Highly-multiplexed and thermally-controllable DNA extension method and application thereof

Abstract

The methods, compositions and kits described herein provide a signal amplification method for the detection of target biomolecules, significantly improving the sensitivity of detection using target binding ligand molecules. These methods include the in-situ cycling addition of nucleic acid repeats to, for example, a target binding molecule, thereby providing a plurality of landing pads for the labeled probe. These methods are well suited for multiplex detection, so that multiple targets can be sensitively detected in one detection. The methods and compositions can be applied to the fields of imaging, flow cytometry, CyTOF, immunoblotting and the like, and provide additional tools for research and diagnosis purposes.

Description

[0001] Cross-references

[0002] This application claims the benefit of U.S. Provisional Application Serial No. 63 / 390,948, filed on July 20, 2022, under 35 U.S. Patent Act, § 119(e), the contents of which are incorporated herein by reference in their entirety.

[0003] Government support

[0004] This invention was made with government support from the National Institutes of Health (NIH) under Grants MH128861, MH124606, and CA235421 and from the Office of Naval Research (NAVY / ONR) under Grant N00014-18-1-2549. The government has certain rights in this invention. Technical Field

[0005] The technology described herein relates to methods for amplifying signals when detecting, quantifying, and imaging biomolecules. Background Art

[0006] Methods for detecting, quantifying and / or imaging biological target molecules of interest are central to a wide range of research and diagnostic methods. There is a need in the art for compositions and methods that allow for more sensitive detection of target biomolecules, and for example, detection of multiple such target biomolecules in the same assay - so-called multiplexed detection.

[0007] Nucleic acid amplification allows the study of many different pathways in biological systems. This amplification can be used for imaging, flow cytometry, time-of-flight cytometry (CyTOF), immunoblotting and other techniques. The ability to amplify multiple nucleic acid sites simultaneously allows different responses and pathways to be examined simultaneously, which is suitable for research and diagnostic testing. Summary of the invention

[0008] The technology described in the present invention generally relates to signal amplification, and the signal is a signal related to the in situ or ectopic binding of a target binding ligand to its target. Such signal amplification can provide the same high sensitivity and resolution of readings as single molecule detection. In various embodiments, the technology involves the in situ generation of concatemers of nucleic acid sequences on target binding ligand molecules, wherein the repeated sequence provides hybridization sites for multiple labeled nucleic acid probes of each target binding ligand molecule, thereby amplifying the signal related to the binding of the target molecule in a cell or tissue sample by the target ligand binding molecule. The methods, compositions and kits described herein have applications in a variety of situations, but are particularly relevant to quantifying the abundance of target molecules and detecting the cellular or subcellular location of target molecules in situ in cells and tissue samples.

[0009] In one aspect, the present invention provides a method for labeling a target binding ligand, the method comprising: (a) providing a conjugate of a target binding ligand and a first oligonucleotide primer; (b) in a reaction mixture, under conditions allowing the first oligonucleotide primer to hybridize and extend using the first single-stranded extension template, contacting the conjugated first oligonucleotide primer with a first single-stranded extension template and a nucleic acid polymerase; (c) heating the reaction mixture of step (b) to separate the first single-stranded extension template from the extended first oligonucleotide primer produced in step (b); (d) repeating steps (b) and (c) at least once, thereby producing a concatemer comprising repeating units of the oligonucleotide primer sequence, wherein the concatemer is conjugated to the target binding ligand.

[0010] In one embodiment of this aspect and any other aspect described herein, the steps are performed in the order shown. It is noteworthy that, although the in-situ application of technology described herein is particularly interesting, the signal amplification method based on concatemers can also be applied in vitro, such as in a test tube or other container without a cell or tissue sample. As a non-limiting example, concatemer generation methods can be used to label antibodies or other target binding partners (e.g., members of affinity pairs (affinity pairs), such as biotin / streptavidin, etc.), for example, Western blot or other immunoassays. In these embodiments, concatemers can be produced by extending oligonucleotide primers to the target binding partner, substantially as described herein, but the target binding partner does not need to be combined with its target. It can also be envisioned that, for example, a pre-formed connector (straight or branched chain as further described below) can be extended by thermal cycles as described herein, and then combined with the target binding partner, such as by hybridizing and selectively cross-linking with the oligonucleotides on the target binding partner.

[0011] In another embodiment, the first single-stranded extension template comprises a concatemer of at least two copies of a sequence complementary to the first oligonucleotide primer ligated in a head-to-tail orientation and a 3' chain terminator such that the extension template is not extended by a polymerase.

[0012] In another embodiment, after repeating steps (b)-(d) at least once, the method further comprises the following steps: (i) adding a second oligonucleotide primer and a second single-stranded extension template oligonucleotide, wherein the second oligonucleotide primer comprises a sequence complementary to the first oligonucleotide primer and a sequence complementary to the second single-stranded extension template in a 5' to 3' order, wherein the second single-stranded extension template comprises a 3' chain terminator; (ii) heating the reaction mixture to separate the nucleic acid chains and cooling to allow the second oligonucleotide primer to hybridize with the concatemers produced in steps (b)-(d) and to allow the second single-stranded extension template to hybridize with the second oligonucleotide primer; (iii) extending the second oligonucleotide primer using the second single-stranded extension template oligonucleotide as a template; and (iv) repeating steps (ii) and (iii) at least once, thereby producing a concatemer comprising a second oligonucleotide primer repeating unit.

[0013] In another embodiment, the method further comprises, after step (d): (i) adding a nucleic acid under conditions allowing hybridization, wherein the nucleic acid comprises: (1) a second concatemer consisting of repeating units of a second oligonucleotide sequence; and (2) a sequence complementary to the first oligonucleotide primer, such that multiple molecules of the nucleic acid comprising the second concatemer hybridize to the concatemer formed in step (d) through the sequence complementary to the first oligonucleotide primer.

[0014] In another embodiment, the method is performed in situ on a cell or tissue sample comprising or for detecting a target ligand.

[0015] In another embodiment, the cells or tissue sample are fixed.

[0016] In another embodiment, the tissue sample is paraffin embedded.

[0017] In another embodiment, the method further comprises contacting the concatemer produced in the previous step with a labeled nucleic acid probe. In one embodiment, the nucleic acid probe comprises a sequence complementary to the concatemer repeating unit. In another embodiment, the nucleic acid probe comprises a sequence complementary to the first oligonucleotide primer.

[0018] In another embodiment, the method further comprises contacting the concatemer consisting of the second oligonucleotide primer repeating units with a labeled nucleic acid probe, wherein the nucleic acid probe comprises a sequence complementary to the second oligonucleotide primer.

[0019] In another embodiment, the method further comprises contacting the concatemer consisting of repeating units of the second oligonucleotide sequence with a labeled nucleic acid probe, wherein the nucleic acid probe comprises a sequence complementary to the second oligonucleotide sequence.

[0020] In another embodiment, a plurality of labeled nucleic acid probe molecules hybridize to a plurality of concatemer repeat units.

[0021] In another embodiment, the target binding ligand comprises an antibody or antigen-binding fragment thereof.

[0022] In another embodiment, multiple different target binding ligands are multiplex labeled by different orthogonal oligonucleotide primer / single-stranded extension template pairs.

[0023] In another aspect, the present invention provides a method for in situ detection of a target molecule in a preparation of a cell or tissue, the method comprising: (a) contacting the preparation of a cell or tissue with a target binding ligand bound to a first oligonucleotide primer under conditions that allow the target binding ligand to specifically bind to the target molecule; (b) adding a reaction mixture comprising a first single-stranded extension template and a nucleic acid polymerase to the preparation of the cell or tissue under conditions that allow the first oligonucleotide primer to hybridize and extend with the first single-stranded extension template; (c) heating the reaction mixture of step (b) to separate the first single-stranded extension template from the extended first oligonucleotide primer produced in step (b); (d) repeating steps (b) and (c) at least once, thereby producing a concatemer comprising a repeating unit of the first oligonucleotide primer sequence, wherein the concatemer is coupled to the target binding ligand; (e) contacting the concatemer produced in step (d) with a labeled nucleic acid probe; and (f) detecting the labeled nucleic acid probe, wherein detection of the labeled nucleic acid probe indicates the presence and location of the target molecule in the preparation of the cell or tissue.

[0024] In one embodiment of this aspect, the first single-stranded extension template comprises a concatemer of at least two copies of a sequence complementary to the first oligonucleotide primer ligated in a head-to-tail orientation and a 3' chain terminator such that the extension template is not extended by a polymerase.

[0025] In another embodiment, the steps are performed in the order shown.

[0026] In another embodiment, the target binding ligand comprises an antibody or antigen-binding fragment thereof.

[0027] In another embodiment, the labeled nucleic acid probe comprises a sequence that is complementary to the first oligonucleotide primer.

[0028] In another embodiment, the labeled nucleic acid probe comprises a sequence complementary to a concatemer repeat unit.

[0029] In another embodiment, a plurality of labeled nucleic acid probe molecules are hybridized to a plurality of concatemer repeat units that are coupled to a target binding ligand.

[0030] In another embodiment, after step (d) and before step (e), the method further comprises the steps of: (i) adding a second oligonucleotide primer and a second single-stranded extension template oligonucleotide, wherein the second oligonucleotide primer comprises a sequence complementary to the first oligonucleotide primer in 5' to 3' order, and a sequence complementary to the second single-stranded extension template, wherein the second single-stranded extension template comprises a 3' chain terminator; (ii) heating the reaction mixture to separate the nucleic acid chains and cooling to allow the second oligonucleotide primer to hybridize with the concatemer produced in step (d), and to allow the second single-stranded extension template to hybridize with the second oligonucleotide primer; (iii) extending the second oligonucleotide primer using the second single-stranded extension template oligonucleotide as a template; and (iv) repeating steps (ii) and (iii) at least once, thereby generating a concatemer comprising a repeating unit of the second oligonucleotide primer.

[0031] In another embodiment, the labeled nucleic acid probe comprises a sequence that is complementary to a second oligonucleotide primer.

[0032] In another embodiment, a plurality of labeled nucleic acid probe molecules are hybridized to a plurality of concatemer repeat units that are coupled to a target binding ligand.

[0033] In another embodiment, after step (d) and before step (e), the method further comprises: (i) adding a nucleic acid under conditions permitting hybridization, wherein the nucleic acid comprises: (1) a second concatemer consisting of repeating units of a second oligonucleotide sequence; and (2) a sequence complementary to the first oligonucleotide primer, so that multiple molecules of the nucleic acid comprising the second concatemer hybridize to the concatemer comprising repeats of the first oligonucleotide primer sequence formed in step (d) through the sequence complementary to the first oligonucleotide primer.

[0034] In another embodiment, the labeled nucleic acid probe comprises a sequence that is complementary to the second oligonucleotide sequence.

[0035] In another embodiment, a plurality of labeled nucleic acid probe molecules are hybridized to a plurality of the second concatemer repeat units.

[0036] In another embodiment, the preparation of cells or tissue is fixed.

[0037] In another embodiment, the preparation of tissue is paraffin embedded.

[0038] In another aspect, the present invention provides a method for labeling a group of target binding ligands, the method comprising: (a) providing a group of orthogonal first oligonucleotide primers and first single-stranded extension template oligonucleotide pairs, wherein the first oligonucleotide primer of each pair has a sequence different from other first oligonucleotide primers in the group and is coupled to different members of a group of target binding ligands; (b) in a reaction mixture, contacting the coupled first oligonucleotide primers with a group of orthogonal pairs of first single-stranded extension templates, and adding a nucleic acid polymerase under conditions that allow the first oligonucleotide primers to hybridize and extend with the first single-stranded extension templates of the group of orthogonal pairs; (c) heating the reaction mixture of step (b) to separate the first single-stranded extension template from the extended first oligonucleotide primer produced in step (a); (d) repeating steps (b) and (c) at least once, thereby generating a concatemer on each member of the group of target ligands, the concatemer comprising a repeating unit of the oligonucleotide primer sequence bound to the target binding ligand.

[0039] In one embodiment, the first single-stranded extension template in each orthogonal first oligonucleotide primer / first single-stranded extension pair includes a concatemer of at least two copies of a sequence complementary to the first oligonucleotide primer linked in a head-to-tail orientation and a 3' chain terminator such that the extension template is not extended by a polymerase.

[0040] In another embodiment, the steps are performed in the order presented.

[0041] In another embodiment, after step (d), the method further comprises the following steps: (i) adding a second set of orthogonal second oligonucleotide primers, second single-stranded extension template oligonucleotide pairs, wherein the second oligonucleotide primers in each pair comprise, in 5' to 3' order, sequences complementary to members of a set of first oligonucleotide primers, and sequences complementary to second single-stranded extension templates of the second set of orthogonal oligonucleotide pairs, wherein each second single-stranded extension template comprises a 3' chain terminator; (ii) heating the reaction mixture to separate the nucleic acid chains and cooling to allow the second oligonucleotide primers to hybridize with the concatemers produced in steps (b)-(d), and to allow the second single-stranded extension templates to hybridize with the second oligonucleotide primers; (iii) extending the second oligonucleotide primer using the second single-stranded extension template oligonucleotide as a template; and (iv) repeating steps (ii) and (iii) at least once, thereby generating concatemers comprising corresponding second oligonucleotide primer repeating units on corresponding members of the set of target binding ligands.

[0042] In another embodiment, the method further comprises contacting the concatemer produced in the previous step with a plurality of labeled nucleic acid probes, wherein each different probe is labeled with a distinguishable detectable label portion, and wherein members of the plurality of labeled nucleic acid probes comprise sequences complementary to corresponding members of the first oligonucleotide primer set.

[0043] In another embodiment, the method further comprises contacting the concatemer produced in step (iv) with a plurality of labeled nucleic acid probes, wherein each different probe is labeled with a distinguishable detectable label portion, and wherein members of the plurality of labeled nucleic acid probes comprise sequences that are complementary to corresponding members of the second oligonucleotide primer set.

[0044] In another embodiment, for each member of the set of target binding ligands, a plurality of labeled nucleic acid probe molecules hybridize to a plurality of concatemer repeat units.

[0045] In another embodiment, the method is performed in situ on a cell or tissue sample comprising or for detecting a target ligand.

[0046] In another embodiment, the preparation of cells or tissue is fixed.

[0047] In another embodiment, the tissue preparation is paraffin embedded.

[0048] In another embodiment, the target binding ligand comprises an antibody or antigen-binding fragment thereof.

