CROSS REFERENCE OF RELATED APPLICATION

[0001] This application claims priority to U.S. Provisional Patent Application No. 63/379,603, filed on October 14, 2022, titled “LABELING KIT INVOLVING NETWORKED CLUSTER FORMATION AND LABELING METHOD AND LABELED TARGET USING THE SAME,” which is herein incorporated by reference in its entirety.

SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted herewith and is hereby incorporated by reference in its entirety. Said ST.26.xml copy, created on October 12, 2023, is named 14815-713600, and is 7,409 bytes in size.

TECHNICAL FIELD

[0003] Described herein are methods and kits useful for identifying, tagging, and analyzing biomolecules. Specifically described are labeling kits involving networked cluster formation and labeling methods using the same.

BACKGROUND

[0004] Characterizing the subcellular environment, within which biomolecules interact with one another, and how the biomolecules function together is very challenging. Biomolecules are small, and they exist in a cell environment with tens of millions of other molecules. The interactions between neighboring biomolecules are frequently weak, and techniques used to study biomolecules can disrupt their interactions. While techniques such as yeast two- hybridization assays and more recently proximity labeling have advanced our understanding of the cell environment, these techniques suffer from various limitations such as nonspecific binding, slow reaction times, and disruption of the natural cell environment, resulting in false positives and missed interactions.

[0005] Existing techniques for selectively identifying biomolecular interactions, e.g., proteinprotein interactions, include enzymatic proximity labeling platforms, such as BioID (proximity dependent biotin identification), APEX (engineered ascorbate peroxidase), and EMARS (Enzyme-Mediated Activation of Radical Source). These technologies involve the use of enzyme-based reactive intermediates that label neighboring proteins on specific amino acid residues, but they also have been identified to have drawbacks. For example, these reactive intermediates may diffuse far from their target and give unreliable or misleading results.

[0006] In recent years, use of a photocatalyst-antibody conjugate to spatially localize carbene generation was disclosed for use in proximity labeling experiments (Jacob B. Geri et. al., Science. 2020 March 06; 367(6482): 1091-1097). This micromapping technology aims to operate at shorter range and with higher precision than enzymatic proximity labeling platforms.

However, to minimize damage to the biological sample and reduce the overall photochemical reaction time, the illumination duration is typically kept short. For example, 100 microseconds may be needed for a specific reaction. Therefore, the efficiency of the photochemical reaction is low, e.g., merely 1-10% of neighboring molecules may be labeled, leading to inconclusive or misleading results in subsequent mass spectrometry analysis. Accordingly, there is an ongoing need for new methods and probes for proximity labeling in biological systems.

SUMMARY OF THE DISCLOSURE

[0007] Described herein are systems, kits, and methods useful for identifying, tagging, and analyzing biomolecules. Specifically described are labeling kits which involve networked cluster formation. These labeling kits can be used with a photosensitive probe and a microscope system for selectively tagging and labeling of biomolecules via selective light illumination through a microscope system.

[0008] One aspect of the disclosure provides a labeling kit for proximity labeling comprising a plurality of first tagged spacer precursors, each of the first tagged spacer precursors represented by the following general structure (II): L _a -Ti, wherein L _a is a first linkable moiety and Ti is a first tag moiety connected covalently to L _a; a plurality of second tagged spacer precursors, each of the second tagged spacer precursors represented by the following general structure (III): Ti-Lb, wherein T2 is a second tag moiety and Lb is a second linkable moiety connected covalently to T2, wherein first linkable moiety L _a and second linkable moiety Lb are configured to form a linkage therebetween to thereby form a dual-tagged spacer; a plurality of tagged enzymes, each of the tagged enzymes having the structure represented by the following general structure (IX): E1-T3, wherein T3 is a tag moiety and Ei is an enzymatic moiety, wherein the enzymatic moiety Ei is configured to catalyze an activatable substrate into an activated substrate ready to bind a neighboring molecule; and a plurality of connectors, each of the connectors including a plurality of binding sites, wherein each binding site is configured to allow affinity binding with one or more of the first tag moiety Ti, the second tag moiety T2, or the third tag moiety T3. [0009] Another aspect of the disclosure provides another labeling kit for proximity labeling including a plurality of dual-tagged spacers, each of the dual-tagged spacers represented by the following general structure (I): T1-L1-T2, wherein Ti is a first tag moiety, T2 is a second tag moiety, and Li is a linker therebetween connecting Ti and T2; a plurality of tagged enzymes, each of the tagged enzymes having the structure represented by the following general structure (IX): E1-T3, wherein the enzymatic moiety Eiis configured to catalyze an activatable substrate into an activated substrate ready to bind to a neighboring molecule; and a plurality of connectors, wherein each of the connectors includes a plurality of binding sites, wherein each binding site is configured to allow affinity binding with one or more of the first tag moiety Ti, the second tag moiety T2, or the third tag moiety T3.

[0010] In this and other embodiments, the first linkable moiety L _a and second linkable moiety

Lb are configured by click chemistry or hybridization to form a linkage therebetween to thereby form a dual-tagged spacer.

[0011] In these and other embodiments, the first linkable moiety L _a and the second linkable moiety Lb each independently include a click chemical group, each click chemical group is independently an alkyne-based group, BCN, DBCO, N3, or another azide-based group.

[0012] In these and other embodiments, the first linkable moiety L _a includes a nucleic acid anchoring strand, and the second linkable moiety Lb includes a nucleic acid probing strand, and wherein the probing strand and the anchoring strand are configured to form a double-stranded structure with each other along a complementary sequence. In these and other embodiments, the anchoring strand and the probing strand include DNA.

[0013] In these and other embodiments, the linker Li includes a moiety of (PEG) _n , a peptide, an amino acid, an oligonucleotide, or a combination thereof, and wherein n is an integer from 1- 20, and the length of each linker Li is less than 2 nm.

[0014] In these and other embodiments, the first tag moiety Ti, the second tag moiety T2, and the third tag moiety T4 are each independently a biotin derivative.

[0015] In these and other embodiments, the first tag moiety Ti, the second tag moiety T2, and the third tag moiety T3 each independently include the moiety or a derivative thereof [0016] In these and other embodiments, the plurality of connectors includes avidin or streptavidin.

[0017] In these and other embodiments, the labeling kits for proximity labeling further includes a plurality of tagged probes having the structure represented by the following general structure (XV): T4-P1, wherein T4 is a tag moiety and Pi is an activatable substrate, and wherein the tagged probes are activatable by the tagged enzymes having the structure represented by the general structure (IX) to thereby facilitate covalent bond formation between activated tagged probes and neighbor molecules in the vicinity thereof. In these and other embodiments, tagged probes are desthiobiotin-phenol or biotin-phenol.

[0018] In these and other embodiments, the labeling kits for proximity labeling further include a plurality of photoreactive tagged probes, each tagged probe represented by the following general structure (XIV): T5-P2, wherein T5 is a tag moiety and P2 is a probe moiety configured to bind a molecule of interest, wherein the tagged probes represented by the general structure (XIV) are photoactivatable at a wavelength ranging from 700 nm to 1100 nm with a two-photon light source, or at a wavelengh ranging from 300 nm to 800 nm with a light source, so as to allow the photoreactive tagged probe to label a molecule of interest Ii in a biological sample to form a tagged probe-target complex represented by the general structure (IV): Ts-Pz’Ii. [0019] In these and other embodiments, the tagged probes and the photoreactive tagged probes are desthiobiotin-phenol or biotin-phenol.

