing the first order extended record tag also evaluates the partial tag.

60. The method of claim 1, wherein: The evaluation of the at least partial sequence of the polypeptide and the at least partial sequence of the moiety is performed after forming a junction structure between the site of the polypeptide and the site of the moiety.

61. The method of claim 1, wherein: The evaluation of at least a partial sequence of the polypeptide and at least a partial sequence of the portion is performed after dissociation of the polypeptide from the portion.

62. The method of claim 36, wherein: Contacting the polypeptide and the moiety with one or more binding agents is performed after forming a linkage structure between the polypeptide and the moiety.

63. The method of claim 36, wherein: Contacting the polypeptide and the moiety with one or more binding agents is performed after dissociation of the polypeptide from the moiety.

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