[0049] In another aspect, the present invention provides a method for in situ detection of a group of target molecules in a preparation of cells or tissues, the method comprising: (a) providing a group of target binding ligand molecules, wherein members of the target binding ligand molecules of the group are coupled to corresponding first oligonucleotide primer members of a group of orthogonal first oligonucleotide primers and first single-stranded extension template oligonucleotide pairs, wherein the first oligonucleotide primer of each pair has a sequence different from other first oligonucleotide primers in the group and is coupled to different members of the group of target binding ligand molecules; (b) contacting the preparation of cells or tissues with the target binding ligands of the group under conditions that allow the target binding ligand molecules to specifically bind to the target molecules present in the preparation of cells or tissues; (c) introducing the target binding ligand molecules of the group into the preparation of cells or tissues under conditions that allow the first oligonucleotide primers to hybridize and extend using the corresponding first single-stranded extension template. (d) heating the reaction mixture of step (c) to separate the first single-stranded extension template from the extended first oligonucleotide primer produced in step (c); (e) repeating steps (c) and (d) at least once, thereby producing concatemers on corresponding members of the set of target binding ligand molecules, the concatemers comprising corresponding first oligonucleotide primer sequence repeating units coupled to the target binding ligand; (f) contacting the concatemers produced in step (e) with a plurality of distinguishably labeled nucleic acid probes; and (g) detecting the labeled nucleic acid probes, wherein detecting the labeled nucleic acid probes indicates the presence and location of the target molecule in the cell or tissue preparation.

[0050] In one embodiment, the steps are performed in the order shown.

[0051] In another embodiment, the first single-stranded extension template comprises a concatemer of at least two copies of a sequence complementary to the first oligonucleotide primer ligated in a head-to-tail orientation and a 3' chain terminator such that the extension template is not extended by a polymerase.

[0052] In another embodiment, after step (e) and before step (f), the method further comprises the steps of: (i) adding a second set of orthogonal second oligonucleotide primers, a second single-stranded extension template oligonucleotide pair, wherein the second oligonucleotide primer comprises, in 5' to 3' order, a sequence complementary to a member of a set of first oligonucleotide primers and a sequence complementary to a second single-stranded extension template of a second set of orthogonal oligonucleotide pairs, wherein each second single-stranded extension template comprises a 3' chain terminator; (ii) heating the reaction mixture to separate the nucleic acid chains and cooling to allow the second oligonucleotide primer to hybridize with the concatemers produced in steps (c)-(e) and to allow the second single-stranded extension template to hybridize with the second oligonucleotide primer; (iii) extending the second oligonucleotide primer using the second single-stranded extension template oligonucleotide as a template; and (iv) repeating steps (ii) and (iii) at least once, thereby generating concatemers comprising corresponding second oligonucleotide primer repeating units on corresponding members of the set of target binding ligands.

[0053] In another embodiment, the plurality of distinguishably labeled nucleic acid probes comprise sequences complementary to corresponding first oligonucleotide primer members of an orthogonal first oligonucleotide primer and first single strand extension template oligonucleotide pair in the set.

[0054] In another embodiment, the plurality of distinguishably labeled nucleic acid probes comprise sequences complementary to corresponding second oligonucleotide primer members of a second set of orthogonal second oligonucleotide primers, second single strand extension template oligonucleotide pairs.

[0055] In another embodiment, the target binding ligand comprises an antibody or antigen-binding fragment thereof.

[0056] In another embodiment, a plurality of labeled nucleic acid probe molecules are hybridized to a plurality of concatemer repeat units that are coupled to a target binding ligand.

[0057] In another aspect, provided herein is a kit comprising reagents for practicing one or more of the methods described herein.

[0058] In another aspect, provided herein is a target binding ligand comprising a concatemer produced by the method described herein. In one embodiment, the target binding ligand comprises an antibody or an antigen-binding fragment thereof.

[0059] In another aspect, the present invention provides a method for producing a nucleic acid chain comprising a concatemer repeating unit of a given sequence, the method comprising: a) providing a first oligonucleotide primer and a first extension template oligonucleotide in a reaction mixture, the first extension template oligonucleotide comprising a concatemer of at least two copies of a sequence connected in a head-to-tail manner, the sequence being complementary to the first oligonucleotide primer, wherein the first extension template comprises a chain terminator at its 3' end; b) incubating the reaction mixture under conditions allowing the first oligonucleotide primer to hybridize with the first extension template oligonucleotide; c) extending the hybridized first primer with a nucleic acid polymerase, thereby producing an extended nucleic acid chain complementary to the first extension template; d) heating the reaction mixture to separate the extended nucleic acid chain complementary to the first extension template from the first extension template; e) cooling the heated product to allow the first extension template oligonucleotide to hybridize with the extended nucleic acid chain complementary to the first extension template produced in step (c); f) extending the extended nucleic acid chain complementary to the first extension template produced in step (c) with a polymerase, thereby producing an extended nucleic acid chain comprising a concatemer repeating unit of the sequence contained in the first oligonucleotide primer.

[0060] In one embodiment, the method further comprises repeating steps (d) to (f) at least once.

[0061] In another embodiment, the steps are performed in the order presented.

[0062] In another embodiment, the method comprises repeating steps (d) to (f) at least n times, wherein each iteration of steps (d) to (f) increases the length of the tandem nucleic acid by one concatemer repeat unit.

[0063] In another embodiment, step (a) comprises providing an orthogonal first oligonucleotide primer and a first extension template oligonucleotide pair such that steps (b)-(f) produce multiple concatemers, each comprising a concatemer repeat unit of a corresponding orthogonal oligonucleotide primer sequence.

[0064] In another embodiment, after one or more repetitions of steps (d) to (f), the method further comprises: i) adding a second oligonucleotide primer and a second extension template oligonucleotide to the reaction, wherein the second oligonucleotide primer comprises a 5' proximal sequence and a 3' proximal sequence, the 5' proximal sequence is complementary to the first oligonucleotide primer sequence, the 3' proximal sequence is complementary to one of the at least two repeating portions of the second extension template oligonucleotide, wherein the second extension template oligonucleotide comprises a sequence of at least two repeating units linked head-to-tail and a chain terminator at its 3' end, the sequence being complementary to the second oligonucleotide primer; ii) reacting the second oligonucleotide primer with the first oligonucleotide primer formed after one or more repetitions of steps (d) to (f); The invention relates to a method for preparing a nucleic acid sequence comprising the steps of: hybridizing one or more concatemer repeating units on the concatemer formed by the step (iv); hybridizing the second extension template oligonucleotide with the second oligonucleotide primer; iv) extending the second oligonucleotide primer with a polymerase to generate an extended nucleic acid chain complementary to the second extension template; v) heating the reaction mixture to separate the extended nucleic acid chain complementary to the second extension template from the second extension template; vi) cooling the heated product to allow the second extension template oligonucleotide to hybridize with the extended nucleic acid chain complementary to the second extension template generated in step (iv); and vii) extending the extended nucleic acid chain complementary to the second extension template generated in step (iv) with a polymerase to generate an extended nucleic acid chain comprising the concatemer repeating units of the sequence contained in the second oligonucleotide primer.

[0065] In another embodiment, the method further comprises repeating steps (v) to (vii) at least once.

[0066] In another embodiment, step (i) comprises providing an orthogonal second oligonucleotide primer and a second extension template oligonucleotide pair such that steps (ii)-(vii) produce multiplex sets of concatemers, each comprising concatemer repeat units of the corresponding orthogonal oligonucleotide primer sequence.

[0067] In another embodiment, the first oligonucleotide primer is bound to the target binding ligand via its 5' end.

[0068] In another embodiment, the target binding ligand is a polypeptide.

[0069] In another embodiment, the polypeptide comprises an antibody or an antigen-binding fragment thereof.

[0070] In another embodiment, the method further comprises contacting the target binding ligand with a preparation of cells or tissue comprising the target, or with a preparation of cells or tissue for detecting the presence of the target.

[0071] In another embodiment, the contacting is performed prior to concatemer formation steps (a)-(f) or (i)-(vii).

[0072] In another embodiment, the method further comprises contacting the extended nucleic acid chain comprising the concatemer repeat units produced according to steps (a)-(f) and / or (i)-(vii) with a nucleic acid probe comprising a complementary sequence to the concatemer repeat unit and a detectable label.

[0073] In another embodiment, the method generates a linear concatemer comprising the first oligonucleotide primer sequence.

[0074] In another embodiment, the method generates branched concatemers comprising a concatemer of a first oligonucleotide primer sequence complexed with a concatemer of a second oligonucleotide primer sequence.

[0075] In another embodiment, the method is performed in contact with a preparation of cells or tissue.

[0076] In another embodiment, the preparation of cells or tissue is fixed.

[0077] In another aspect, provided herein is a method for labeling a target binding ligand, the method comprising: performing the method of any one of the aforementioned embodiments, wherein the first oligonucleotide primer is coupled to the target binding ligand.

[0078] In one embodiment, the target binding ligand comprises an antibody or antigen-binding fragment thereof.

[0079] In another embodiment, the first oligonucleotide primer is coupled to the target binding ligand in a manner that allows extension of the primer from its 3' end.

[0080] In another embodiment, the method is performed in contact with a preparation of cells or tissue.

[0081] In another embodiment, the preparation of cells or tissue is fixed.

[0082] In another embodiment, the method further comprises contacting the concatemer produced in any of the preceding claims with a labeled nucleic acid probe and detecting the labeled nucleic acid probe attached to the concatemer, wherein detection of the labeled nucleic acid probe indicates the presence and location of the target molecule.

[0083] In another embodiment, the nucleic acid polymerase is a thermostable nucleic acid polymerase.

[0084] In one aspect, described herein is a method for producing a nucleic acid molecule, the method comprising: (a) providing a first oligonucleotide primer; (b) contacting the first oligonucleotide primer with a first single-stranded extension template and a nucleic acid polymerase in a reaction mixture under conditions that allow hybridization and extension of the first oligonucleotide primer using the first single-stranded extension template, wherein the first single-stranded extension template comprises a first extension template element, a sequence complementary to the first oligonucleotide primer, and a 3' chain terminator in the 5' to 3' direction, such that the extension template is not extended by the polymerase; (c) heating the reaction mixture of step (b) to separate the first single-stranded extension template from step (c) (d) contacting the extended first oligonucleotide primer with a second single-stranded extension template under conditions that allow hybridization and extension of the first extension oligonucleotide primer generated in step (b) using the second single-stranded extension template, wherein the second single-stranded extension template comprises, in a 5' to 3' direction, a second extension template element, a copy of the first extension template element, and a 3' chain terminator; wherein steps (a)-(d) produce a nucleic acid molecule comprising, in a 5' to 3' order, the first oligonucleotide primer sequence, the complement of the first extension template element, and the complement of the second extension template element.

[0085] In one embodiment, the method further comprises repeating steps (d) to (f) at least once.

[0086] In another embodiment, the steps are performed in the order presented.

[0087] In another embodiment, the method comprises repeating steps (d) to (f) at least n times, wherein each iteration of steps (d) to (f) increases the length of the tandem (concatemer ic) nucleic acid by one concatemer repeat unit.

[0088] In another embodiment, step (a) comprises providing an orthogonal first oligonucleotide primer and a first extension template oligonucleotide pair such that steps (b)-(f) produce multiple concatemers, each comprising a concatemer repeat unit of a corresponding orthogonal oligonucleotide primer sequence.

[0089] In another embodiment, after one or more repetitions of steps (d) to (f), the method further comprises: i) adding a second oligonucleotide primer and a second extension template oligonucleotide to the reaction, wherein the second oligonucleotide primer comprises a 5' proximal sequence complementary to the first oligonucleotide primer sequence and a 3' proximal sequence complementary to one of the at least two repeating portions of the second extension template oligonucleotide, wherein the second extension template oligonucleotide comprises a sequence of at least two repeating units linked head-to-tail, and a chain terminator at its 3' end, the sequence being complementary to the second oligonucleotide primer; ii) ligating the second oligonucleotide primer to one of the concatemers formed after one or more repetitions of steps (d) to (f); The invention relates to a method for preparing an extended nucleic acid chain comprising the plurality of concatemer repeating units of the sequence contained in the second oligonucleotide primer; the method comprises the steps of: step (i) hybridizing the second extension template oligonucleotide with the second oligonucleotide primer; ii) hybridizing the second extension template oligonucleotide with the second oligonucleotide primer; iv) extending the second oligonucleotide primer with a polymerase, thereby generating an extended nucleic acid chain complementary to the second extension template; v) heating the reaction mixture to separate the extended nucleic acid chain complementary to the second extension template from the second extension template; vi) cooling the heated product to allow the second extension template oligonucleotide to hybridize with the extended nucleic acid chain complementary to the second extension template generated in step (iv); and vii) extending the extended nucleic acid chain complementary to the second extension template generated in step (iv) with a polymerase, thereby generating an extended nucleic acid chain comprising the plurality of concatemer repeating units of the sequence contained in the second oligonucleotide primer.

[0090] In another embodiment, the polymerase is a thermostable polymerase.

[0091] In another embodiment, the method further comprises repeating steps (b) and (c) at least once prior to step (d), wherein each repetition of steps (b) and (c) results in the production of a nucleic acid molecule extended by one copy of the complementary sequence of the first extension template element.

[0092] In another embodiment, the method further comprises at least one iteration of the following steps: (e) heating the reaction mixture of step (d) to separate the second single-stranded extension template from the extended first oligonucleotide primer produced in step (d); and (f) repeating step (d), wherein each iteration of steps (e) and (f) extends the generated nucleic acid molecule by one copy of the complementary sequence of the second extension element.

[0093] In another embodiment, the method further comprises heating the reaction mixture after step (d) and contacting the nucleic acids produced in steps (a) to (d) with at least one additional single-stranded extension template under conditions that allow hybridization and extension of the produced nucleic acid molecules, wherein the additional single-stranded extension template comprises in the 5' to 3' direction an additional extension template element, a copy of the extension template element from a previous round of extension, and a 3' chain terminator, wherein the generated nucleic acid molecules are extended to include the complementary sequence of the additional extension template element.

[0094] In another embodiment, the first oligonucleotide is conjugated to a target binding ligand.

[0095] In another embodiment, the method is performed in contact with a cell or tissue sample.

[0096] In another embodiment, the method further comprises contacting the generated nucleic acid molecules with a labeled nucleic acid probe comprising one or more extension template element sequences.

[0097] In another aspect, provided herein is a kit for performing one or more methods described herein, the kit comprising: a) a first oligonucleotide primer or a set of first oligonucleotide primers; and b) a single-stranded extension template or a set of single-stranded extension templates, or an ordered extension template nucleic acid molecule.

[0098] In one embodiment, the first oligonucleotide primer or set of first oligonucleotide primers and the single strand extension template or set of single strand extension templates are orthogonal sets.

[0099] In another embodiment, orthogonal sets are optimized to avoid primer / extender cross-talk, primer dimer formation, and off-target hybridization.

[0100] In another embodiment, the single-stranded extension template or set of single-stranded extension templates is suitable for performing a concatemer generation method.

[0101] In another embodiment, the kit further comprises packaging materials for the various components, and optionally instructions for use.

[0102] In another embodiment, the kit further comprises one or more target binding ligand molecules or reagents for coupling the first oligonucleotide to the target binding ligand.

[0103] In another embodiment, the kit further comprises a thermostable polymerase, nucleotides, reaction buffer components, and reagents for labeling nucleic acid probe molecules.

[0104] In another embodiment, the kit further comprises a probe molecule complementary to an element of the concatemer repeat unit.

[0105] In another embodiment, the probe molecule may be complementary to one or more elements in the generated ordered extension products.