[0020] Another aspect of the disclosure provides methods for proximity labeling of neighboring molecules in the vicinity of a molecule of interest in a biological sample by using the labeling kits described herein.

[0021] Another aspect of the disclosure provides methods method for proximity labeling of neighboring molecules in the vicinity of a molecule of interest in a biological sample, including one or more of the steps of (a) delivering a plurality of first connectors Ci to the biological sample, wherein the biological sample includes a plurality of tagged probe-molecule of interest complexes, each tagged probe-molecule of interest complex represented by the following general structure (IV): Ts-P ’Ii, wherein T5 is a tag moiety, P2 is a probe moiety, and Ii is a molecule of interest, wherein each of the first connectors Ci includes a plurality of binding sites with each binding site configured to allow affinity binding with the tag moiety T5 of the tagged probemolecule of interest complex; (b) capturing, with each tag portion T5 of each tagged probemolecule of interest complex, one of the plurality of first connector binding sites to thereby attach each first connector Ci to a corresponding tagged probe-molecule of interest complex to form a complex represented by the general structure (V): Ci — tagged probe-molecule of interest complex; (c) delivering a plurality of first tagged spacer precursors to the biological sample, each of the first tagged spacer precursors represented by the following general structure (II): L _a -Ti, wherein L _a is a first linkable moiety and Ti is a first tag moiety covalently connected to L _a ; (d) binding the first tag moiety Ti of each first tagged spacer precursor to an available first connector binding site on each Ci-tagged probe-molecule of interest complex to form a complex represented by the general structure (VI): L _a -Ti — Ci — tagged probe-molecule of interest complex; (e) delivering a plurality of second tagged spacer precursors to the biological sample, each of the second tagged spacer precursors represented by the following general structure (III): T2-L1,, wherein T2 is a second tag moiety and b is a second linkable moiety covalently connected to T2, wherein the first linkable moiety L _a and the second linkable moiety Lb are configured to form a linkage therebetween and form a dual-tagged spacer; (f) forming a linkage between the first linkable moiety L _a of each complex represented by the general structure (VI) and the second linkable moiety Lb of each second tagged spacer precursor to thereby form a complex represented by the general structure (VII): T2-Lb’L _a -Ti — Ci — tagged probe-molecule of interest complex; (g) delivering a plurality of second connectors C2 to the biological sample, wherein each of the second connectors C2 includes a plurality of binding sites, each second connector binding site configured to affinity bind with the second tag moiety T2 of the dual-tagged spacers; (h) binding each second connector binding site to one second tag moiety T2 in the complex having the general structure (VII) to form a complex represented by the general structure (VIII): (C2 — T2-Lb ^, L _a -Ti) _q — Ci — tagged probe-molecule of interest complex; (g) delivering a plurality of tagged enzymes to the biological sample, each enzymic tagged enzyme represented by the general structure (IX): E1-T3, wherein T3 is a third tag moiety and Ei is an enzymatic moiety; (h) binding the third tag moiety T3 of each tagged enzyme with an available binding site on an available binding site on a connector C2 of the complex represented by the general structure (VIII) to form a complex represented by the general structure (X): E1-T3 — (C2 — T2-Lb*L _a -Ti) _q — Ci — tagged probemolecule of interest complex; (i) delivering a plurality of enzymic tagged probe having an activatable substrate to the biological sample; and (j) catalyzing, with the tagged enzyme in the complex represented by the general structure (X), the activatable substrate into an activated substrate, and covalently binding the activated substrate to a neighbor molecule in the vicinity of the complex represented by the general structure (X).

[0022] Another aspect of the disclosure provides method for proximity labeling of neighboring molecules in the vicinity of a molecule of interest in a biological sample, including: (a) delivering a plurality of first connectors Ci to the biological sample, wherein the biological sample includes a plurality of tagged probe-molecule of interest complexes, each tagged probemolecule of interest complex represented by the following general structure (IV): Ts-Pz’li, wherein T5 is a tag moiety, P2 is a probe moiety, and Ii is a molecule of interest, wherein each of the first connectors Ci includes a plurality of binding sites with each binding site configured to allow affinity binding with the tag moiety T5 of the tagged probe-molecule of interest complex;

(b) capturing, with each tag portion T3 of each tagged probe-molecule of interest complex, one of the plurality of first connector binding sites to thereby attach each first connector Ci to a corresponding tagged probe-molecule of interest complex to form a complex represented by the general structure (V): Ci — tagged probe-molecule of interest complex; (c) delivering a plurality of dual tagged spacer to the biological sample, each of the dual tagged spacer represented by the following general structure (I): T1-L1-T2, wherein Ti is a first tag moiety, T2 is a second tag moiety, and Li is a linker therebetween connecting Ti and T2; (d) binding the first tag moiety Ti of each dual tagged spacer to an available first connector binding site on each Cl -tagged probemolecule of interest complex to form a complex represented by the general structure (XI): T2-L1- Ti — Ci — tagged probe-molecule of interest complex; (e) delivering a plurality of second connectors C2 to the biological sample, wherein each of the second connectors C2 includes a plurality of binding sites, each second connector binding site configured to affinity bind with the second tag moiety T2 in the complex represented by the general structure (XI); (f) binding each second connector binding site to one second tag moiety T2 in the complex having the general structure (XI) to form a complex represented by the general structure (XII): (C2 — T2- Li- Ti) _q — Ci — tagged probe-molecule of interest complex; (g) delivering a plurality of tagged enzymes to the biological sample, each tagged enzyme represented by the general structure (IX): E1-T3, wherein T3 is a third tag moiety and El is an enzymatic moiety; (h) binding the third tag moiety T3 of each tagged enzyme with an available binding site on an available binding site on the second connector C2 of the complex represented by the general structure (XII) to form a complex represented by the general structure (XIII): Ei- T3 — (C2 — T2- Li-Ti)q — Ci — tagged probemolecule of interest complex; (i) delivering a plurality of enzymic tagged probe having an activatable substrate to the biological sample; and (j) catalyzing, with the tagged enzyme in the complex represented by the general structure (XIII), the activatable substrate into an activated substrate, and covalently binding the activated substrate to a neighbor molecule in the vicinity of the complex represented by the general structure (XIII).

[0023] In these and other embodiments, the biological sample includes a monolayer of cells disposed on a substrate.

[0024] In these and other embodiments, the linker moiety Li includes a moiety of (PEG)n, a peptide, an amino acid, an oligonucleotide, or a combination thereof, and wherein n is an integer from 1-20.

[0025] In these and other embodiments, a length of each linker Li is less than 2 nm. [0026] These and other embodiments may also include the step of forming by click chemistry or hybridization a linkage between the first linkable moiety L _a and second linkable moiety Lb to thereby form the dual-tagged spacer.

[0027] In these and other embodiments, the first linkable moiety L _a and second linkable moiety Lb each independently include a click chemical group.

[0028] In these and other embodiments, each click chemical group is independently an alkyne-based group, BCN, DBCO, N3, or another azide-based group.

[0029] In these and other embodiments, the first linkable moiety L _a includes a nucleic acid anchoring strand, and the second linkable moiety Lb includes a nucleic acid probing strand, step (f) forming a linkage further includes forming a double- stranded structure between the probing strand and the anchoring strand along a complementary sequence.

[0030] In these and other embodiments, the anchoring strand and the probing strand include DNA.

[0031] In these and other embodiments, the enzymatic moiety is either a peroxidase or a biotin ligase.

[0032] In these and other embodiments, the first tag moiety, the second tag moiety, and the third tag moiety are each independently a biotin derivative.