[0106] definition

[0107] As used herein, the term "target binding ligand" refers to a molecule or portion that specifically binds to a given target molecule. Target binding ligands may include, for example, peptides, polypeptides, nucleic acids, aptamers, receptors and / or their cognate ligands, members of affinity binding pairs (including but not limited to biotin / streptavidin), and small molecule agents that specifically bind to target molecules as defined herein. Antibodies and antigen binding fragments or constructs thereof represent a class of target binding ligands that can be used in the methods, compositions, and kits described herein. Nucleic acids comprising complementary sequences to a given target DNA or RNA molecule (including but not limited to mRNA molecules) represent another class of target binding ligands that can be used in the methods, compositions, and kits described herein.

[0108] As used herein, the term "specific binding" refers to a physical interaction between two molecules, compounds, cells and / or particles, wherein the binding of a first entity to a second target entity has a higher specificity and affinity than the binding to a third entity that is a non-target. A reagent specific to a given target is a reagent that exhibits specific binding to the target under the assay conditions used. The specificity of an antibody or its antibody fragment can be determined based on affinity and / or avidity. Affinity is represented by the equilibrium constant (KD) of the dissociation of an antigen and an antigen-binding protein, and is a measure of the binding strength between an antigenic determinant and an antigen-binding protein (such as an antibody or its antigen-binding fragment) on the antigen binding site: the smaller the KD value, the stronger the binding strength between the antigenic determinant and the antigen binding molecule. Alternatively, affinity can also be expressed as an affinity constant (KA), i.e., 1 / KD). Thus, when an antibody or antigen-binding fragment thereof as described herein binds to a first antigen with an affinity that is at least 1000-fold, 10,000-fold or more better than the affinity with which the amino acid sequence or polypeptide binds to another given polypeptide (as described above and appropriately expressed as, for example, a KD value), the antibody or antigen-binding fragment thereof is said to "specifically" or "specifically binds" or "selectively binds" to a first target or antigen as compared to a second target or antigen. In general, a molecule that "specifically binds" a given target, "selectively binds" a given target, or is "specific" for a given target will bind with a KD of 10-5 M (10000 nM) or less, such as 10-6 M, 107 M, 108 M, 109 M, 10-10 M, 10-11 M, 10-12 M, or less. Specific binding may be affected by, for example, the affinity and avidity of the polypeptide agent and the concentration of the polypeptide agent. One of ordinary skill in the art can determine suitable conditions for a polypeptide agent described herein to selectively bind to a target using any suitable method, such as titrating the polypeptide agent in a suitable cell binding assay.

[0109] In this context, it should be understood that, where antibodies or antigen-binding fragments thereof are concerned, specific binding is mediated by the CDRs of the antibody polypeptide, rather than any other portion of the antibody polypeptide. Antibody dissociation constants and affinities can be determined, for example, by surface plasmon resonance-based assays (such as the BIAcore assay described in PCT application publication number WO2005 / 012359); Forte Bio Octe™ assays, enzyme-linked immunosorbent assays (ELISAs); and competition assays (e.g., RIAs).

[0110] As used herein, the term "coupled" refers to, for example, an oligonucleotide and a target binding ligand connected in a stable manner by a thermal cycling step to produce a concatemer or ordered nucleic acid extension product as described herein. The conjugate may include a covalent bond. In various embodiments, the conjugate may include a linker molecule between the target binding ligand and the coupled oligonucleotide.

[0111] As used herein, the term "concatemer" refers to a nucleic acid molecule comprising two or more head-to-tail directional connections of a given sequence, repeated in the 5' to 3' direction. Concatemers can include two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, 15 or more, 20 or more, 25 or more, 30 or more, 35 or more, 40 or more, 45 or more, 50 or more repeating units of a given sequence. The number of repetitions of the concatemers produced as described herein is determined by the number of cycles of the chain separation and primer extension adopted. The length of the concatemer repeat unit as described herein can determine the multiple degree achievable by the orthogonal group of repetitions of a given length. The length of the repeat units in the concatemers as described herein can be, for example, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, 20 or more, 25 or more, 30 or more, 35 or more, 40 or more, 45 or more, or 50 or more nucleotides.

[0112] As used herein, the term "conditions that allow hybridization and extension" for a given molecule (such as an oligonucleotide molecule) refers to conditions where the temperature, salts, buffers, and other reaction components are sufficient for a template-directed polymerase-mediated primer extension reaction. The exact conditions for a given polymerase extension vary depending on the selected enzyme, but are known in the art and / or can be determined without undue experimentation. Hybridization conditions, such as the conditions for hybridization of the primers described herein with the single-stranded extension templates described herein, are generally salt, buffer, and other reaction component conditions suitable for the selected polymerase, and the annealing or hybridization temperature is determined primarily by the length of the concatemer repeat unit and the degree of multiplexing in a given reaction by those skilled in the art. Therefore, in a single (uniplex) reaction, when a given molecule is labeled as a single concatemer sequence in the reaction, the annealing or hybridization temperature can be determined based on the Tm of the specific concatemer repeat sequence-usually, the annealing temperature in the cyclic reaction is about 5°C lower than the Tm of the repeat sequence hybridizing to its complementary sequence at the optimal salt and reaction component concentrations of the selected enzyme. When performed in multiplex, the length of the repeat unit becomes more important because the Tm of various repeat sequences will vary. Primer / single-strand extension template design can take into account sequence variation to design primers and single-strand extension templates with relatively close Tm values, typically within 5-7°C for all members of the primer / extension template sequence set. In these cases, a single annealing or hybridization temperature in a cyclic reaction can allow for efficient hybridization and extension of the group members in multiplexing. In various embodiments of the method including the step of contacting a nucleic acid molecule with a single-strand extension template under conditions that allow hybridization and extension of nucleic acid molecules, the reaction can be incubated at the annealing temperature for a period of time (typically a few seconds to a few minutes) before the temperature is raised to the optimal extension temperature of the polymerase. It should be understood that contacting a nucleic acid molecule with a single-strand extension template under conditions that allow hybridization and extension of nucleic acid molecules can include such annealing / extension temperature changes. In some embodiments, annealing or hybridization occurs effectively at a temperature at which the polymerase is sufficiently active without requiring such a temperature change; whether such a shift is required in a given case will be apparent to those of ordinary skill in the art, depending on the enzyme used, and can also be determined empirically without having to perform excessive experiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0113] Figures 1A-1C . Thermally controllable DNA extension scheme. Figure 1A shows that thermal cycle extension (TCE) can be used to generate concatemers with repetitive sequences. Figure 1B shows that multiplexed concatemer generation is achieved using TCE. Figure 1C shows that TCE can also be used for primer exchange reactions.

[0114] Figure 2The maximum multiplex times for different primer lengths are depicted, including 9nt: 93 times; 10nt: 170 times; 11nt: 315 times; 12nt: 585 times; 13nt: 1092 times; 14nt: 2048 times; and 15nt: 3855 times.

[0115] Figure 3 Highly controlled amplification is shown. Two randomly selected sequences were used for the TCE reaction. 30, 60, 90, 120 amplification cycles were performed. The size of the concatemers corresponds to the number of amplification cycles.

[0116] Figure 4 High amplification uniformity is shown. The amplification uniformity of 50 different 9bp primers was tested in a test tube. 5nM of each primer was added to different test tubes containing 1μM of the corresponding single-stranded extension body ("ssEXTer"). Each test tube was amplified for 80 cycles. The two dotted lines are 350bp and 500bp respectively.

[0117] Figure 5 Highly multiplexed amplification is shown. 50 primers are added here and their ssEXTers are placed in a test tube. The sample is amplified for 120 cycles and sent for next generation sequencing. After each extension repetition, the frequency of all 50 sequences for each primer is counted. The results show that 45 ssEXTers have a specificity of >99.5% and 48 ssEXTrs have a specificity of >99%.

[0118] Figure 6 Schematic diagram depicting the principle of multiplexed imaging by in situ extension.

[0119] Figure 7A-7B Figure 7A shows in situ extension (ISE) signal amplification on a protein target. Figure 7B shows ISE signal amplification on an RNA target. Signal amplification was adjusted by varying the number of thermal cycles.

[0120] Figure 8 It is indicated that branched ISE imaging resulted in signal amplification >100-fold. Contrast was adjusted to avoid oversaturation of the images.

[0121] Fig. 9 Protein expression levels were identified by multiplexed ISE imaging on Py2T model cells.

[0122] Figures 10A-10B Figure 10A shows co-imaging of protein and RNA in Hela cells using ISE imaging. Figure 10B shows co-imaging of protein and RNA in fresh frozen sections of mouse brain using ISE imaging.

[0123] Fig.11The ISE protocol was examined, which enables labeling of neuronal markers in thick (0.5 mm) mouse brain tissue.

[0124] Figures 12A-12B Figure 12A examines an ISE that shows a high signal-to-noise ratio and low nonspecific fluorescence background, and enables single-molecule imaging, where most of the signal spots in the cell were observed to co-localize with areas of high tubulin-GFP expression. Figure 12B examines an ISE that shows a high signal-to-noise ratio and low nonspecific fluorescence background, and enables single-molecule imaging, where signal amplification by fluorophore-labeled secondary antibodies results in high nonspecific background signals in areas lacking tubulin-GFP expression.

[0125] Figures 13A-13B Figure 13A is a schematic diagram of epitope detection using conventional mass cytometry antibodies. Figure 13B is a schematic diagram of epitope detection using an amplification method using cyclic extension amplification of DNA oligomers (ACED).

[0126] Fig.14 Depicted is CNvK-based photocrosslinking, which allows hybridized detection strands to remain intact during mass cytometry sample acquisition.

[0127] Figures 15A-15C .Figure 15A shows that transient transfection is used to produce the expression gradient of GFP in the cell group, and the cell group is subsequently targeted by the anti-GFP antibody coupled with ACED oligonucleotide.By 1-500 times thermal cycle, the signal amplification of ACED (y axis) is compared with the counterstaining using anti-rat secondary antibody, to show the specificity of ACED.Figure 15B examines the data in Figure 15A, and divides it into 10 equal width boxes (bin) according to the GFP expression level shown by the secondary antibody.Box 1 represents untransfected cells as internal control.Box 10 shows the cells with the highest GFP expression level.Figure 15C examines the box median value and signal-to-noise ratio (that is, the median value of box 1-9 is compared with the median value in box 1) in 1-500 thermal cycles, as shown in the left figure, indicating that the amplification intensity is 13 times, and the signal-to-noise ratio is enhanced by 6 times.The right side draws the multiple change of each box in 1-500 cycles in the form of a bar graph.

[0128] Figures 16A-16C Signal amplification with one or two rounds of branching was compared to linear amplification without branching. A 17-fold amplification was observed with each branching round.

[0129] Fig.17 Pairwise analysis of six extended bodies and metal chains identified how ACED exhibits high orthogonality.

[0130] Fig.18ACED mass cytometry analysis of Py2T cells undergoing EMT was examined. Samples that were untreated or treated with TGFβ for 3, 5, or 7 days were analyzed. Thirty EMT markers, including E-cadherin, vimentin, Zeb1, Snail / Slug, Smad2 / 3, and Smad4, were also analyzed to reveal the EMT status.

[0131] Fig.19 Shown is how ACED is able to detect RNA targets in cells.

[0132] Fig. 20 A scheme for multiplex flow cytometry and flow sorting with TCE amplification is depicted.

[0133] Figures 21A-21B Figure 21A depicts the scheme of GFP transfection and flow cytometry experiments. Figure 21B examines the flow cytometry results of TCE-Flow with secondary antibody staining. Signal-to-noise ratio (GFP high / GFP low): 130 TCE cycles: 333.35; 260 cycles TCE: 359.1; Secondary antibody: 97.80.

[0134] Fig. 22 Multiplex immunoblotting scheme of TCE amplification is shown.

[0135] Fig.23 Immunoblot of GFP protein with TCE amplification is depicted. Left lane: protein ladder. Right lane: sample. Arrows indicate target lane. GFP is a 28 kD protein.

[0136] Fig.24 Shown is a schematic diagram describing the simultaneous amplification of RNA and protein signals in cell and tissue samples using ISE.

[0137] Fig.25 is a more general schematic diagram showing the simultaneous amplification of biomolecules in cell and tissue samples using ISE.

[0138] Fig.26 Depicted are multiplexed protein and RNA imaging of a cell line (Py2T cells) treated or not with TGFβ. ISE validates changes in targets during epithelial to mesenchymal transition.

[0139] Fig. 27 Depicted are multiplexed protein and RNA imaging of a PFA-fixed tissue sample (mouse brain). 79-plex (43 RNAs and 36 proteins) thin tissue imaging (14 μm, sagittal section of mouse brain).

[0140] Fig.28Shown is 16-plex protein imaging in a fresh frozen sample of a human cortical region. 16-plex imaging of thin tissue (12 μm, human cortical region).

[0141] Fig.29 8-plex protein imaging of cleared thick mouse tissue (3x4x1 mm) is shown. The ISE is compatible with various types of clearing protocols for thick tissue imaging.

[0142] Fig.30 4-plex protein imaging of cleared thick human tissue (3x3x1 mm, w / high autofluorescence) is shown. The sample was photobleached by LED to reduce autofluorescence. DETAILED DESCRIPTION

[0143] Technology described herein relates to the improvement of the ability of in-situ detection of target molecules in a manner that retains information about the location of such target molecules (usually subcellular). More specifically, technology described herein provides methods, compositions and kits for marking and detecting target ligand binding molecules, wherein marking and detection are carried out in-situ on cells or tissue samples contacted with ligand binding molecules. For example, oligonucleotides, particularly oligonucleotides with a length exceeding about 40 nucleotides and antibodies can affect the diffusion characteristics of antibodies and may cause non-specific binding. By generating tandem oligonucleotides in situ, the target binding ligand has been combined with its target, and the methods described herein provide significant signal amplification while avoiding these problems. Methods described herein also provide compositions, methods and kits, which allow for multiple ways of marking and / or detecting target binding ligand binding molecules, and in a manner that allows for distinguishable detection of signals from multiple different target binding molecules in multiple ways.

[0144] In one embodiment, the technology described herein involves the in situ generation of concatemers of nucleic acid sequences on target binding ligand molecules, wherein the repeating sequence provides multiple hybridization sites for labeled nucleic acid probes for each target ligand binding molecule, thereby amplifying the signal associated with the target ligand binding molecule binding to the target molecule in a cell or tissue sample. The multiple hybridization sites provided by the concatemer can be considered as "landing pads" for labeled probe molecules, which hybridize with corresponding monomers such as in the concatemer. In the case where there are multiple such monomers in each concatemer connected to the target ligand binding molecule, the signal from each such target ligand binding molecule is amplified, thereby facilitating the detection, quantification and / or imaging of the target molecule when it occurs in situ.

[0145] In another embodiment, the in situ concatemer generation method is very suitable for in situ multiple labeling, detection and / or imaging. As discussed in further detail below, a multiplex method uses different target ligand binding molecule groups, each molecule is specific for a different target ligand, and each molecule is coupled to a different single-stranded oligonucleotide primer. When the corresponding primers are multiply extended to form concatemers of different repeat sequences by one or more methods described herein, the different repeats on the corresponding members of the target ligand binding molecule group provide different, highly selective binding sites or landing pads for a group of different, distinguishably labeled probes, which allow multiple detection, quantification and / or imaging of members of a group of target molecules in situ.

[0146] The following describes various considerations involved in implementing the techniques disclosed herein.

[0147] Preparation of cell or tissue samples

[0148] Samples prepared for histology, including cell and / or tissue samples, are fixed to avoid deterioration during subsequent staining and detection steps. Samples can be fixed as soon as possible after collection. Many different types of fixatives are known in the art. Exemplary fixatives include, but are not limited to, 4% paraformaldehyde, 4% formaldehyde, 10% neutral buffered formalin, Bonn's solution, methanol, acetone, glutaraldehyde, and the like. Cell or tissue samples can be treated or processed to minimize nucleic acid degradation, for example, where RNA is the target molecule. One of ordinary skill in the art will be able to determine the most suitable fixative for a cell or tissue sample and the technique to be performed. In some embodiments, the fixative that can be used is 4% paraformaldehyde.

[0149] In some embodiments, fixed cells can be in suspension, for example, to allow for sorting or detection of cells based on the presence or amount of one or more target ligands using flow cytometry.

[0150] In some embodiments, fixed cells or tissues can be embedded in culture medium, for example, to facilitate sections for histology and / or imaging. Paraffin embedding of fixed samples is well known in the art. However, in brief, tissue samples, after fixation, are pre-embedded in paraffin to replace the water content of the sample with paraffin. Paraffin embedding involves dehydrating the tissue in the presence of increasing concentrations of alcohol, and then gradually replacing the alcohol with a paraffin solvent. Examples of paraffin solvents include xylene. After pre-embedding, the sample is embedded in the melted paraffin using a mold and hardened. Substitutes for paraffin include, but are not limited to, epoxy resins, acrylic resins, agar, gelatin, and collodion. One of ordinary skill in the art will be able to determine applicable embedding parameters.

[0151] After hardening, paraffin-embedded tissue samples are sectioned, where the sample is cut into thin slices or sections that are placed on glass slides. These sections are typically about 5 μm thick, but they can be thinner or thicker depending on parameters such as tissue type, target molecule, and marker / marker detection used. One of ordinary skill in the art will be able to determine how thick the sections need to be. After the sections are cut, they are transferred to a warm water bath and placed on charged slides. The slides are dried to remove excess wax.

[0152] In some embodiments, after the sample is placed on a slide, the sample can be stained. The staining contrasts with the tissue section, making it easier to observe the structure of the sample. Exemplary dyes include, but are not limited to, Alcian blue, aldehyde fuchsin, alkaline phosphatase, Bielshowsky stain, Congo red, crystal violet, eosin, FontanaMasson, Giemsa, hematoxylin, Luna stain, Nissl stain, periodic acid Schiff (PAS), Oil Red 3, reticular fiber dye, Sudan black, toluidine blue and vanGieson. Those of ordinary skill in the art will be able to determine which staining is most effective for the sample and the technology to be performed. The term "staining" is also used to refer to the detection of specific target molecules, for example, using target binding ligands or molecules applied to cells or tissue samples as described herein. More details about histology can be found in Ross, MH et al. Histology: a text and atlas with correlated cell and molecular biology (7th ed.) Wolters Kluwer. (ISBN: 978-1451187427).

[0153] Target binding ligand

[0154] In some embodiments of any of the aspects, the oligonucleotide primer strand is attached to the target binding ligand molecule.

[0155] As used herein, a "target binding ligand" is a molecule or moiety that binds (e.g., specifically binds) to a target molecule of interest. A target binding ligand can be a synthetic or natural molecule. A target binding ligand can be a biomolecule, such as a polypeptide or a polynucleotide. In some embodiments, a target binding ligand is a polypeptide. In some embodiments, a target binding ligand is a protein or a fragment thereof. Non-limiting examples of target binding ligands include peptides, polypeptides, antibodies and antibody derivatives, oligonucleotides, aptamers, and receptors.

[0156] In some embodiments of any aspect, the target binding ligand binds to a target molecule, i.e., the target molecule is a molecule selected from the non-limiting group of lipids, sugars, oligosaccharides or polysaccharides, amino acids, peptides or polypeptides, nucleotides, nucleotides, oligonucleotides or polynucleotides, hormones, vitamins, small molecules, miRNA, metabolites, and any combination thereof.

[0157] In some embodiments of any of the aspects, the target binding ligand binds to a DNA or RNA barcode molecule.

[0158] In some embodiments of any aspect, the target binding molecule is an antibody. The term "antibody" refers to a protein comprising at least one immunoglobulin variable domain or immunoglobulin variable domain sequence that binds to a target molecule. For example, an antibody can include an immunoglobulin heavy chain (H) variable region (abbreviated herein as VH) and an immunoglobulin light chain (L) variable region (abbreviated herein as VL). In another example, an antibody includes two heavy chain (H) variable regions and two light chain (L) variable regions. An antibody can include structural features of IgA, IgG, IgE, IgD, IgM (and its subtypes). The term antibody used herein includes any of a variety of different constructs that mediate binding to a target molecule using one or more antigen binding domains or fragments of an antibody. Thus, in addition to complete IgA, IgG, IgE, IgD or IgM antibodies, antibodies also include, but are not limited to, antigen-binding fragments of antibodies (e.g., single-chain antibodies, Fab and sFab fragments, F(ab')2, Fd fragments, Fv fragments, scFv, domain antibodies (dAb) (de Wildt et al., Eur J Immunol. 1996; 26(3): 629-39) and nanobodies. In the methods, compositions and kits described herein, antibodies using non-antibody scaffolds to support multiple target binding domains can also be used as target binding ligands.

[0159] As used herein, "antigen binding fragment" refers to the part of an antibody that is necessary and sufficient to bind to a given antigen. At a minimum, an antigen binding fragment of a conventional antibody will contain six complementarity determining regions (CDRs) derived from the heavy and light chain polypeptides of the antibody arranged on a scaffold, enabling it to selectively bind to the antigen. Commonly used antigen binding fragments include the VH and VL domains of an antibody, which may be connected by a portion of the constant domains of the heavy and light chains of the antibody, or by a linker, such as a peptide linker. Unconventional antibodies, such as camelids and short antibodies, have only heavy chain sequences, such as VHH. These can be used in a manner similar to that containing VH / VL antigen binding fragments. Non-limiting examples of antibody fragments encompassed by the term antigen-binding fragment include: (i) a Fab fragment having VL, CL, VH and CH1 domains; (ii) a Fab' fragment, which is a Fab fragment having one or more cysteine ​​residues at the C-terminus of the CH1 domain; (iii) a Fd fragment having VH and CH1 domains; (iv) a Fd' fragment having VH and CH1 domains and one or more cysteine ​​residues at the C-terminus of the CH1 domain; (v) a Fv fragment having the VL and VH domains of a single arm of an antibody; (vi) a dAb fragment consisting of a VH domain (Ward et al., Nature 341, 544-546 (1989)); (vii) a F(ab')2 fragment, a bivalent fragment comprising two Fab' fragments linked by a disulfide bond at the hinge region; (viii) a single-chain antibody molecule (e.g., single-chain Fv; scFv) (Bird et al., Science 341, 544-546 (1989)). 242:423-426 (1988); and Huston et al., PNAS (USA) 85:5879-5883 (1988)); (ix) "diabodies" with two antigen-binding sites, which comprise a heavy chain variable region (VH) connected to a light chain variable domain (VL) in the same polypeptide chain (see, e.g., EP 404,097; WO 93 / 11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); and "linear antibodies" comprising a pair of tandem Fd fragments (VH-CH1-VH-CH1), which together with complementary light chain polypeptides form a pair of antigen-binding regions (Zapata et al., Protein Eng. 8(10):1057-1062 (1995); and U.S. Pat. No. 5,641,870). Fv, scFv or diabody molecules can be stabilized by incorporating disulfide bonds linking the VH and VL domains. Minibodies comprising scFv fragments linked to a CH3 domain can also be obtained.Other examples of binding fragments are Fab', which differs from Fab fragments by the addition of a few residues at the carboxyl terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region, and Fab'-SH, a Fab' fragment in which the cysteine ​​residues of the constant domains carry a free thiol group.

[0160] Oligonucleotides

[0161] Target binding ligands as described herein include single-stranded oligonucleotide primers that allow concatemers to be produced according to the method. Single-stranded oligonucleotide primers can be attached to the target binding ligand by any of a variety of methods known in the art. Note that although the single-stranded oligonucleotide can be DNA, RNA, or a modified form thereof, for ease of reference and proof of principle, DNA oligonucleotides are used in the following description and examples.

[0162] Oligonucleotides and oligonucleotide synthesis are well known in the art, and many different commercial sources provide customized oligonucleotide synthesis, so that oligonucleotides with essentially any sequence can be easily obtained. Oligonucleotides are commercially available from Eurofins Genomics (Louisville, Kentucky), ThermoFisher (Waltham, Massachusetts), Integrated DNA Technologies (Coralville, Iowa), TriLink Bioechologies (San Diego, California), etc. for custom design and use. In addition, it is known in the art how to produce oligonucleotides including modified nucleotides and / or modified connections, and oligonucleotides including any of many such modifications are also commercially available. It should be clear that an oligonucleotide "primer" is an oligonucleotide that can be extended by a template-dependent nucleic acid polymerase when hybridized with a template nucleic acid molecule (e.g., by hydrogen bond base pairing). The single-stranded extension template oligonucleotide described herein has a 3' modification or portion (also referred to as a "termination" portion) that blocks or excludes template-dependent extension. Such modifications may also be referred to as chain terminators, and include, for example, dideoxynucleosides, 3'-CPR IICPG, 3'-phosphoCPG, 5'-Ome-dT-CE phosphoramidite, 5'-amino-dT-CE phosphoramidite, 2'3'-ddA-CE phosphoramidite, 2'3-ddC-CE phosphoramidite, 2'3-ddG-CE phosphoramidite, 2'3'-ddT-CE phosphoramidite, 3'-dA-CPG, 3'-ddC-CPG, 3'-dC-CPG, 3'-dT CPG, 3'Spacer C3 CPG, etc. In some embodiments, the oligonucleotide may also include, for example, a nucleotide modified to include a crosslinking moiety. Incorporation of a crosslinking moiety, including but not limited to a photocrosslinking moiety, may allow, for example, the oligonucleotide to be crosslinked to the target sequence so that the oligonucleotide does not dissociate during a subsequent process, such as thermal cycling to produce a concatemer. Thus, in some embodiments of any aspect, the oligonucleotide chain comprises a photocrosslinking moiety, e.g., a photocrosslinking moiety selected from 3-cyanovinylcarbazole (CNVK) nucleotides, 5-bromodeoxycytosine, 5-iododeoxycytosine, 5-bromodeoxyuridine, 5-iododeoxyuridine, and nucleotides comprising aryl azide (AB-dUMP), benzophenone (BP-dUMP), perfluorinated aryl azide (FAB-dUMP) or diazirine (DB-dUMP), psoralen, 4-thio-dT (S4dT), and the like.

[0163] Oligonucleotides useful in the methods, compositions and kits described herein are typically at least 9 nucleotides in length, but can vary in length from 5 to 100 or more nucleotides. Thus, oligonucleotides useful in the methods, compositions, or kits described herein can be, for example, between 5-100 nucleotides in length, between 5-95 nucleotides in length, between 5-90 nucleotides in length, between 5-85 nucleotides in length, between 5-80 nucleotides in length, between 5-75 nucleotides in length, between 5-70 nucleotides in length, between 5-65 nucleotides in length, between 5-60 nucleotides in length, between 5-55 nucleotides in length, between 5-50 nucleotides in length, between 5-45 nucleotides in length, between 5-40 nucleotides in length, between 5-35 nucleotides in length, between 5-30 nucleotides in length, between 5-25 nucleotides in length, between 5-20 nucleotides in length, between 5-15 nucleotides in length, between 5-10 nucleotides in length, between 5-9 nucleotides in length, 9-100 nucleotides in length, 9-95 nucleotides in length, 9-90 nucleotides in length, 9-85 nucleotides in length, 9-80 nucleotides in length, 9-75 nucleotides in length, 9-70 nucleotides in length, 9-65 nucleotides in length, 9-60 nucleotides in length, 9-55 nucleotides in length, 5-50 nucleotides in length, 9-45 nucleotides in length, 9-40 nucleotides in length, 9-35 nucleotides in length, 9-30 nucleotides in length, 9-25 nucleotides in length, 9-20 nucleotides in length, 9-15 nucleotides in length, 10-100 nucleotides in length, 10-90 nucleotides in length, 10-80 nucleotides in length, 10-70 nucleotides in length,10-60 nucleotides in length, 10-50 nucleotides in length, 10-40 nucleotides in length, 10-30 nucleotides in length, 10-20 nucleotides in length, 11-100 nucleotides in length, 11-90 nucleotides in length, 11-80 nucleotides in length, 11-70 nucleotides in length, 11-60 nucleotides in length, 11-50 nucleotides in length, 11-40 nucleotides in length, 11-30 nucleotides in length, 11-20 nucleotides in length, 12-100 nucleotides in length, 12-90 nucleotides in length, 12-80 nucleotides in length, 12-70 nucleotides in length, 12-60 nucleotides in length, 12-50 nucleotides in length, 12-40 nucleotides in length, 12-30 nucleotides in length, 12-20 nucleotides in length, 13-100 nucleotides in length, 13-90 nucleotides in length, 13-80 nucleotides in length, 13-70 nucleotides in length, 13-60 nucleotides in length, 13-50 nucleotides in length, 13-40 nucleotides in length, 13-30 nucleotides in length, 13-20 nucleotides in length, 14-100 nucleotides in length, 14-90 nucleotides in length, 14-80 nucleotides in length, 14-70 nucleotides in length, 14-60 nucleotides in length, 14-50 nucleotides in length, 14-40 nucleotides in length, 14-30 nucleotides in length, 14-20 nucleotides in length, 15-100 nucleotides in length, 15-90 nucleotides in length, 15-80 nucleotides in length, 15-70 nucleotides in length, 15-60 nucleotides in length, 15-50 nucleotides in length, 15-40 nucleotides in length, 15-30 nucleotides in length, or 15-20 nucleotides in length.

[0164] Methods for combining oligonucleotides with antibodies are known in the art. For example, see Dugal-Tessier et al., J.Clin.Med.10:838 (2021) for a review of various methods. Kits can be used for coupling oligonucleotides to antibodies; for example, see Abcam Oligonucleotide Coupling Kit (ab218260), which allows coupling to the 5' or 3' end of oligonucleotides 10 to 120 nucleotides long. The methods suitable for coupling oligonucleotides to non-antibody target binding ligands vary depending on the ligand, but are known to those of ordinary skill in the art. The coupling mode of a single-stranded oligonucleotide primer to a target binding ligand allows a template-guided nucleic acid polymerase to extend from the 3' end of the primer. Although various primer coupling methods can be used, it is important that the method used maintains the openness of the 3' nucleotide for extension. Examples include coupling by a connecting portion connected to or near the 5' end of an oligonucleotide primer.