[0033] In these and other embodiments, the first tag moiety, the second tag moiety, and the third tag moiety each independently include the moiety or a derivative thereof.

[0034] In these and other embodiments, the first connector and the second connector independently include avidin or streptavidin.

[0035] In these and other embodiments, the tagged probes are desthiobiotin-phenol or biotinphenol.

[0036] These and other methods can further include, prior to step (a), the steps of (i) delivering a plurality of tagged probes to the biological sample, each tagged probe represented by the following general structure (XIV): T5-P2, wherein T5 is a tag moiety and P2 is a probe moiety configured to bind the molecule of interest and (ii) photoactivating each tagged probe represented by the general structure (XIV) at a wavelength ranging from 700 nm to 1100 nm with a two- photon light source, to thereby label a molecule of interest L in the biological sample to form the tagged probe-target complex represented by the general structure (IV): Ts-Pi’Ii. [0037] These and other methods can further include, prior to step (a), the steps of (i) delivering a plurality of tagged probes to the biological sample, each tagged probe represented by the following general structure (XIV): T5-P2 wherein T5 is a tag moiety and P2 is a probe moiety configured to bind the molecule of interest and (ii) photoactivating each tagged probe represented by the general structure (XIV) at a wavelength ranging from 300 nm to 800 nm with a light source, to thereby label a molecule of interest Ii in the biological sample to form the tagged probe-target complex represented by the general structure (IV): Ts-Pi’Ii.

[0038] These and other methods can further include, prior to step of delivering a plurality of tagged enzymes, forming multilayers of dual-tagged spacers and connectors C2 on the (C2 — T2- La«L _b -Ti) _q — Ci — tagged probe-molecule of interest complex, such that q is a number from 2 to 30.

[0039] These and other methods can further include, prior to step of delivering a plurality of tagged enzymes, forming multilayers of dual-tagged spacers and connectors C2 on the (C2 — T2- Li) _q — Ci — tagged probe-molecule of interest complex, such that q is a number from 2 to 30.

[0040] In these and other embodiments, the activatable substrate is desthiobiotin-phenol or biotin-phenol.

[0041] Accordingly, the present disclosure provides labeling kits and methods thereof utilize tagging system, e.g., the biotin-(strept)avidin system, and tyramide signal amplification (TSA) to form networked cluster that may be useful to enhance the detection of low-abundance targets.

BRIEF DESCRIPTION OF THE DRAWINGS

[0042] A better understanding of the features and advantages of the methods and apparatuses described herein will be obtained by reference to the following detailed description that sets forth illustrative embodiments, and the accompanying drawings of which:

[0043] FIG. 1 shows a schematic depiction of a system for photoselective spatial tagging and labeling of cells on a substrate.

[0044] FIGS. 2A-2B schematically illustrates a labeling process for labeling molecules of interest using a networked cluster formation process. FIG. 2A schematically illustrates selective illumination of a small region of a substrate to label biomolecules of interest in the illuminated area. FIG. 2B schematically illustrates steps in a networked cluster formation process and further labeling of biomolecules proximal to the biomolecule of interest.

[0045] FIG. 3 schematically illustrates another labeling process for labeling molecules of interest using a networked cluster formation process.

[0046] FIGS. 4A-4E show examples of click chemical groups that can be used as the first and second linkable moieties of the first and second tagged spacers described herein. FIG. 4A illustrates an alkyne-based group. FIG. 4B illustrates DBCO (dibenzocyclooctyne). FIG. 4C illustrates BCN (bicyclo[6.1. 0]non-4-yne). FIG. 4D illustrates an IM (azide) group. FIG. 4E illustrates an azide-based group.

[0047] FIGS. 5A-5B (SEQ ID NOs: 1-7) shows examples of nucleic acid anchoring strand sequences and nucleic acid probing strand sequences that can be used as the first and second linkable moieties of the first and second tagged spacers described herein. FIG. 5A illustrates examples of nucleic acid anchoring strand sequences. FIG. 5B illustrates examples of nucleic acid probing strand sequences.

[0048] FIGS. 6A-6C shows examples of biotin derivatives that can be used as tag moieties in the first tagged spacer precursor, the second tagged spacer precursor, the dual-tagged spacers, the tagged enzymes, or other tags as described herein. FIG. 6A illustrates biotin derivatives. FIG. 6B illustrates desthiobiotin derivatives. FIG. 6C illustrates iminobiotin.

DETAILED DESCRIPTION

[0049] The embodiments of the invention will be apparent from the following detailed description, which proceeds with reference to the accompanying drawings, wherein the same reference characters relate to the same elements.

[0050] The ranges set forth herein may be interpreted as being inclusive of their endpoints, and open-ended ranges may be interpreted to include only commercially practical values. Similarly, lists of values may be considered as inclusive of intermediate values unless the context indicates the contrary. The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range.

[0051] Methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise indicated otherwise context. The use of examples, or exemplary language (e.g., “such as”) provided with respect to certain embodiments herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed.

[0052] Described herein are labeling kits, and labeling methods useful for identifying, tagging, obtaining, and analyzing biomolecules of interest and their neighboring biomolecules. To solve the aforementioned technical problem, the labeling kits provide by the various embodiments of this present invention can be used for tyramide signal amplification via networked cluster of biotin/ streptavidin interaction after in situ tagging of biomolecules such as proteins inside cells or tissues. The tagged biomolecules and their neighboring biomolecules can be further analyzed by analytical techniques such as mass spectrometry and sequencing. Moreover, the proximity labeling method provide by the various embodiments of this present invention may be especially useful for performing omics studies, such as genomics, proteomics, and transcriptomics, and for finding relevant biomarkers for diagnosis and treatment.

[0053] Abbreviations and Definitions:

[0054] The term “biotin” or “biotin derivative” refers to a biotin moiety, including biotin and variations of biotin, such as biotin with an open ring or substitutions. Typically, a biotin derivative is easily detectable with a biotin-binding entity or protein, such as avidin or streptavidin.

[0055] The term “biotin ligase” (e.g., biotin-protein ligase, BirA, BirA*, BirZD, BioID2, miniTurbo, Turbo ID) refers to an enzyme that activates a biotin moiety and ligates the activated biotin moiety to a target biotin acceptor. A target biotin acceptor can be, for example, a lysine amino acid, a peptide, or a protein.

[0056] The term "click chemistry" refers to a chemical approach that easily joins molecular building blocks (e.g., click chemical groups). Typically, click chemistry reactions are efficient, high-yielding, reliable, create few or no byproducts, and are compatible with an aqueous environment or without an added solvent. An example of click chemistry is cycloaddition, such as the copper(I)-catalyzed [3+2]- Huisgen 1,3-dipolar cycloaddition of an alkyne and azide leading to the formation of 1,2, 3 -triazole or Diels-Adler reaction. Click chemistry also includes copper free reactions, such as a variant using substituted cyclooctyne (see e.g., J. M. Baskin et al., Proc. Natl. Acad. Sci. U.S.A. 2007 Oct. 23, 104 (43), 16793-16797.) Other examples of click chemistry are nucleophilic substitutions; additions to C-C multiple bonds (e.g., Michael addition, epoxidation, dihydroxylation, aziri dination); and nonaldol like chemistry (e.g., N- hydroxysuccinimide active ester couplings). Click chemistry reactions can be bioorthogonal reactions, but do not need to be.

[0057] The term “tagged enzyme” refers to an enzyme with a tag attached thereto. In some embodiments, a tagged enzyme can be configured to catalyze an activatable substrate into an activated substrate ready to bind to a neighboring molecule. Examples of tagged enzymes include tagged biotin ligase-based enzymes and peroxidase-based enzymes, such as horseradish peroxidase and (engineered) ascorbate peroxidase (APEX). Examples of activable substrates for tagged enzymes include biotin, biotin derivatives, biotin-phenol, and hydrogen peroxide.