[0165] The oligonucleotides described herein include nucleic acid probe molecules. In general, a nucleic acid probe molecule is an oligonucleotide that includes a label that can be detected directly or indirectly to provide a signal. Non-limiting examples of labels can include fluorescent labels or metals, such as lanthanide metals that allow detection in mass spectrometry flow cytometers. Other examples of detectable labels include, for example, isotopes (such as 32 P. 35 S, etc.), quantum dots, organic dyes, polymer nanoparticles, metal nanoparticles, and Raman dots, etc. Methods for preparing labeled oligonucleotide probes with any of a variety of different detectable moieties are known to those skilled in the art. Multiple copies of nucleic acid probes that bind to the repeated sequences in the concatemers generated as described herein can be combined with such concatemers so that each target molecule bound by the target binding ligands described herein has multiple labeled moieties associated therewith, thereby greatly amplifying the signal in situ so that the presence, location, and / or amount of a given target can be determined with single molecule sensitivity.

[0166] A variety of fluorescent reporter dyes are known in the art. Typically, the fluorophore is an aromatic or heteroaromatic compound, which may be pyrene, anthracene, naphthalene, acridine, stilbene, indole, benzindole, oxazole, thiazole, benzothiazole, cyanine, carbocyanine, salicylate, anthranilate, coumarin, fluorescein, rhodamine, or other similar compounds.

[0167] Exemplary fluorophores include, but are not limited to, 1,5IAEDANS, 1,8-ANS, 4-methylumbelliferone, 5-carboxy-2,7-dichlorofluorescein, 5-carboxyfluorescein (5-FAM), 5-carboxynaphthofluorescein (pH 10), 5-carboxytetramethylrhodamine (5-TAMRA), 5-FAM (5-carboxyfluorescein), 5-hydroxytryptamine (HAT), 5-ROX (carboxy-X rhodamine), 5-TAMRA (5-carboxytetramethylrhodamine), 6-carboxyrhodamine 6G, 6-CR 6G, 6-JOE, 7-amino-4-methylcoumarin, 7-aminoactinomycin D (7-AAD), 7-hydroxy-4-methylcoumarin, 9-amino-6-chloro-2-methoxyacridine, ABQ, acid fuchsin, ACMA (9-amino-6-chloro-2-methoxyacridine), acridine orange, acridine red, acridine yellow, Acriflavin, Acriflavin FeulgenSITSA, aequorin (Photoprotein), AlexaFluor 350 TM Alexa Fluor 430 TM Alexa Fluor 488 TM Alexa Fluor 532 TM Alexa Fluor 546 TM Alexa Fluor 568 TM Alexa Fluor 594 TM Alexa Fluor 633 TM Alexa Fluor 647 TM Alexa Fluor 660 TM Alexa Fluor 680 TM , Alizarin aminocarboxylate complex, Alizarin red, Allophycocyanin (APC), AMC, AMCA-S, AMCA (aminomethylcoumarin), AMCA-X, Aminoactinomycin D, Aminocumarin, Aniline blue, Anthracene stearate, APC-Cy7, APTS, Basic Red 14, Basic Orange 22, Vat Red 6B, Astrazone Cationic Yellow 7GLL, Atabrine, ATTO-TAG TM CBQCA, ATTO-TAG TMFQ, Auramine, Aurophosphine G, Aurophosphine, BAO 9 (Bisaminophenyloxadiazole), BCECF (high pH), BCECF (low pH), berberine sulfate, β-lactamase, BFP blue shifted GFP (Y66H), BG-647, Bimane, Bisbenzamide, Blancophor FFG, Blancophor SV, BOBO TM -1.BOBO TM -3. Body 492 / 515, Bodipy493 / 503, Bodipy500 / 510, Bodipy505 / 515, Bodipy530 / 550, Bodipy542 / 563, Bodipy558 / 568, Bodipy564 / 570, Bodipy576 / 589, Bodipy581 / 591, Bodipy630 / 650-X, Bodipy650 / 665-X, Bodipy665 / 676, BodipyFl, BodipyFLATP, Bodipy Fl Ceramide, BodipyR6G SE, Bodipy TMR, Bodipy TMR-X Conjugate, Bodipy TMR-X SE, Bodipy TR, Bodipy TRATP, Bodipy TR-X SE, BO-PRO TM -1. BO-PRO TM -3, Brilliant Sulphoflavin FF, Calcein, Calcein Blue, Calcium Crimson TM , Calcium Green, Calcium Green-1Ca2+ Dye, Calcium Green-2Ca2+, Calcium Green-5N Ca2+, Calcium Green-C18 Ca2+, Calcium Orange, Calcium Fluorescent White, Carboxy-X Rhodamine (5-ROX), Cascade Blue TM , CascadeYellow, Catecholamine, CFDA, CFP cyan fluorescent protein; Chlorophyll, Chromomycin A, Chromomycin A, CMFDA, Coelenterazine, Coelenterazine cp, Coelenterazine f, Coelenterazine fcp, Coelenterazine h, Coelenterazine hcp, Coelenterazine ip, Coelenterazine O, CoumarinPhalloidin, CPM methylcoumarin, CTC, Cy2 TM , Cy3.18, Cy3.5 TM , Cy3 TM , Cy5.18, Cy5.5 TM 、Cy5 TM 、Cy7TM , Cyan GFP, cyclic AMP fluorescent sensor (FiCRhR), d2, Dabcyl, Dansyl, Dansylamide, Dansylcadaverine, Dansyl chloride, DansylDHPE, Dansyl fluoride, DAPI, Dapoxyl, Dapoxyl 2, Dapoxyl 3, DCFDA, DCFH (2',7'-dichlorodifluorofluorescein phosphophenol ester), DDAO, DHR (dihydrorhodamine 123), Di-4-ANEPS, Di-8-ANEPPS (non-ratio), DiA (4-Di-16-ASP), DIDS, dihydrorhodamine 123 (DHR), DiO (DiOC18 (3)), DiR, DiR (DiIC18 (7)), dopamine, DsRed, DTAF, DY-630-NHS, DY-635-NHS, EBFP, ECFP, EGFP, ELF 97, Eosin, Erythrosin, Erythrosin ITC, Ethidium-1 (EthD-1), Acridine Orange (Euchrysin), Europium (III) chloride, Europium, EYFP, Fast Blue, FDA, Felgren (pararosaniline), FITC, FL-645, Flazo Orange, Fluoro-3, Fluoro-4, Fluorescein diacetate, Fluoro-Emerald; Fluoro-Gold (hydroxyamidine), Fluor-Ruby, FluorX, FM 1-43 TM 、Table 4-46、FuraRed TM(high pH), Fura-2 (high calcium), Fura-2 (low calcium), Genacryl Brilliant Red B, Genacryl Brilliant Yellow 10GF, Genacryl Pink 3G, Genacryl Yellow 5GF, GFP (S65T), GFP red shifted (rsGFP), GFP wild type, non-UV excited (wtGFP), GFP wild type, UV excited (wtGFP), GFPuv, Gloxalic Acid, GranularBlue, Hematoporphyrin, Hoechst 33258, Hoechst 33342, Hoechst 34580, HPTS, Hydroxycoumarin; Hydroxystilbene (FluoroGold), Serotonin, Indole dicarbocyanine (DiD), Indole tricarbocyanine (DiR), Intrawhite Cf, JC-1, JO-JO-1, JO-PRO-1, LaserPro, Laurodan, LDS 751, LeucophorPAF, Leucophor SF, Leucophor WS, Lissamine Rhodamine, Lissamine Rhodamine B, LOLO-1, LO-PRO-1, Lucifer Yellow, Mag Green, Magdala Red (Root Skin Red B), Magnesium Green, Magnesium Orange, Malachite Green, Marina Blue, Maxilon Brilliant Flavin 10GFF, Maxilon Brilliant Flavin 8GFF, Merocyanin, Methoxycoumarin, Mitotracker Green FM, Mitotracker Orange, Mitotracker Red, Mitramycin, Monobromodiamine (Monobromobimane), Monobromodiamine (mBBr-GSH); Monochlorodiamine (Monochlorobimane), MPS (Methyl Green-Pyronine), NBD, NBD Amine, Nile Red, Nitrobenzoxazole, Norepinephrine, Nuclear Fast Red, Nuclear Yellow, Nylosan Brilliant Iavin E8G, Oregon Green TM , Oregon Green488-X, Oregon Green TM 488、Oregon Green TM 500、Oregon Green TM514, Pacific Blue, Pararosaniline (Fulgen), PE-Cy5, PE-Cy7, PerCP, PerCP-Cy5.5, PE-TexasRed (Red 613), Root Red B (Sudan Red), Phorwite AR, Phorwite BKL, Phorwite Rev, Phorwite RPA, Phosphine 3R, Photoresist, Phycoerythrin B [PE], Phycoerythrin R [PE], PKH26, PKH67, PMIA, Pontochrome Blue Black, POPO-1, POPO-3, PO-PRO-1, PO-PRO-3, Primuline, Procion Yellow, Propidium Iodide (PI), PyMPO, Pyrene, Pyronine, Pyronine B, Pyrozal BrilliantFlavin 7GF, QSY 7, Quinacridone, Resorufin, RH 414, Rhod-2, Rhodamine, Rhodamine 110, Rhodamine 123, Rhodamine 5-GLD, Rhodamine 6G, Rhodamine B540, Rhodamine B 200, Rhodamine B (Rhodamine B extra), Rhodamine BB, Rhodamine BG, Rhodamine Green, Rhodamine Phallicidine, Rhodamine labeled phalloidin, Rhodamine Red, Rhodamine WT, Rose Bengal, R-phycoerythrin (PE), red-shifted GFP (rsGFP, S65T), S65A, S65C, S65L, S65T, Sapphire GFP, Serotonin, Sevron Brilliant Red 2B, Sevron Brilliant Red 4G, Sevron Brilliant Red B, Sevron Orange, Sevron Yellow L, sgBFP TM sgBFP TM (super luminescent BFP), sgGFP TM sgGFP TM (superluminescent GFP), SITS, SITS(Primuline), SITS(stilbene isothiosulfate), SPQ(6-methoxy-N-(3-sulfopropyl)quinoline), Stilbene, Sulphorhodamine B can C, Sulphorhodamine G Extra, Tetracycline, Tetramethylrhodamine, Texas Red TM 、Texas Red-X TMConjugate, thiadicarbocyanine (DiSC3), thiazine red R, thiazole orange, thioflavin 5, thioflavin S, thioflavin TCN, Thiolyte, thiazole orange, Tinopol CBS (calcofluor fluorescent brightener), TMR, TO-PRO-1, TO-PRO-3, TO-PRO-5, TOTO-1, TOTO-3, TriColor (PE-Cy5), TRITC (tetramethylrhodamine isothiocyanate), blueing agent, TruRed, Ultralite, Uranine B, Uvitex SFC, wt-GFP, WW 781, XL665, X-rhodamine, XRITC, xylenol orange, Y66F, Y66H, Y66W, yellow GFP, YFP, YO-PRO-1, YO-PRO-3, YOYO-1, and YOYO-3. Fluorescent labeling methods for nucleic acids are known in the art. For multiplex approaches, probes labeled with fluorophores having distinguishable excitation and / or emission spectra can be used.

[0168] Several embodiments of the concatemer generation methods described herein are shown in FIG. 1 .

[0169] The single-stranded oligonucleotide primer will contain or consist of a unit sequence that will be repeated in the concatemer generated using the method described herein. The concatemer repeat element is represented as "a" in Figure 1A. In fact, the actual concatemer repeat sequence will be determined by the sequence of the single-stranded extension template described herein. That is, the oligonucleotide primer coupled to the target binding ligand may include a 5' sequence in addition to the sequence containing the concatemer repeat sequence, but the 3' part of the original oligonucleotide primer that is complementary to the sequence repeated by the head-to-tail directional connection in the single-stranded extension template will determine the repeat sequence. For example, this can be illustrated by reference to Figure 1A, which shows an oligonucleotide primer "a" and an extension "a*a*", where a* is a complementary sequence to a. The 5' end of the primer optionally includes an additional sequence (not shown) that is not necessarily complementary to a*, which is essentially a linker and will not be replicated in the concatemer according to the a* copy of the extension template. The single-stranded extension template will be discussed in more detail below and demonstrated in the examples.

[0170] As discussed further below, the advantages of the methods, compositions and kits described herein are that concatemer-labeled target binding ligands can be generated in a multiplex manner (and ultimately detected). The length of the concatemer repeat sequence directly affects the multiplex degree that can be achieved in the methods, compositions and kits described herein. Generally, the longer the repeat unit, the greater the multiplex degree that can be achieved using an orthogonal primer of this length. For example, for a repeat unit length of 9 nucleotides, there may be up to 93 orthogonal primers. For a repeat unit length of 10nt, the maximum number of primers increases to 170. For a repeat unit length of 11nt, the maximum value is 315. For 12nt, the maximum value is 585. For 13nt, the maximum value is 1092. For 14nt, the maximum value is 2048, and for 15nt, the maximum value is 3855. For multiple applications, it is preferred but not absolutely required that the concatemer repeat unit length on each target binding ligand is the same. Similar repeat unit lengths can provide similar efficiencies for the cycles of extension body annealing and primer extension.

[0171] In one embodiment, the target molecule itself can be an RNA molecule, including but not limited to an mRNA molecule. The hybridization of one or more single-stranded oligonucleotides comprising a 5' region complementary to the sequence on the target mRNA and a 3' region comprising at least one sequence to be extended to a concatemer provides a starting site for producing concatemers using the single-stranded extension template oligonucleotide described herein. In this way, nucleic acids comprising 5' and 3' regions of this type can be used as target binding ligand molecules used herein. When the target molecule is RNA, the single-stranded target binding ligand oligonucleotide can be hybridized with its RNA target in situ, and then cross-linked to maintain its binding to the target RNA through subsequent concatemer generation steps. For example, cross-linking can be achieved by incorporating a photocrosslinker described herein or known in the art into a single-stranded target binding ligand oligonucleotide (preferably only incorporating the oligonucleotide portion hybridized with the target RNA). After hybridization and removal of unhybridized oligonucleotides, the sample is irradiated with ultraviolet light to cross-link the oligonucleotides with their RNA targets.

[0172] In one embodiment, the target molecule itself can be a DNA molecule, or a sequence on such a molecule, including but not limited to a chromosome or free DNA sequence. The hybridization of one or more single-stranded oligonucleotides provides a starting site for producing concatemers using the extended oligonucleotides described herein, and the single-stranded oligonucleotides include a 5' region complementary to the sequence on the target DNA and a 3' region including at least a sequence to be extended to a concatemer. In this way, nucleic acids comprising 5' and 3' regions of this type can be used as target binding ligand molecules used herein. In the case where the target molecule is a DNA molecule of a given sequence, the single-stranded target binding ligand oligonucleotide can be hybridized in situ to its DNA target by a target complementary sequence located at 5' of the 3' sequence, extended as a concatenated repeat element, and then cross-linked (after removing unhybridized oligonucleotides), such as known in the art or described herein, so as to maintain its binding to the target DNA by the subsequent steps of concatemer generation.