[0058] The term “linker” refers to a structure which connects two or more substructures. A linker may have one (or more than one) uninterrupted chain of atoms extending between the substructures. The atoms of a linker are connected by chemical bonds, which in some embodiments are covalent bonds. In some variations, a linker may include two (or more than two) moi eties joined specifically together, such as by hybridization therebetween. The moieties of a linker joined by hybridization are connected by chemical bonds, typically non-covalent bonds (e.g., hydrogen bonds).

[0059] The term “proximity molecule” or “neighboring molecule” refers to a molecule that is near to a biomolecule of interest. A “proximity molecule” or “neighboring molecule” may be bound (e g., covalently or non-covalently) to the biomolecule of interest or may be close by and not bound to the biomolecule of interest.

[0060] The term “tagging” refers to the process of adding a tag to a structure (to a functional group, compound, molecule, substituent, or the like). The term “tagged” refers to a functional group, compound, molecule, substituent, or the like that has a tag attached. A tag can be attached to a structure covalently or non-covalently (e.g., conjugated). Examples of tagged structures include tagged probes, tagged enzymes, dual-tagged spacers (with two or more tags attached thereto), and tagged biomolecules. In some embodiments, a tag is biotin or a biotin derivative and can be a biotin conjugate. In some embodiments, tagged biomolecules refers to biotinylation of the biomolecules.

[0061] The term “tyramide signal amplification” refers to a catalyzed reporter deposition (CARD). Tyramide signal amplification is an enzyme-mediated detection method that utilizes catalytic activity of an enzyme (e.g., horseradish peroxidase (HRP)) to catalyze inactive tyramide molecules to highly active tyramide molecules ready to be deposited. Each enzyme can catalyze many tyramide molecules (amplification) and amplification can take place in the presence of low concentrations of hydrogen peroxide (H2O2) to activate phenolic compounds (tyramide) for phenoxy radical reaction with nucleic acids or proteins (such as on tyrosine amino acids). In some embodiments herein, tyramide can be bound to (e g., conjugated with) a biotin or biotin derivative before reaction to achieve peroxidase-based biotinylation of neighboring molecules. [0062] Throughout the detailed description and examples of the disclosure the following general shorthand will be used:

[0063] General structure (I): T1-L1-T2

[0064] General structure (II): L _a -Ti

[0065] General structure (III): T2-Lb

[0066] General structure (IV): Ts-Pi’Ii

[0067] General structure (V): Ci — tagged probe-molecule of interest complex

[0068] General structure (VI): L _a -Ti — Ci — tagged probe-molecule of interest complex [0069] General structure (VII): T2-Lb*L _a -Ti — Ci — tagged probe-molecule of interest complex

[0070] General structure (VIII): (C2 — T2-Lb*L _a -Ti) _q — Ci — tagged probe-molecule of interest complex [0071] General structure (IX): E1-T3

[0072] General structure (X): E1-T3 — (C2 — T2-Lb*L _a -Ti) _q — Ci — tagged probe-molecule of interest complex

[0073] General structure (XI): T2- L1-T1 — Ci — tagged probe-molecule of interest complex [0074] General structure (XII): (C2 — T2-Li-Ti) _q — Ci — tagged probe-molecule of interest complex

[0075] General structure (XIII): E1-T3 — (C2 — T2-Li-Ti) _q — Ci — tagged probe-molecule of interest complex

[0076] General structure (XIV): T5-P2

[0077] General structure (XV): T4-P1

[0078] The shorthand refers to a covalent bond.

[0079] The shorthand “ — ” refers to a non-covalent bond.

[0080] The shorthand refers to a molecular binding, which can be either covalent bond or non-covalent bond.

[0081] Terms not specifically defined herein are given their normal meaning in the art.

[0082] Described herein are labeling kits that may be useful for performing proximity labeling, methods described herein, e.g., for analyzing, tagging, and labeling biomolecules. In a first aspect, the labeling kits can include a plurality of first tagged spacer precursors, each of the first tagged spacer precursors represented by the following general structure (II): L _a -Ti, wherein L _a is a first linkable moiety and Ti is a first tag moiety connected covalently to L _a; a plurality of second tagged spacer precursors, each of the second tagged spacer precursors represented by the following general structure (III): T2-Lb, wherein T2 is a second tag moiety and Lb is a second linkable moiety connected covalently to T2, wherein first linkable moiety L _a and second linkable moiety Lb are configured to form a linkage therebetween to thereby form a dual-tagged spacer; a plurality of tagged enzymes, each of the tagged enzymes having the structure represented by the following general structure (IX): E1-T3, wherein T3 is a tag moiety and Ei is an enzymatic moiety, wherein the enzymatic moiety Ei is configured to catalyze an activatable substrate into an activated substrate ready to bind a neighboring molecule; and a plurality of connectors, each of the connectors including a plurality of binding sites, wherein each binding site is configured to allow affinity binding with one or more of the first tag moiety Ti, the second tag moiety T2, or the third tag moiety T3. [0083] Another aspect of the disclosure provides labeling kits including a plurality of dual-tagged spacers, each of the dual-tagged spacers represented by the following general structure (I): T1-L1-T2, wherein Ti is a first tag moiety, T2 is a second tag moiety, and Li is a linker therebetween connecting Ti and T2; a plurality of tagged enzymes, each of the tagged enzymes having the structure represented by the following general structure (IX): E1-T3, wherein the enzymatic moiety Ei is configured to catalyze an activatable substrate into an activated substrate ready to bind to a neighboring molecule; and a plurality of connectors, wherein each of the connectors includes a plurality of binding sites, wherein each binding site is configured to allow affinity binding with one or more of the first tag moiety Ti, the second tag moiety T2, or the third tag moiety T3.

[0084] The kits may additionally include wash solutions, such as blocking agents, detergents, salts (e.g., sodium chloride, potassium chloride, phosphate buffer saline (PBS)) for one or more steps (e.g., after sample fixation). The kits may include variations of wash solutions, such as concentrates of wash buffers configured to be diluted before use or components to use for making one or more wash solutions) and other reagents routinely used for the practice of a particular method. The kits may include fixatives and other sample preparation materials (e.g., ethanol, methanol, formalin, paraffin, etc.)

[0085] In some embodiments, the sample is fixed. For example, a cell or tissue sample may be fixed with e.g., acetic acid, acetone, formaldehyde (4%), formalin (10%), methanol, glutaraldehyde, or picric acid. A fixative may be a relatively strong fixative and may crosslink molecules or may be weaker and not crosslink molecules. A cell or tissue sample for analysis may be frozen, such as using dry ice or flash frozen, prior to analysis. A cell or tissue sample may be embedded in a solid material or semi-solid material such as paraffin or resin prior to analysis. In some embodiments, a cell or tissue sample for analysis may be subject to fixation followed by embedding, such as formalin fixation and paraffin embedding (FFPE).

[0086] Example 1 : Photolabeling of neighbor molecules proximal to a biomolecule of interest

[0087] The kits described herein can advantageously be used with a photosensitive probe and a microscope system, such as the systems described herein and in U.S. Patent No. 11,265,449, which is herein incorporated by reference in its entirety, to enable automatic labeling of hundreds of thousands of cellular biomolecules proximal to a biomolecule of interest. FIG. 1 shows a schematic depiction of a system useful for photoselective spatial tagging and labeling. The bottom part of FIG. 1 shows substrate 106, such as a microscope stage, and a monolayer of plurality of cells 108 disposed on the substrate. In some embodiments, the surface of an entire substrate, or a portion of the substrate, can be analyzed using an automated microscope system to identify a region of interest. For example, a sample can be stained or labeled to identify a region of interest. The top part of FIG. 1 shows an expanded view of cell 108a, one of the plurality of cells 108. The cell 108a has a nucleus 116 and a plurality of various types of organelles 112, such as cell membranes, mitochondria, ribosomes, and vacuoles. Microscope system 102 selectively shines narrow band of light 104 onto region of interest (ROI) 118 for illumination of the region of interest 118. The illumination can be selective, and large regions 114 of the cell and substrate are not illuminated.

[0088] FIG. 2A schematically illustrates a close-up of photoselective spatial tagging on the region of interest 118 (as shown in FIG. 1). Prior to performing the illumination, a sample (e.g., a cell or tissue sample) containing a biomolecule of interest 210 (protein will be used herein by way of example, but other biomolecules could instead be analyzed) on the substrate 209 is analyzed and a region of interest identified. The sample can be pretreated, such as fixed and stained. For example, a sample can be fixed and stained with a cell stain (e.g., hematoxylin and eosin (H &E); Masson’s tri chrome stain), identified with an immunofluorescent labeled antibody recognizing a protein of interest or by other methods. Once the region of interest is identified, photosensitive probes 201 and photoreactive tagged probes 202 are added to the sample, then the microscope system 102 illuminates the region of interest 118.

[0089] The photosensitive probes 201 include a photocatalyst 221 coupled to a labeling moiety 222, wherein the labeling moiety is configured to selectively bind the biomolecule of interest 210 (e.g., a primary antibody) or to selectively bind the above immunofluorescent labeled antibody (e.g., a secondary antibody), and the photocatalyst 221 can have an electronic structure permitting energy transfer to the photoreactive tagged probe 202 to form an activated photoreactive tagged probe 202’. The type of the photocatalyst 221 is not particularly limited and may be, for example, any one of those described in WO2023/196986, which is herein incorporated by reference in its entirety. In some embodiments of the present invention, the photocatalyst may be ruthenium-based photocatalyst.

[0090] The photoreactive tagged probes 202 (non-activated type) include a tag portion Ts and a phenol group P2, wherein the phenol group P2 can be activated (e.g., oxidized) by photocatalyst 221 to generate a phenoxyl radical resulting in the labelling of nearby neighbor molecules (while the phenolic group represented by P2 is present in the structure represented by 202”, for simplicity in the drawings, it is omitted from bound tagged probe 202” such as shown in e.g., FIG. 2A). In some embodiments, the tag portion T5 may be biotin or desthiobiotin.

[0091] The reactive tagged probe 202’ reacts or crosslinks with neighboring biomolecules 211 (the “target” of the photoreactive tagged probe) within a diffusion radius of the reactive tagged probe 202’. Therefore, after illumination of narrow band of light 104, the proximity labeling of one neighboring biomolecule 211 with bound tagged probe 202” is completed to form a tagged probe-target complex 230 (indicated by the dotted circle area). However, because illumination time of each point is very short (e g., laser-exposure time within 100-1000 microseconds), the efficiency of photoreaction is usually poor, and the number of neighboring molecules labeled can be limited. For example, there may have thousands of neighboring molecules bound to or nearby one biomolecule of interest, but merely 10-100 tagged probe-target complexes may be generated. To address this or other problems, the present disclosure provides labeling kits useful for signal amplification. Certain exemplary embodiments according to the present disclosure are described as below.

[0092] Example 2: Proximal labeling signal amplification by a labeling kit

[0093] This disclosure provides an embodiment of labeling kits for signal amplification. As illustrated in FIG. 2B diagram (i), after illumination with narrow band of light 104, a tagged probe-target complex 230 is formed in the region of interest 108. Excess probe (unconjugated probe) can be washed away in a wash step with wash solution, and a first connector 203 can be delivered to the sample and captured by the tag portion Ts of the tagged probe-target complex 230, as shown in the diagram (ii) of FIG. 2B. In this example, the first connector 203 can include a plurality of tag-binding sites and may be avidin, streptavidin, neutravidin, or the like for conjugation with a plurality of biotin-based tags. For simplicity in the figures, photosensitive probe 201 is omitted and not shown in FIG. 2B and FIG. 3; however it is present (as illustrated in FIG. 2A).

[0094] In diagram (iii) as shown in FIG. 2B, the dual-tagged spacers 204, each including a first tag moiety Ti, a second tag moiety T2, and a linker Li therebetween connecting the first tag moiety Ti and the second tag moiety T2, are delivered to the sample and conjugated to the (conjugated( first connector 203. As the first connector 302 in this example includes multiple tag-binding sites, the first connector 203 can capture more than one (e.g., two, three, four) dualtagged spacers 204 through affinity binding between the first tag moiety Ti of the dual -tagged spacers 204 and the tag-binding site of the first connector 203. After allowing remaining tagbinding sites of the first connector 203 to be occupied by the dual-tagged spacers 204, unconjugated dual-tagged spacers 204 can be washed away with wash solution.

[0095] In the diagram (iv) as shown in FIG. 2B, second connectors 205 are delivered to the sample, each of the second connectors 205 including a plurality of binding sites, each second connector binding site configured to allow affinity binding with the second tag moiety T2 of the dual-tagged spacers 204. In some embodiments of the present disclosure, the second connector 205 may be the same as or may be different from the first connector 203. For example, both first connector 203 and second connector 205 can be avidin, streptavidin, neutravidin or another connector with a plurality of tag binding sites. Any specific detail described herein for the first connector 203 may also be applicable for the second connector 205 (unless context indicates otherwise). In some embodiments of the present disclosure, the linker Li of the dual-tagged spacers 204 can include a polyethylene glycol ((PEG)n), peptide, amino acid, oligonucleotide, other polymers, or a combination thereof, and wherein n is independently is an integer of 1-20. In some embodiments, the linker Li is a polymeric linker such as polypropylene glycol, polyethylene, polypropylene, polyamides, and polyesters. The linker Li can be a linear molecule in a chain of at least one or two atoms and can include more.

[0096] Continuing with this example, as shown in the diagram (v) as shown in FIG. 2B, tagged enzymes 206, each including a third tag moiety T3 and an enzymatic moiety Ei, are delivered to the sample and the third tag moiety T3 is conjugated to (binds to) the second connectors 205. As the second connectors 205 also include multiple tag-binding sites, each second connector 205 can capture multiple tagged enzymes 206 through affinity binding between the third tag moiety T of the tagged enzymes 206 and the tag-binding site of the second connectors 205. Subsequently, as shown in the diagram (vi) of FIG. 2B, a plurality of enzymic tagged probes 207 (also referred to as “activatable substrates”) is delivered to the sample, wherein each enzymic tagged probe includes a fourth moiety T4 and a phenol group Pi With the presence of H2O2, the enzymatic moiety Ei of the tagged enzyme 206 can activate the phenol group Pi to allow covalent bond formation between the activated tagged probes 207’ and a plurality of neighbors molecules 214 proximal the tagged probe-target complex 230 (Pi is omitted in 207”). [0097] By attaching the tagged enzyme 206, such as tagged peroxidase, tagged ligase or the like to the tagged complex, the peroxidase or ligase and its associated reaction can be localized to a region with a radius of < e g., 100 nm. In some variations, a larger region (e.g., with a radius up to about 200 nm, up to about 300 nm, up to about 400 nm, up to about 500 nm, up to about 1 pm, up to about 2 pm, up to about 5 pm) could be labeled.