[0173] As previously described, an important advantage of the methods, compositions, and kits described herein is the ability to perform a concatemer generation step in a multiplex, so that a variety of different target binding ligands have different concatemer probe landing pads generated in situ in a multiplex. For example, as shown in FIG1B , an orthogonal primer having a repeating sequence "a", "b" ... "n" is extended using a single-stranded extension template, wherein the single-stranded extension template has at least two repeats or copies of a complementary sequence "a*", "b*", ... "n*". Once generated, different concatemers provide landing pads for orthogonal complementary probes, which can also detect various target ligands in multiplexes. Various methods for detecting and distinguishing probes in multiplexes are known in the art and / or discussed elsewhere herein.

[0174] As described herein, the target binding ligand with oligonucleotide primers can be contacted with the cell or tissue sample preparation according to methods known in the art. For example, when the target binding ligand is an antibody or its antigen binding fragment, the sample can be stained using a method widely used in immunohistochemistry to detect a given target ligand. Therefore, the method for contacting the cell or tissue sample with the target binding ligand can be parallel to the method for, for example, fluorescently labeled antibodies or antibody fragments. When performed in a multiplex manner, contact or staining can include adding a set of target binding ligands, each ligand comprising different oligonucleotide primers. In the case where the target binding ligand is not an antibody or its antigen binding fragment, the method for contacting the preparation of the cell or tissue with the target binding ligand can be applicable to a given ligand known in the art. In particular, when the target molecule is a DNA or RNA molecule comprising a given sequence, conditions such as for in situ hybridization can be used to allow the target binding ligand oligonucleotide to bind to the target sequence. In some embodiments, it may be beneficial to remove unbound target binding ligands before subsequent concatemer generation or detection steps. For example, this can be accomplished by removing a solution containing the target binding ligand from the cell or tissue sample, followed by one or more wash steps in an appropriate solution lacking the target binding ligand.

[0175] In situ generation of concatemers

[0176] In one embodiment, described herein is a method for labeling a target binding ligand with a concatemer that provides sites for binding of multiple copies of a labeled nucleic acid probe that is complementary to a concatemer repeat sequence. The generation of concatemers on a target binding ligand is described below and can be performed in situ, for example, in a cell or tissue sample where the target binding ligand is bound to its target molecule. These methods typically use an oligonucleotide bound to a target binding ligand and a single-stranded extension template, and repeat cycles of annealing, polymerase extension, and hot strand separation, wherein each cycle adds another concatemer repeat unit to the oligonucleotide bound to the target binding ligand. In one embodiment, this method of labeling a target binding ligand includes (a) providing a conjugate of a target binding ligand and a first oligonucleotide primer; (b) contacting the conjugated first oligonucleotide primer with a first single-stranded extension template and a nucleic acid polymerase in a reaction mixture under conditions that allow hybridization and extension of the first oligonucleotide primer using the first single-stranded extension template; (c) heating the reaction mixture of step (b) to separate the first single-stranded extension template from the extended first oligonucleotide primer produced in step (b); and (d) repeating steps (b) and (c) at least once, thereby producing a concatemer comprising repeating units of the oligonucleotide primer sequence, which is bound to the target binding ligand.

[0177] The conjugates of target binding ligands and oligonucleotide primers are discussed above. The exact composition of the reaction mixture effective for template-mediated primer extension will depend on the specific polymerase used, such as the temperature of the extension reaction, in terms of extension template and nucleotide concentrations, buffers, salts, divalent cations and other cofactors and their concentrations. Those of ordinary skill in the art can adjust these parameters for a given polymerase.

[0178] The melting temperature, Tm, of a nucleic acid duplex is a measure of the thermal stability of the duplex and is defined as the temperature at which half of the DNA strands are in a single-stranded or dissociated state. The Tm of any given nucleic acid duplex is determined by parameters including its length, nucleotide composition, nucleic acid concentration, and salt conditions in a given reaction. The empirical formula for calculating the Tm of short oligonucleotides is Tm = 2x(A+T) + 4x(G+C), but other formulas can provide more accurate estimates, such as Tm = 81.5 + 0.41(%G+C) - 675 / N, where N is the total number of bases. The optimal annealing temperature for a primer extension reaction can be determined empirically by one of ordinary skill in the art, but for a given primer / template duplex, it will usually be about 5°C lower than the Tm. When performed in a multiplex format, the annealing temperature is preferably about 5°C lower than the Tm for the primer / template duplex with the lowest Tm, but the primers should be designed to minimize Tm differences. Primer design for multiplex primer extension reactions is well established in the art and takes into account other parameters including, but not limited to, minimizing the formation of primer dimers (where one primer has sufficient complementarity with another primer in the same reaction to allow extension using the other primer as a template) and minimizing off-target extension or amplification. Software tools for multiplex primer design are known in the art, including, for example, OligoPerfect™ Primer Designer available from ThermoFisher Scientific, and the like.

[0179] Therefore, in a given concatemer generation scheme, in a reaction mixture suitable for the polymerase used, the oligonucleotide primer coupled to the target binding ligand is contacted in situ with the single-stranded extension template oligonucleotide at a temperature, which is determined, for example, based on the Tm of the primer-template hybridization double strand. This allows the single-stranded extension template to be annealed with the primer, and when incubated at the extension temperature of the polymerase, the annealed primer is extended by the selected polymerase. Heat is applied to separate the extended chain from the single-stranded extension template, cool to allow the single-stranded extension template to anneal, and incubate at the extension temperature of the polymerase, thereby producing a concatemer with a repeat of the oligonucleotide primer sequence defined by the single-stranded extension template. When performed in a multiplex manner, concatemers of different orthogonal repeat sequences on different target binding ligands are produced, and these ligands are in situ bound to respective targets. Subsequent detection based on distinguishable labeled probes hybridized with concatemers allows in situ detection, quantification and / or imaging of target molecules.

[0180] Single strand extension template

[0181] The use of single-stranded extension templates is central to the methods, compositions, and kits described herein. Single-stranded extension templates useful for the methods, compositions, and kits described herein comprise a concatemer of at least two copies of a sequence linked head-to-tail, the concatemer being bound to a target binding ligand, the sequence being complementary to an oligonucleotide primer. The single-stranded extension template also includes a 3' blocking or chain termination modification so that the single-stranded extension template itself cannot be used to generate polymerase extension of the concatemer. The design of the single-stranded extension template provides a method in which one copy of the concatemer repeat sequence complementary sequence hybridizes to an oligonucleotide bound to a target binding ligand, and at least one additional copy of the concatemer repeat sequence complementary sequence provides a template for the extension of an oligonucleotide bound to a target ligand. As a representative illustration, please refer again to Figure 1A (extension a*a* with a 3' "stopper" or chain terminator). Because the single-stranded extension template must include a sequence complementary to the desired repeat sequence of the concatemer to be generated, the sequence of the single-stranded extension template is determined by the desired repeat sequence. As discussed above with respect to primer design, the sequence of the desired concatemer repeat unit is determined by parameters that allow multiplex target detection while minimizing primer interactions and off-target hybridization.

[0182] In a multiplex experiment, a set of different single-stranded extension templates can be used, orthogonal to a set of oligonucleotide primers, which are coupled to corresponding members of a set of different target binding ligands. As with the single-stranded extension templates used alone, each single-stranded extension template in a set comprises a concatemer of at least two copy sequences connected head-to-tail, the concatemer being bound to the target binding ligand, the sequence being complementary to different oligonucleotide primers, for example, as described herein. Each single-stranded extension template in the set also includes a 3' blocking or chain termination modification, which prevents polymerase extension of the extension template. For example, as shown in FIG. 1B , this multiplex approach (including extensions of a*a*, b*b*, and n*n*, each having a 3' terminator (stoppers)) produces concatemers with different repeat unit sequences on each member of the target binding ligand set.

[0183] The polymerase used to extend the oligonucleotide primer and generate the concatemer can be any of a variety of template-dependent nucleic acid polymerases. In one embodiment, the polymerase is thermostable so that it can withstand heating to a temperature and time sufficient to denature or dissociate the double-stranded nucleic acid, and maintains template-dependent polymerization activity when the reaction mixture is cooled to a temperature that allows the extension template to anneal to the oligonucleotide primer and primer extension. Different thermostable polymerases have different reaction buffers and extension temperature optima; these parameters are known to those of ordinary skill in the art and / or described in the product literature for a given polymerase. Non-limiting examples of thermostable polymerases that can be used in the methods, compositions, and kits described herein include the following. Examples of polymerases that can be used in the methods described herein include, but are not limited to: standard Taq DNA polymerase (Cat. No. 10342053, Invitrogen, Carlsbad, CA), Platinum II Taq Hot Start DNA Polymerase (Cat. No. 14966001, Invitrogen, Carlsbad, CA), Platinum SuperFi II DNA Polymerase (Cat. No. 12361010, Invitrogen, Carlsbad, CA), USB™ CycleSeq™ Thermostable DNA Polymerase (Cat. No. 792001000UN, Applied Biosystems, Waltham, MA); Taq DNA polymerase (Cat. No. EP0402, ThermoScientific, Waltham, MA); HoTaq DNA polymerase (HT-200, McLab, San Francisco, CA); I-5 Hi-Fi DNA polymerase (PDP-100, McLab, San Francisco, California); I-5 hot start DNA polymerase (I5HD-100, McLab, San Francisco, California); DNA polymerase, Thermotoga neoapollogenes (DPTN-100, McLab, San Francisco, California); Pfu DNA polymerase (AD-200, McLab, San Francisco, California); Pfu DNA polymerase (Cat. No. 600135, Agilent Technologies, Wood Dale, Illinois); PfuTurbo DNA polymerase (Cat. No. 600252, Agilent Technologies, Wood Dale, Illinois), etc. One of ordinary skill in the art can identify other polymerases that work in the methods, compositions, and kits described herein, and can adjust the reaction conditions as needed for any given polymerase.

[0184] Branched chain concatemer

[0185] One option for further increasing the number of probe binding sites on the target binding ligand is to introduce branches so that the initial linear concatemer of the target binding ligand provides sites to generate additional concatemers that branch off the initial linear concatemer. It is important to note that in some embodiments, branching can be performed multiple times, using earlier branches as sites for generating additional concatemers, and each round of branching increases the number of concatemer repeat units, thereby increasing the number of potential probe binding sites on a given target binding ligand. In some embodiments, branches are generated in situ on linear concatemers generated as described herein. In other embodiments, preformed concatemers are added to a reaction mixture that hybridizes to repeat sequences in the concatemers on the target ligand. These methods will be discussed further below.

[0186] In one embodiment, to generate branched concatemers in situ on the target binding ligand, a second oligonucleotide primer and a second single-stranded extension template are added to the reaction mixture after the linear concatemers are generated in situ as described herein. The considerations for designing the second oligonucleotide primers for single and multiple branched concatemer generation are the same as those for designing the first oligonucleotide primers in terms of avoiding crosstalk, primer dimers and off-target hybridization. As with the first oligonucleotide primer, any of a number of different software packages can assist in this design.

[0187] In this embodiment, the second oligonucleotide primer comprises a sequence complementary to the first oligonucleotide primer and a sequence complementary to the second single-stranded extension template in a 5' to 3' order, wherein the second single-stranded extension template comprises a 3' chain terminator. The second oligonucleotide primer molecule is allowed to anneal, via its sequence complementary to the first oligonucleotide primer, to at least one, preferably a plurality of, concatemer repeat units of the first extended concatemer oligonucleotide coupled to the target binding ligand.

[0188] The sequence complementary to the first oligonucleotide primer may include a (photo) cross-linking moiety, such that after annealing, irradiation or other appropriate treatment of the reaction mixture, the second oligonucleotide primer will be cross-linked to the extended first oligonucleotide primer, such that the second oligonucleotide primer remains bound to the extended first oligonucleotide primer during a subsequent hot strand separation step.

[0189] For concatemer generation, a second single-stranded extension template is ligated to a complementary sequence on a second oligonucleotide primer, and the second oligonucleotide primer is extended, thereby adding new copies of the second concatemer repeat unit to new branches of the first concatemer. Repeated cycles of hot strand separation and second single-stranded template-directed primer extension produce concatemers containing a repeat sequence that includes the second oligonucleotide primer sequence. When multiple second oligonucleotide primers hybridize to multiple concatemer repeat units on the first extended concatemer oligonucleotide, the first extended oligonucleotide actually provides a trunk with many branches, each branch including multiple repeat units of the second concatemer repeat sequence. Additional branching cycles using a third, fourth, fifth, or more oligonucleotide primers and their orthogonal single-stranded extension templates (similar in design to the second oligonucleotide primer / second single-stranded extension template) can further amplify the number of probe binding sites associated with a given target binding ligand.

[0190] Another branching method produces concatemers of a second repeating sequence, which contains a sequence complementary to the first concatemer repeating unit at its 5' end. These second concatemer molecules are then allowed to hybridize with the repeating sequence in the concatemer connected to the first target binding ligand through the complementary sequence to provide branched concatemers. The sequence complementary to the first concatemer repeating unit may include a (photo) cross-linking portion that allows the second concatemer to be cross-linked to the first concatemer. It is expected that the second concatemer molecule itself may include branches with a third, fourth, fifth or more repeating elements to further amplify the number of probe binding sites connected to a given target binding ligand.

[0191] Any of the alternative branching approaches described above can be used in the multiplex reactions described herein.

[0192] Ordered extension and nucleic acid generation

[0193] In another aspect, the single-stranded extension template method can allow the generation of nucleic acids having a desired sequence or set of ordered sequence elements. In this regard, a set of different single-stranded extension elements can be used, each of which includes a sequence complementary to a previous extension element and a new extension element template. Repeated cycles of hot strand separation and extension using continuous single-stranded extension templates allow the generation of nucleic acids having a desired sequence or set of ordered sequence elements.

[0194] In one embodiment, such as shown in FIG. 1C , an ordered extension method for producing a nucleic acid molecule of a desired sequence includes providing a first oligonucleotide primer and contacting the first oligonucleotide primer with a first single-stranded extension template and a nucleic acid polymerase in a reaction mixture under conditions that allow the first oligonucleotide primer to hybridize and extend using a first single-stranded extension template. The polymerase can be a thermostable polymerase, such as a polymerase described herein or known in the art. The first single-stranded extension template comprises a first extension template element, a sequence complementary to the first oligonucleotide primer, and a 3' chain terminator in a 5' to 3' direction or orientation. This step extends the first oligonucleotide primer by complementation of the first extension template element. It is noteworthy that this ordered extension method can add continuous single-stranded extension templates in continuous steps, or, in another case, a whole set of single-stranded extension templates can be added at once, and an ordered extension is established in a single pot reaction by continuous thermal cycling, which is based on the specific hybridization of the appropriate single-stranded extension body with each continuously added extension body.

[0195] After the extension step, the mixture is heated to separate the strands, and under conditions that allow the use of the second single-stranded extension template to hybridize and extend the first extension oligonucleotide primer, the second single-stranded extension template is contacted with the extension oligonucleotide primer produced in the first extension step. The second single-stranded extension template includes a second extension template element, a copy of the first extension template element, and a 3' chain terminator in a 5' to 3' direction or orientation. This step adds a sequence complementary to the second extension template element to the first extension oligonucleotide primer. Chain separation and contact with the continuous single-stranded extension template can be performed in successive rounds, each of which includes a new extension template element in a 5' to 3' direction or orientation, a copy of the previous extension template element, and a 3' chain terminator. The design of the continuous single-stranded extension template allows the assembly of nucleic acid molecules of substantially any desired sequence, which are composed of sequence elements complementary to the continuous extension template element. In other words, the ordered extension method can be used to produce nucleic acid molecules that sequentially include the first oligonucleotide primer sequence, followed by the complementary sequence of each corresponding extension template element. If desired, this approach can be used to barcode a surface, target binding ligand, or other moiety to which the first oligonucleotide is coupled, among other uses.

[0196] If desired, the ordered extension method can include a single-stranded extension template as described above, for the generation of concatemers for the reaction, so that one or more extension elements are repeated in concatemers in the generated nucleic acid molecules. This can be done before or after one or more rounds of single-stranded extension template switching, which introduces new sequence elements into the growing extension oligonucleotide. Therefore, the repeating units added in this way can provide signal amplification as described herein.

[0197] Ordered extension methods can also be performed in multiplexes. Primer design considerations are similar to those described herein for designing primers for multiplex concatemer generation protocols.

[0198] The ordered extension method can be performed with a first oligonucleotide primer in solution, or on the surface of the nucleic acid molecule to be constructed or coupled thereto. The method can also be performed wherein the oligonucleotide primer is coupled to a target binding ligand as described herein. The method can also be performed in contact with a cell or tissue sample so that the nucleic acid molecule is combined with the cell or tissue sample to be generated in situ, for example, to a target molecule in a cell or tissue sample that is bound by a target binding ligand.

[0199] The ordered extension method may further comprise contacting the generated nucleic acid molecules with a labeled nucleic acid probe comprising one or more extension template element sequences. Detection of the label attached to the probe may provide information on the presence, amount and / or location of the target attached to the first oligonucleotide primer.

[0200] In another approach, a method is also considered to add a long DNA chain and extend it only once, rather than performing multiple rounds of thermal cycling with short DNA chains. The long DNA chain extended in one step is a variation of the schematic of Figure 1C - considering a2* as a repetition of imaging probe binding (imagerbinding). For the one-step extension scheme, instead of using heat to melt the long DNA chain to allow imaging probe binding and CNVK cross-linking, uracil bases are incorporated into the long DNA chain and digested with uracil DNA glycosidase (e.g., uracil DNA glycosidase; M0280S, New England Biolabs), and the long DNA chain is fragmented after one-step extension to allow imaging probe binding and CNVQ cross-linking.

[0201] In situ detection of target molecules

[0202] Among other uses, the compositions, methods and kits described herein can be used to detect target molecules in situ. Once a concatemer sequence is generated on a target binding ligand that binds to its target in situ, a labeled nucleic acid probe (or a set of probes when performing multiplex detection) can be contacted with and hybridized to the concatemer repeat sequence of the target binding ligand. The label attached to the target binding ligand in this manner can then be detected in a manner appropriate for a given label.

[0203] If multiple targets are detected in a single assay, the labels on a single probe should be distinguishable from other labels used in the same assay. For example, if the labels are fluorescent, they should have excitation and / or emission spectra that allow the user to distinguish them in the same assay. It is contemplated that repeated detections of different fluorophores with non-overlapping excitation spectra could be performed using different wavelengths. Similarly, detection of non-overlapping emission spectra of different fluorophores could be used to detect multiple different fluorophores associated with multiple different targets. It is also contemplated that a first round of fluorescently labeled probes could be applied and detected, followed by photobleaching of the fluorophores, and then application of a second round of different probes labeled with one or more of the same fluorophores as the first round.

[0204] When mass cytometry is used for detection, each lanthanide metal can be distinguished by time-of-flight mass spectrometry, so that probes labeled with different metal ions can be multiplexed to detect different target molecules in the same assay.

[0205] Given that concatemeric repeat sequences on target binding ligands provide a high degree of signal amplification, detection down to single-molecule sensitivity can be achieved in multiplexed assays using the methods described herein.

[0206] Mass cytometry:

[0207] Mass cytometry, also known as time-of-flight mass cytometry Provides a tool for high-dimensional and high-throughput single-cell analysis. Mass spectrometry flow cytometry was first introduced in 2009 (Bandura et al., Anal. Chem. 81: 6813-6822 (2009)), and due to its high parameter capabilities, has been widely used in the analysis of immune cell function / activation and other processes. By using metal ion labels instead of fluorescent labels commonly used in fluorescence-based flow cytometry, mass spectrometry flow cytometry overcomes the problem of overlapping emission spectra, thereby reducing the number of different targets or parameters that can be analyzed in a single assay.

[0208] For example, Iyer et al. (Front. Immunol. 13, Article 815828 (2022)) reviewed the methods, applications, and precautions for performing mass cytometry. In short, in mass cytometry, cells are typically incubated with a mixture of antibodies labeled with unique non-radioactive heavy metal (most commonly lanthanide) isotopes. A single cell suspension is atomized in such a way that each droplet contains a single cell. The individual cells pass through an argon (Ar) plasma to atomize and ionize the sample. This process converts each cell into a cloud containing elemental ions present in or on the cell. High-pass optics (quadrupoles) remove low-mass (mainly biological) ions (i.e., ions with a mass below 75Da) from each cloud, thereby forming a cloud containing only ions from isotope-coupled probes. In the time-of-flight (TOF) chamber, ions are separated by mass-to-charge ratio. Upon encountering the detector, the ion count is amplified and converted into an electrical signal. In theory, many more parameters corresponding to different targets are possible, but in current practice, approximately 60 different parameters can be distinguished in a mass cytometry panel. When adapted for use with the concatemer-based labeling / signal amplification approach described herein, highly multiplexed, high-sensitivity detection of a large number of even low-abundance targets is possible.

[0209] As described above, the signal amplification method based on concatemers described herein can be easily applied to other biomolecular target detection or analysis methods. In addition to flow cytometry, mass spectrometry flow cytometry and, for example, protein blotting, non-limiting examples include, but are not limited to, imaging mass spectrometry flow cytometry methods (see, for example, Giesen et al., Nat. Methods 11: 417-422 (2014), see also Hyperion + TM imaging system (Fluidigm Co., Ltd.; see World Wide Web fluidigm.com / products-services / technologies / imaging-mass-cytometry), multiple ion beam imaging (MIBI; see, for example, Angelo et al., Nat. Med. 20: 436-442 (2014)) and time-of-flight multiple ion beam imaging (MIBI-TOF; see, for example, Keren et al., Sci. Adv. 5: eaax5851 (2019)) etc.

[0210] It is noteworthy that the concatemer-based methods and compositions described herein, as well as the cross-linking of nucleic acids to which probes are attached to target binding ligands, can be applied to or combined with other methods for detecting target molecules. For example, the concatemer methods described herein can be used to further amplify RNAScope TM(See Wang, H. et al. (2015). Multiplex Fuorescent RNA In Situ Hybridization Via RNAscope. In: Hauptmann, G. (eds) In Situ Hybridization Methods. Neuromethods, vol 99 (2015). Humana Press, New York, NY). Another alternative is applicable to branched DNA FISH or InSituPlex TM (Ultivue) imaging. It should also be noted that the crosslinking methods described herein, including but not limited to CNVK-mediated crosslinking, can be applied to these and other methods of crosslinking labeled probes to single or repeated nucleic acid sequences attached to target molecules or target binding ligand molecules. In this method, the probes described herein are bound to and bound to a given target RNA species (e.g., in RNAScope TM Nucleic acid crosslinking (e.g., using CNVK or other crosslinking agents described herein coupled to probes) to a molecule that is hybridized or bound (e.g., in branched DNA FISH) can be used to maintain the binding of the probe to the nucleotides to which the molecule is attached.

[0211] In addition to the concatemer generation methods described above and demonstrated in the embodiments herein, other repetitive generation methods can also be used to generate multiple probe binding sites on the target molecule. Rolling circle amplification is a non-limiting example of this additional method for generating multiple probe binding sites on the target binding ligand (see, for example, Mohsen, MG et al. The Discovery of Rolling Circle Amplification and Rolling Circle Transcription. Acc. Chem. Res. 49: 2540-2550 (2016)). This method can also be combined with cross-linking as described herein, including but not limited to CNVK-mediated cross-linking, to attach probe molecules to repeat sequences in a stable manner, otherwise nucleic acid probes may be dissociated. In the case of providing an advantage, CNVK-mediated cross-linking can also be reversed (e.g., irradiated with a separate wavelength).

[0212] Signal amplification by exchange reaction (SABER; see, e.g., Treangen TJ et al., Repetitive DNA and next-generation sequencing: computational challenges and solutions. Nat Rev Genet. 13: 36-46 (2011)) can also be adjusted in a similar manner by using CNVK cross-linking chemistry. For example, long repeat sequences generated according to the SABER method can be hybridized and CNVK cross-linked with oligonucleotides on a target binding ligand (e.g., an antibody or other target binding ligand), and nucleic acid probe molecules can be hybridized to these repeat sequences and similarly CNVK cross-linked to provide signal amplification (e.g., in a manner such that the repeat sequence and the probe do not dissociate from the target in a subsequent step). In the case of generating repeat sequences, the same method can be applied, for example, by hybridization chain reaction (HCR; see, Hybridization chain reaction: a versatile molecular tool for biosensing, bioimaging, and biomedicine. Chem. Soc. Rev. 46: 4281-4298 (2017)).

[0213] In a further modification, the concatemer approach as described above and demonstrated in the examples can be adapted to use click chemistry, rather than CNVK cross-linking, to covalently bind, for example, repeat-containing nucleic acids to a target binding ligand and / or covalently bind nucleic acid probe molecules to repeats on the target binding ligand to amplify the signal; for example, see ClampFISH (Tavakoli, S. et al., Chapter Twenty: Click-chemistry-based amplification and detection of endogenous RNA and DNA molecules in situ using clampFISH probes. 641: 459-476 (2020)).

[0214] Reagent test kit

[0215] In various embodiments, a kit for performing one or more methods described herein is provided. Such a kit may include, for example, a first oligonucleotide primer or a first oligonucleotide primer set as described herein, and a single-stranded extension template or a single-stranded extension template set, suitable for performing the concatemer generation method and / or ordered extension or nucleic acid molecule / sequence assembly described herein. The kit may include packaging materials for various components, such as instructions for use.

[0216] The kit can include, for example, an orthogonal set of a first oligonucleotide primer and a single-stranded extension template, allowing for use in multiplexing. This orthogonal set can be optimized to avoid primer / extension crosstalk, primer dimer formation, and off-target hybridization in multiple labeling and / or detection reactions. The kit can also include one or more target binding ligand molecules and / or reagents for coupling the first oligonucleotide to the target binding ligand. The kit can also include a polymerase, such as a thermostable polymerase described herein or known in the art, and nucleotides and reaction / buffer components suitable for primer extension using a given polymerase. The kit can also further include one or more labeled nucleic acid probe molecules and / or reagents for labeling nucleic acid probe molecules. The probe molecule can be complementary to, for example, a multiple repeat element, or complementary to one or more elements in an ordered extension product generated as described herein. The label can include any label described herein or known in the art, including but not limited to fluorescent labels, metal ion labels, etc.

[0217] It should be understood that the present disclosure is not limited to the specific methods, protocols and reagents provided herein, etc., and therefore may vary. The terms used herein are only used to describe specific embodiments and are not intended to limit the scope of the present disclosure, which is defined only by the claims. The present invention is further illustrated by the following examples, which should not be construed as further limitations.

[0218] The technology may be as described in any one of the following numbered embodiments:

[0219] Embodiment 1: A method for labeling a target binding ligand, the method comprising: (A) providing a conjugate of the target binding ligand and a first oligonucleotide primer; (b) in a reaction mixture, under conditions that allow the first oligonucleotide primer to hybridize and extend using the first single-stranded extension template, contacting the conjugated first oligonucleotide primer with a first single-stranded extension template and a nucleic acid polymerase; (c) heating the reaction mixture of step (b) to separate the first single-stranded extension template from the extended first oligonucleotide primer produced in step (b); (d) repeating steps (b) and (c) at least once, thereby producing a concatemer comprising repeating units of an oligonucleotide primer sequence coupled to a target binding ligand.

[0220] Embodiment 2: The method of embodiment 1, wherein the first single-stranded extension template comprises a concatemer of at least two copies of the sequence linked head-to-tail and a 3' chain terminator so that the extension template is not extended by a polymerase, and the sequence is complementary to the first oligonucleotide primer.

[0221] Embodiment 3: The method of embodiment 1 or embodiment 2, after at least one repetition of steps (b)-(d), further includes the following steps: (i) adding a second oligonucleotide primer and a second single-stranded extension template oligonucleotide, wherein the second oligonucleotide primer comprises a sequence complementary to the first oligonucleotide primer and a sequence complementary to the second single-stranded extension template in 5' to 3' order; (ii) heating the reaction mixture to separate the nucleic acid chains and cooling to allow the second oligonucleotide primer to hybridize with the concatemers produced in steps (b)-(d) and to allow the second single-stranded extension template to hybridize with the second oligonucleotide primer; (iii) extending the second oligonucleotide primer using the second single-stranded extension template oligonucleotide as a template; and (iv) repeating steps (ii) and (iii) at least once, thereby producing a concatemer comprising a repeating unit of the second oligonucleotide primer.

[0222] Embodiment 4: The method of embodiment 1 or embodiment 2 further comprises, after step (d): (i) adding nucleic acid under conditions allowing hybridization, wherein the nucleic acid comprises: (1) a second multimer composed of repeating units of a second oligonucleotide sequence; and (2) a sequence complementary to the first oligonucleotide primer, so that multiple molecules of the nucleic acid comprising the second multimer hybridize with the multimer formed in step (d) through the sequence complementary to the first oligonucleotide primer.

[0223] Embodiment 5: The method of any one of Embodiments 1-4, wherein the method is performed in situ on a cell or tissue sample containing the target ligand or for detecting the target ligand.

[0224] Embodiment 6: The method of Embodiment 5, wherein the cell or tissue sample is fixed.

[0225] Embodiment 7: The method of Embodiment 5 or Embodiment 6, wherein the tissue sample is paraffin-embedded.

[0226] Embodiment 8: The method of any one of Embodiments 1-7, further comprising contacting the concatemers produced in the previous step with a labeled nucleic acid probe.

[0227] Embodiment 9: The method of Embodiment 8, wherein the nucleic acid probe comprises a sequence complementary to the concatemer repeat unit.

[0228] Embodiment 10: The method of Embodiment 8 or Embodiment 9, wherein the nucleic acid probe comprises a sequence complementary to the first oligonucleotide primer.

[0229] Embodiment 11: The method of any one of Embodiments 3 or 5-7 further comprises contacting the concatemer consisting of the second oligonucleotide primer repeating units with a labeled nucleic acid probe, wherein the nucleic acid probe comprises a sequence complementary to the second oligonucleotide primer.