[0098] Accordingly, as shown in the exemplary FIG. 2B, it assumes that one proximal labeling - the tagged probe-target complex 230, may be obtained in Example 1. By implementing a method of the disclosure, the number of tagged neighbor molecules (e g., biotinylated proteins if the tag is biotin, depicted as the symbol of B in a circle) has increased The diagram (vi) of FIG. 2B is for illustration purpose only. In practice the labeling capacity can be increased by 1 to 100 times or more. In some embodiments, the labeling capacity can be increased approximately 5 to 10 times.

[0099] Example 3: Proximal labeling signal amplification by another labeling kit of the disclosure [0100] FIG. 3 schematically shows another aspect of labeling process, which is similar to that illustrated in FIG. 2B, except that the delivery of the dual-tagged spacers 204 (shown in FIG. 2B) is replaced with delivery of first tagged spacer precursors 304-1 and second tagged spacer precursors 304-2 (shown in FIG. 3) to the sample. Each first tagged spacer precursor 304-1 includes a first linkable moiety L _a and a first tag moiety Ti connected covalently to the first linkable moiety L _a . and each second tagged spacer precursor 304-2 includes a second linkable moiety Lb and a second tag moiety Tz connected covalently to the second linkable moiety Lb. According to the disclosure, the first linkable moiety L _a and the second linkable moiety Lb are configured to form a linkage therebetween to thereby form a dual-tagged spacer 304. A difference between dual-tagged spacer 204 and dual-tagged spacer 304 lies in the structure of the linker that connects the first tag moiety Ti and the second tag moiety T2, but in some embodiments dualtagged spacer 204 and dual-tagged spacer 304 can have similar functions.

[0101] In one embodiment, the first linkable moiety L _a and the second linkable moiety Lb described herein can each independently include a click chemical group (wherein the click chemical groups are complementary to each other) to allow a click chemistry reaction and form a linkage between the first tagged moiety and second tagged moiety. The click chemical group may be, for example, an alkyne-based group as shown in FIG. 4A, DBCO (dibenzocyclooctyne) as shown in FIG. 4B, BCN (bicyclo[6.1. 0]non-4-yne) as shown in FIG. 4C, N3 (azide) as shown in FIG. 4D, or an azide-based group as shown in FIG. 4E, that can be used as the first and second linkable moieties of the first and second tagged spacers precursors, respectively.

[0102] In some embodiments, the first linkable moiety L _a and the second linkable moiety Lb described herein may include an anchoring strand and a probing strand, respectively, to allow hybridization for the linkage between the first and second tagged spacers precursors. FIGS. 5A-B show examples of nucleic acid anchoring strand sequences (SEQ ID NO: 1-5) and nucleic acid probing strand sequences (SEQ ID NO: 6-7) that can be used as the first and second linkable moieties of the first and second tagged spacers to allow formation of a double-stranded structure with each other along complementary sequences.

[0103] It should be noted that the elements of FIG. 3 include the same reference numerals as that of FIG. 2B, and description of the same components is not repeated here for the sake of simplicity.

[0104] Diagram (i) of FIG. 3, as described in Example 2, shows the proximity labeling of one neighboring biomolecule 211 is completed to form a tagged probe-target complex 230 (shown as dotted circle area). As shown in diagram (ii) of the FIG. 3, the first connector 203 is delivered to the sample and captured by the tag portion T5 of the tagged probe-target complex 230.

[0105] In diagram (iii) as shown in FIG. 3, the dual-tagged spacers precursors (first tagged spacer precursors 304-1) are delivered to the sample, each of the first tagged spacer precursors 304-1 including a first linkable moiety L _a and a first tag moiety Ti covalently connected to L _a , and the first tag moiety Ti is configured to affinity bind one of the binding sites of the first connector 203. Unbound first tagged spacer precursors 304-1 can be washed away with a wash solution. After washing away the unbound first tagged spacer precursors, a step of delivering a plurality of second tagged spacer precursors 304-2 to the biological sample can be perform, each of the second tagged spacer precursors 304-2 including a second tag moiety T2 and a second linkable moiety Lb covalently connected to T2, and the first linkable moiety L _a and the second linkable moiety Lb are configured to form a linkage therebetween and form a dual -tagged spacer 304, as shown in diagram (iv) of the FIG. 3.

[0106] Next, as illustrated in diagram (v) of FIG. 3, second connectors 205 are delivered to the sample and conjugated to the dual-tagged spacers 304 through affinity binding between the second connector 205 and the second tag moiety T2 of the dual-tagged spacer 304.

[0107] Continuing with this example, as shown in diagram (vi) in FIG. 3, tagged enzymes 206, each including a third tag moiety T3 and an enzymatic moiety Ei, are delivered to the sample and the third tag moiety T3 is conjugated to bind the second connectors 205. As the second connectors 205 can also include multiple tag-binding sites, the second connectors 205 can capture multiple tagged enzymes 206 through affinity binding between the third tag moiety T3 of the tagged enzymes 206 and the tag-binding site of the second connectors 205. Subsequently, as shown in diagram (vii) of FIG. 3, a plurality of enzymic tagged probes 207 (also referred to as “activatable substrates”) is delivered to the sample, wherein each enzymic tagged probes includes a fourth moiety T4 and a phenol group Pi. With the presence of H2O2, the enzymatic moiety Ei of the tagged enzyme 206 can activate the phenol group P2 to allow covalent bond formation between the activated tagged probes 207’ and a plurality of neighbor molecules 214 proximal the tagged probe-target complex 230 to tag the plurality of neighbor molecules 214 with tagged probes 207” (Pi is omitted in 207”).

[0108] When streptavidin or avidin is used as the first connector 203 of the disclosure, it can bind (conjugate) up to four biotins with high affinity and selectivity. However, a dual -tagged spacer with a single structure (T1-L1-T2) may occupy two binding sites of a first connector or two binding sites of a second connector, etc. The sites are then blocked. As a result, the degree of signal amplification can be limited. In order to solve this and/or other problems, one option is to shorten the length of the linker LI to less than the distance between two adjacent binding sites of the first connector, e.g., less than 2 nm. Another option is shown in FIG. 3, by delivering two independent precursors in two separate steps, each precursor will only bind one site on a connector (e g., first tag moiety Ti binds one binding site of the first connector).

[0109] Example 4: Method (I) for proximity labeling of neighboring molecules by forming a networked cluster

[0110] A method (I) of the disclosure can include several steps as follows and with reference to FIG. 2 A and FIG. 2B. Step (a) can include delivering a plurality of first connectors Ci (203) to the biological sample, wherein the biological sample includes a plurality of tagged probemolecule of interest complexes (230), each tagged probe-molecule of interest complex represented by the following general structure (IV): T5-P2*II, wherein Ts is a tag moiety, P2 is a probe moiety, and Ii is a molecule of interest, wherein each of the first connectors Ci includes a plurality of binding sites with each binding site configured to allow affinity binding with the tag moiety T5 of the tagged probe-molecule of interest complex 230. Step (b) can include the step of capturing, with each tag portion T3 of each tagged probe-molecule of interest complex 230, one of the plurality of first connector binding sites to thereby attach each first connector Ci to a corresponding tagged probe-molecule of interest complex 230 to form a complex represented by the general structure (V): Cl — tagged probe-molecule of interest complex. Step (c) can include the step of delivering a plurality of dual tagged spacers 204 to the biological sample, each of the dual tagged spacers 204 represented by the following general structure (I): T1-L1-T2, wherein Ti is a first tag moiety, T2 is a second tag moiety, and Li is a linker therebetween connecting Ti and T2. Step (d) can include binding the first tag moiety Ti of each dual tagged spacer 204 to an available first connector binding site on each Ci-tagged probe-molecule of interest complex to form a complex represented by the general structure (XI): T2-L1-T1 — Ci — tagged probemolecule of interest complex. Step (e) can include delivering a plurality of second connectors C2 (205) to the biological sample, wherein each of the second connectors C2 includes a plurality of binding sites, each second connector binding site configured to affinity bind with the second tag moiety T2 in the complex represented by the general structure (XI). Step (f) can include binding each second connector binding site to one second tag moiety T2 in the complex having the general structure (XI) to form a complex represented by the general structure (XII): (C2 — T2- Li-Ti) _q — Ci — tagged probe-molecule of interest complex. Step (g) can include delivering a plurality of tagged enzymes 206 to the biological sample, each tagged enzyme 206 represented by the general structure (IX): E1-T3, wherein T3 is a third tag moiety andEi is an enzymatic moiety. Step (h) includes binding the third tag moiety T3 of each tagged enzyme 206 with an available binding site on an available binding site on a connector C2 of the complex represented by the general structure (XII) to form a complex represented by the general structure (XIII): E1-T3 — (C2 — T2- Li-Ti) _q — Ci — tagged probe-molecule of interest complex. Step (i) can include delivering a plurality of enzymic tagged probe 207 having an activatable substrate Pi to the biological sample. Step (j) can include catalyzing, with the tagged enzyme 206 in the complex represented by the general structure (XIII), the enzymic tagged probes 207 into an activated tagged probes 207’, and covalently binding the activated tagged probes 207’ ’ to a neighbor molecule 211 in the vicinity of the complex represented by the general structure (XIII).

[0111] In this example, the molecule of interest Ii can be the biomolecule of interest 210 labeled by a primary antibody or secondary antibody conjugated to biotin or a biotin derivatives, or the molecule of interest Ii can be the neighbor molecules 211 labeled by the reactive tagged probe 202’ as shown in FIG. 2A.

[0112] As mentioned above, the networked cluster may include one or multiple layers. If q = 1, the cluster may have one (e.g., a maximum of one) layer represented by the general structure (XIII): E1-T3 — (C2 — T2- L1-T1) — Ci — tagged probe-molecule of interest complex, as shown in the diagram (vii) of FIG. 2B. If q = 2, two layers are represented by the general structure: E1-T3— -(C2 — T2- LI-TI)2 — Ci — tagged probe-molecule of interest complex. With each increase in the number of layers in the network cluster, the number of tagged enzymes that can be attached to the complex can also increases, thus it can be reasonably anticipated that the number of neighboring molecules available for labeling by the activated substrate 207’ will increase.

Therefore, the effect of the signal amplification can be achieved. The number of layers (the value of q) can be at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100 and/or least 1, not more than 2, not more than 3, not more than 4, not more than 5, not more than 6, not more than 7, not more than 8, not more than 9, not more than 10, not more than 20, not more than 30, not more than 40, not more than 50, not more than 60, not more than 70, not more than 80, not more than 90, not more than 100 In some embodiments, the number of q ranges from 2 to 30.

[0113] Example 5: Method (II) for proximity labeling of neighboring molecules by forming a networked cluster

[0114] The method (II) of the disclosure can include several steps as follows and referring to FIG. 3. Step (a) can include delivering a plurality of first connectors Ci (203) to the biological sample, wherein the biological sample includes a plurality of tagged probe-molecule of interest complexes 230, each tagged probe-molecule of interest complex 230 represented by the following general structure (IV): Ts-Pz’Ii, wherein T5 is a tag moiety, P2 is a probe moiety, and Ii is a molecule of interest, wherein each of the first connectors Ci includes a plurality of binding sites with each binding site configured to allow affinity binding with the tag moiety T5 of the tagged probemolecule of interest complex. Step (b) can include capturing, with each tag portion T5 of each tagged probe-molecule of interest complex 230, one of the plurality of first connector binding sites to thereby attach each first connector Ci to a corresponding tagged probe-molecule of interest complex to form a complex represented by the general structure (V): Ci — tagged probe-molecule of interest complex. Step (c) can include delivering a plurality of first tagged spacer precursors 304-1 to the biological sample, each of the first tagged spacer precursors 304-1 represented by the following general structure (II): L _a -Ti, wherein L _a is a first linkable moiety and Ti is a first tag moiety covalently connected to L _a . Step (d) can include binding the first tag moiety Ti of each first tagged spacer precursor to an available first connector binding site on each Ci-tagged probemolecule of interest complex to form a complex represented by the general structure (VI): L _a -Ti- — Ci — tagged probe-molecule of interest complex. Step (e) can include delivering a plurality of second tagged spacer precursors 304-2 to the biological sample, each of the second tagged spacer precursors 304-2 represented by the following general structure (III): T2.Lt>, wherein T2 is a second tag moiety and Lb is a second linkable moiety covalently connected to T2, wherein the first linkable moiety L _a and the second linkable moiety Lb are configured to form a linkage therebetween and form a dual -tagged spacer 304. Step (f) can include forming a linkage between the first linkable moiety L _a of each complex represented by the general structure (VI) and the second linkable moiety Lb of each second tagged spacer precursor to thereby form a complex represented by the general structure (VII): T2-Lb*L _a -Ti — Ci — tagged probe-molecule of interest complex. Step (g) can include delivering a plurality of second connectors C2 (205) to the biological sample, wherein each of the second connectors C2 includes a plurality of binding sites, each second connector binding site configured to affinity bind with the second tag moiety T2 of the dual-tagged spacers Step (h) can include binding each second connector binding site to one second tag moiety T2in the complex having the general structure (VII) to form a complex represented by the general structure (VIII): (C2 — T ₂ -Lb-L _a -Ti) _q — Ci — tagged probe-molecule of interest complex. Step (i) can include delivering a plurality of tagged enzymes 206 to the biological sample, each tagged enzyme 206 represented by the general structure (IX): E1-T3, wherein T3 is a third tag moiety and Ei is an enzymatic moiety. Step (j) can include binding the third tag moiety T3 of each tagged enzyme 206 with an available binding site on an available binding site on the second connector C2 of the complex represented by the general structure (VIII) to form a complex represented by the general structure (X): E1-T3 — (C2 — T ₂ -L _b *L _a -Ti) _q — Ci — tagged probe-molecule of interest complex; Step (i): delivering a plurality of enzymic tagged probe 207 having an activatable substrate Pi to the biological sample. Step (k) can include catalyzing, with the tagged enzyme 206 in the complex represented by the general structure (X), the activatable enzymic tagged probes 207 into an activated tagged probes 207’, and covalently binding the tagged probes 207” to a neighbor molecule in the vicinity of the complex represented by the general structure (X).

[0115] In this example, the molecule of interest Ii can be the biomolecule of interest 210 labeled by a primary antibody conjugated to biotin or biotin derivatives, or the neighbor molecules 211 labeled by the reactive tagged probe 202’ as shown in FIG. 2A.

[0116] As mentioned above, the networked cluster may include one or multiple layers. If q = 1, it is one layer represented by the general structure (X): E1-T3 — (C2 — T2-Lb*L _a -Ti) — Ci — tagged probe-molecule of interest complex, as shown in the diagram (vi) of FIG. 3.

If q = 2, it is two layers represented by the general structure: E1-T3 — (C2 — T2-Lb ^, L _a -Ti)2 — Ci — tagged probe-molecule of interest complex. With increase in the number of layers, the quantity of tagged enzymes that can be attached also increases, thus it can be reasonably anticipated that the number of neighboring molecules available for labeling by the activated substrate 207’ will increase. Therefore, the effect of the signal amplification is achieved. Preferably, the number of q ranges from 2 to 30.

[0117] Complementary regions of nucleic acid anchoring strands (L _a ) and nucleic acid probing strands (Lb) of Nucleic acid anchoring strand (L _a > and nucleic acid probing strand (Lb) can independently include DNA or RNA. Thus, after linkage, a linker generated from hybridization of first tagged spacer precursors 304-1 and second tagged spacer precursors 304-2 can be a DNA-DNA linker, an RNA-RNA linker, or a DNA-RNA linker. Complementary portions of nucleic acid anchoring strand (L _a > and nucleic acid probing strand (Lb) can be single stranded before being hybridized/linked to each other so that each and both complementary portions (C _a of L _a and Cb or Lb) are available for binding to each other. Complementary portions of nucleic acid anchoring strand (L _a > and nucleic acid probing strand (Lb) can be designed to be single stranded by, for example, minimizing formation of (very stable) secondary structures. In some variations, one or more than one material used in a method for proximity labeling of neighboring molecules can be heated (and/or cooled) to facilitate single stranded character especially in the nucleic acid anchoring strand (L _a > and nucleic acid probing strand (Lb). Heat can be applied while performing a method of proximity labeling as described herein. For example, heat can be applied to one or more than one of a plurality of first tagged spacer precursors (represented by the general structure (II): L _a -Ti), a plurality of second tagged spacer precursors (represented by the general structure (III): T2-Lb), and a biological sample. Heat can be applied above the melting temperature(s) of the nucleic acid anchoring strand (L _a ) and nucleic acid probing strand (Lb) to create single stranded character in at least in the complementary regions to facilitate binding of nucleic acid anchoring strand (L _a ) and nucleic acid probing strand (Lb). Heat can be applied above 30°C, above 35°C, above 40°C, above 45°C, above 50°C, above 55°C, above 60°C, above 65°C, above 70°C, above 75°C, above 80°C, above 90°C, above 95°C. Heat can be applied below a desired maximum temperature, such as below 95°C, below 90°C, below 85°C, below 80°C, below 75°C, below 70°C, below 65°C, below 60°C, below 55°C, below 50°C, below 45°C, below 40°C, below 35°C. Heat can be applied between any of these values, such as above 45°C and below 60°C or above 85°C and below 95°C. The temperature of applied heat can be decided based on, for example sample composition, sample target, primary sequence of the nucleic acid anchoring strand (L _a j and nucleic acid probing strand (Lb), etc. In some variations, a method for proximity labeling of neighboring molecules can be performed at room temperature (and without heating).

[0118] First and second tagged spacers precursors can be a suitable size for performing a labeling reaction. Complementary regions of nucleic acid anchoring strands (L _a ) and nucleic acid probing strands (Lb) can be sufficiently long to readily hybridize to one another and to stay hybridized. In some embodiments, complementary regions of nucleic acid anchoring strands (L _a ) and nucleic acid probing strands (Lb) can be sufficiently short to minimize tangling or to keep a region of labeling within a desired area. For example, complementary regions of nucleic acid anchoring strands (L _a ) and nucleic acid probing strands (Lb) can be at least 5 nucleotides in length, at least 10 nucleotides in length, at least 15 nucleotides in length, at least 20 nucleotides in length, at least 25 nucleotides in length, at least 30 nucleotides in length, at least 40 nucleotides in length, at least 50 nucleotides in length, or at least 100 nucleotides in length. For example, complementary regions of nucleic acid anchoring strands (L _a ) and nucleic acid probing strands (Lb) can be fewer than 5 nucleotides in length, fewer than 10 nucleotides in length, fewer than 15 nucleotides in length, fewer than 20 nucleotides in length, fewer than 25 nucleotides in length, fewer than 30 nucleotides in length, fewer than 40 nucleotides in length, fewer than 50 nucleotides in length, or fewer than 100 nucleotides in length. For example, complementary regions of nucleic acid anchoring strands (L _a ) and nucleic acid probing strands (Lb) can be between these values (e.g., at least 10 nucleotides in length and not more than 100 nucleotides in length, at least 15 nucleotides in length and not more than 40 nucleotides in length, etc.). Typically, complementary regions of nucleic acid anchoring strands (L _a ) and nucleic acid probing strands (Lb) are complementary along their entire length. In some variations, mismatches may be present (e.g., 100% complementarity, 99% complementarity, 98% complementarity, etc ). In some variations, anchoring strand and probing strand have at least 6 complementary nucleotides in a continuous row (at least 7, at least 8, at least 9, at least 10, etc.) and at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, etc. complementary nucleotides in total. A melting temperature Tm of the double-stranded structure is from 52°C to 60°C. A melting temperature Tm of the double- stranded structure can be at least 35°C, at least 40°C, at least 45°C, at least 50°C, or at least 55°C.

[0119] In some variations, one or both of first tagged spacers precursors and second tagged spacers precursors have a double stranded region as well as a single stranded region (i.e., a single stranded complementary region). A double stranded region may be useful as, for example, a spacer to manage how far away from a biomolecule of interest a neighboring molecule can be labeled. For example, a double stranded region (and/or a single stranded region) can be 100 um or less in length, 10 um or less in length, 1 um or less in length, 200 nm or less in length (e.g., 20 nm or less in length, 10 nm or less in length, 5 nm or less in length, 2 nm or less in length, 0.5 nm or less in length).

[0120] In some embodiments, a nucleic acid anchoring strand (L _a > and a nucleic acid probing strand (Lb) are artificial (i.e., they are different from naturally occurring nucleic acid sequences). In some variations, a nucleic acid anchoring strand (L _a j and a nucleic acid probing strand (Lb) are different from nucleic acid sequences naturally occurring in an organism for which the sequences may be used in. Nucleic acid anchoring strand (L _a > and nucleic acid probing strand (Lb) can be complementary along part, most, or all of their length.

[0121] According to the methods described in Example 4 and Example 6, the proximity labeling includes proximity labeling a region less than 5 pm, less than 2 pm, less than 1pm, less than 500 nm, less than 300 nm, less than 200 nm, or less than 100 nm in diameter.

[0122] As used in the description herein and throughout the claims that follow, the meaning of “a”, “an”, and “the” includes plural reference unless the context clearly dictates otherwise. Also, as used in the description herein, the meaning of “in” includes “in” and “on” unless the context clearly dictates otherwise.

[0123] Although the terms “first”, “second”, “third” and “fourth” may be used herein to describe various features/ elements (including steps), these features/elements should not be limited by these terms, unless the context indicates otherwise. These terms may be used to distinguish one feature/element from another feature/element. Thus, a first feature/element discussed below could be termed a second feature/element, and similarly, a second feature/element discussed below could be termed a first feature/element without departing from the teachings of the present invention.

[0124] Although various illustrative embodiments are described above, any of a number of changes may be made to various embodiments without departing from the scope of the invention as described by the claims. For example, the order in which various described method steps are performed may often be changed in alternative embodiments, and in other alternative embodiments one or more method steps may be skipped altogether. Optional features of various device and system embodiments may be included in some embodiments and not in others. Therefore, the foregoing description is provided primarily for exemplary purposes and should not be interpreted to limit the scope of the invention as it is set forth in the claims.