[0230] Embodiment 12: The method of any one of Embodiments 4 or 5-7, further comprising contacting the concatemer consisting of repeating units of the second oligonucleotide sequence with a labeled nucleic acid probe, wherein the nucleic acid probe comprises a sequence complementary to the second oligonucleotide primer.

[0231] Embodiment 13: The method of any one of Embodiments 8-12, wherein a plurality of labeled nucleic acid probe molecules hybridize to a plurality of concatemer repeat units.

[0232] Embodiment 14: The method of any one of Embodiments 1-8, wherein the target binding ligand comprises an antibody or an antigen-binding fragment thereof.

[0233] Embodiment 15: The method of any one of Embodiments 1-14, wherein a plurality of different target binding ligands are multiplexed labeled by different orthogonal oligonucleotide primer / single-stranded extension template pairs.

[0234] Embodiment 16: A method for in situ detection of a target molecule in a preparation of cells or tissues, the method comprising: (a) contacting the preparation of cells or tissues with a target binding ligand coupled to a first oligonucleotide primer under conditions that allow the target binding ligand to specifically bind to the target molecule; (b) adding a reaction mixture comprising a first single-stranded extension template and a nucleic acid polymerase to the preparation of cells or tissues under conditions that allow the first oligonucleotide primer to hybridize and extend using the first single-stranded extension template; (c) heating the reaction mixture of step (b) to separate the first single-stranded extension template from the extended first oligonucleotide primer produced in step (b); (d) repeating steps (b) and (c) at least once, thereby producing a concatemer comprising repeats of the first oligonucleotide primer sequence coupled to the target binding ligand; (e) contacting the concatemer produced in step (d) with a labeled nucleic acid probe; and (f) detecting the labeled nucleic acid probe, wherein detection of the labeled nucleic acid probe indicates the presence and location of the target molecule in the preparation of cells or tissues.

[0235] Embodiment 17: The method of embodiment 16, wherein the first single-stranded extension template comprises a concatemer of at least two copy sequences connected in a head-to-tail direction and a 3' chain terminator, wherein the 3' chain terminator prevents the extension template from being extended by a polymerase, and the sequence is complementary to the first oligonucleotide primer.

[0236] Embodiment 18: The method of Embodiment 16 or Embodiment 17, wherein the target binding ligand comprises an antibody or an antigen-binding fragment thereof.

[0237] Embodiment 19: The method of any one of Embodiments 16-18, wherein the labeled nucleic acid probe comprises a sequence complementary to the first oligonucleotide primer.

[0238] Embodiment 20: The method of any one of Embodiments 16-19, wherein the labeled nucleic acid probe comprises a sequence complementary to the concatemer repeat unit.

[0239] Embodiment 21: The method of any one of Embodiments 16-20, wherein a plurality of labeled nucleic acid probe molecules are hybridized to a plurality of concatemer repeat units that bind to a target binding ligand.

[0240] Embodiment 22: The method of any one of claims 16-18, after step (d) and before step (e), further comprising the steps of: (i) adding a second oligonucleotide primer and a second single-stranded extension template oligonucleotide, wherein the second oligonucleotide primer comprises a sequence complementary to the first oligonucleotide primer and a sequence complementary to the second single-stranded extension template in 5' to 3' order, wherein the second single-stranded extension template comprises a 3' chain terminator; (ii) heating the reaction mixture to separate the nucleic acid chains and cooling to allow the second oligonucleotide primer to hybridize with the concatemer produced in step (d) and to allow the second single-stranded extension template to hybridize with the second oligonucleotide primer; (iii) extending the second oligonucleotide primer using the second single-stranded extension template oligonucleotide as a template; and (iv) repeating steps (ii) and (iii) at least once, thereby producing a concatemer comprising a second oligonucleotide primer repeating unit.

[0241] Embodiment 23: The method of Embodiment 22, wherein the labeled nucleic acid probe comprises a sequence complementary to the second oligonucleotide primer.

[0242] Embodiment 24: The method of embodiment 22 or 23, wherein a plurality of labeled nucleic acid probe molecules are hybridized to a plurality of concatemer repeat units that bind to a target binding ligand.

[0243] Embodiment 25: The method described in any one of Embodiments 16-18 further comprises, after step (d) and before step (e): (i) adding a nucleic acid under conditions permitting hybridization, wherein the nucleic acid comprises: (1) a second multimer composed of repeating units of a second oligonucleotide sequence; and (2) a sequence complementary to the first oligonucleotide primer, so that multiple molecules of the nucleic acid comprising the second multimer hybridize with the multimer comprising repeating units of the first oligonucleotide primer sequence formed in step (d) through a sequence complementary to the first oligonucleotide primer.

[0244] Embodiment 26: The method of Embodiment 25, wherein the labeled nucleic acid probe comprises a sequence complementary to the second oligonucleotide sequence.

[0245] Embodiment 27: The method of Embodiment 25 or 26, wherein a plurality of labeled nucleic acid probe molecules are hybridized to a plurality of second concatemer repeat units.

[0246] Embodiment 28: The method of any one of Embodiments 16-27, wherein the preparation of cells or tissue is fixed.

[0247] Embodiment 29: The method of Embodiment 28, wherein the tissue preparation is paraffin embedded.

[0248] Embodiment 30: A method for labeling a group of target binding ligands, the method comprising: (a) providing an orthogonal first oligonucleotide primer and a first single-stranded extension template oligonucleotide pair group, wherein the first oligonucleotide primer of each pair has a different sequence from other first oligonucleotide primers in the group and is coupled to different members of the target binding ligands of the group; (b) in a reaction mixture, under conditions allowing the first single-stranded extension template of the orthogonal pair group to hybridize and extend, the coupled first oligonucleotide primer is contacted with the first single-stranded extension template of the orthogonal pair group and a nucleic acid polymerase; (c) heating the reaction mixture of step (b) to separate the first single-stranded extension template from the extended first oligonucleotide primer produced in step (b); (d) repeating steps (b) and (c) at least once, thereby generating a concatemer on each member of the group of target ligands, the concatemer comprising a repeating unit of the oligonucleotide primer sequence coupled to the target binding ligand.

[0249] Embodiment 31: The method described in Embodiment 30, wherein the first single-stranded extension template in each orthogonal first oligonucleotide primer / first single-stranded extension pair includes a multiplex of at least two copies of a sequence connected head-to-tail and a 3' chain terminator so that the extension template is not extended by a polymerase, and the sequence is complementary to the first oligonucleotide primer.

[0250] Embodiment 32: The method described in embodiment 30 or embodiment 31, after step (d), further includes the following steps: (i) adding a second group of orthogonal second oligonucleotide primers and second single-stranded extension template oligonucleotide pairs, wherein the second oligonucleotide primer in each pair comprises a sequence complementary to one member of the first oligonucleotide primer group in 5' to 3' order, and a sequence complementary to the second single-stranded extension template of the second group of orthogonal oligonucleotide pairs; wherein each second single-stranded extension template comprises a 3' chain terminator; (ii) heating the reaction mixture to separate the nucleic acid chains and cooling to allow the second oligonucleotide primer to hybridize with the concatemers produced in steps (b)-(d), and to allow the second single-stranded extension template to hybridize with the second oligonucleotide primer; (iii) extending the second oligonucleotide primer using the second single-stranded extension template oligonucleotide as a template; and (iv) repeating steps (ii) and (iii) at least once, thereby generating concatemers comprising corresponding second oligonucleotide primer repeating units on corresponding members of the group of target binding ligands.

[0251] Embodiment 33: The method described in embodiment 30 or 31 also includes contacting the multimer produced in the previous step with a plurality of labeled nucleic acid probes, wherein each different probe is labeled with a distinguishable detectable label portion, and wherein members of the plurality of labeled nucleic acid probes include sequences complementary to corresponding members of the first oligonucleotide primers of the group.

[0252] Embodiment 34: The method described in Embodiment 32 also includes contacting the multimer produced in step (iv) with a plurality of labeled nucleic acid probes, wherein each different probe is labeled with a distinguishable detectable label portion, and wherein members of the plurality of labeled nucleic acid probes contain sequences that are complementary to corresponding members of the second oligonucleotide primer of the group.

[0253] Embodiment 35: The method of embodiment 33 or 34, wherein, for each member of the target binding ligand set, a plurality of labeled nucleic acid probe molecules hybridize to a plurality of concatemer repeat units.

[0254] Embodiment 36: The method of any one of Embodiments 30-35, wherein the method is performed in situ on a cell or tissue sample comprising or for detecting a target ligand.

[0255] Embodiment 37: The method of Embodiment 36, wherein the preparation of cells or tissue is fixed.

[0256] Embodiment 38: The method of Embodiment 37, wherein the tissue preparation is paraffin embedded.

[0257] Embodiment 39: The method of any one of Embodiments 30-38, wherein the target binding ligand comprises an antibody or an antigen-binding fragment thereof.

[0258] Embodiment 40: A method for in situ detection of a group of target molecules in a preparation of cells or tissues, the method comprising: (a) providing a group of target binding ligand molecules, wherein members of the target binding ligand molecules of the group are coupled to corresponding first oligonucleotide primer members and first single-stranded extension template oligonucleotide pairs in a group of orthogonal first oligonucleotide primers, wherein the first oligonucleotide primer of each pair has a different sequence from other first oligonucleotide primers in the group and is coupled to different members of the target binding ligand molecules of the group; (b) contacting the preparation of cells or tissues with the target binding ligands of the group under conditions that allow the target binding ligand molecules to specifically bind to the target molecules present in the preparation of cells or tissues; (c) introducing the target binding ligands of the group into the preparation of cells or tissues under conditions that allow the first oligonucleotide primers to hybridize and extend using the corresponding first single-stranded extension templates. (d) heating the reaction mixture of step (c) to separate the first single-stranded extension template from the extended first oligonucleotide primer produced in step (c); (e) repeating steps (c) and (d) at least once, thereby producing concatemers on corresponding members of the target binding ligand molecules of the group, the concatemers comprising corresponding first oligonucleotide primer sequence repeating units coupled to the target binding ligand molecules; (f) contacting the concatemers produced in step (e) with a plurality of distinguishably labeled nucleic acid probes; and (g) detecting the labeled nucleic acid probes, wherein detection of the labeled nucleic acid probes indicates the presence and location of the target molecule in the preparation of cells or tissues.

[0259] Embodiment 41: The method of embodiment 40, wherein the first single-stranded extension template comprises a concatemer of at least two copies of the sequence linked in a head-to-tail orientation and a 3' chain terminator, wherein the 3' chain terminator prevents the extension template from being extended by a polymerase, and the sequence is complementary to the first oligonucleotide primer.

[0260] Embodiment 42: The method described in embodiment 40 or embodiment 41, after step (e) and before step (f), further includes the steps of: (i) adding a second group of orthogonal second oligonucleotide primers and second single-stranded extension template oligonucleotide pairs, wherein the second oligonucleotide primer in each pair comprises, in 5' to 3' order, a sequence complementary to a member of the group of first oligonucleotide primers, and a sequence complementary to the second single-stranded extension template of the second group of orthogonal oligonucleotide pairs; (ii) heating the reaction mixture to separate the nucleic acid chains and cooling to allow the second oligonucleotide primers to hybridize with the concatemers produced in steps (c)-(e), and to allow the second single-stranded extension template to hybridize with the second oligonucleotide primers; (iii) extending the second oligonucleotide primer using the second single-stranded extension template oligonucleotide as a template; and (iv) repeating steps (ii) and (iii) at least once, thereby generating concatemers comprising corresponding second oligonucleotide primer repeating units on corresponding members of the group of target binding ligands.

[0261] Embodiment 43: The method of any one of Embodiments 40-42, wherein the plurality of distinguishably labeled nucleic acid probes comprise sequences complementary to corresponding first oligonucleotide primer members of an orthogonal first oligonucleotide primer set and a first single-stranded extension template oligonucleotide pair.

[0262] Embodiment 44: The method of Embodiment 42, wherein the plurality of distinguishably labeled nucleic acid probes comprise sequences complementary to corresponding second oligonucleotide primers of a second set of orthogonal second oligonucleotide primers, second single-stranded extension template oligonucleotide pair members.

[0263] Embodiment 45: The method of any one of Embodiments 40-44, wherein the target binding ligand comprises an antibody or an antigen-binding fragment thereof.

[0264] Embodiment 46: The method of any one of Embodiments 40-45, wherein a plurality of labeled nucleic acid probe molecules are hybridized to a plurality of concatemer repeat units coupled to a target binding ligand.

[0265] Embodiment 47: A kit comprising reagents for implementing the method described in any one of embodiments 1-46.

[0266] Embodiment 48: A target binding ligand comprising a concatemer prepared by the method of any one of Embodiments 1-15.

[0267] Embodiment 49: The target binding ligand of Embodiment 48, wherein the target binding ligand comprises an antibody or an antigen binding fragment thereof.

[0268] Embodiment 50: A method for producing a nucleic acid chain comprising a concatemer repeating unit of a given sequence, the method comprising: a) providing a first oligonucleotide primer and a first extension template oligonucleotide in a reaction mixture, the first extension template comprising at least two copies of a concatemer connected in a head-to-tail direction, the sequence being complementary to the first oligonucleotide primer, wherein the first amplification template comprises a chain terminator at its 3' end; b) incubating the reaction mixture under conditions allowing the first oligonucleotide primer to hybridize with the first extension template oligonucleotide; c) extending the hybridized first primer with a nucleic acid polymerase, thereby producing an extended nucleic acid chain complementary to the first extension template; d) heating the reaction mixture to separate the extended nucleic acid chain complementary to the first extension template from the first extension template; e) cooling the heated product to allow the first extension template oligonucleotide to hybridize with the extended nucleic acid chain complementary to the first extension template produced in step (c); f) extending the extended nucleic acid chain complementary to the first extension template produced in step (c) with a polymerase, thereby producing an extended nucleic acid chain comprising concatemer repeating units of the sequence contained in the first oligonucleotide primer.

[0269] Embodiment 51: The method of Embodiment 50 further comprises repeating steps (d) to (f) at least once.

[0270] Embodiment 52: The method described in Embodiment 51 comprises repeating steps (d) to (f) at least n times, wherein each iteration of steps (d) to (f) increases the length of the tandem nucleic acid by one concatemer repeat unit.

[0271] Embodiment 53: The method of any one of Embodiments 50-52, wherein step (a) comprises providing a set of orthogonal first oligonucleotide primers and first extension template oligonucleotide pairs, so that steps (b)-(f) produce multiple sets of concatemers, each concatemer comprising concatemer repeat units of the corresponding orthogonal oligonucleotide primer sequence.

[0272] Embodiment 54: The method described in any one of Embodiments 51-53, further comprising, after one or more repetitions of steps (d) to (f): i) adding a second oligonucleotide primer and a second extension template oligonucleotide to the reaction, wherein the second oligonucleotide primer comprises a 5' proximal sequence complementary to the first oligonucleotide primer sequence and a 3' proximal sequence complementary to one of at least two repeating portions of the second extension template oligonucleotide, wherein the second extension template oligonucleotide comprises a sequence of at least two repeating units linked head-to-tail and a chain terminator at its 3' end, the sequence being complementary to the second oligonucleotide primer; ii) reacting the second oligonucleotide primer with the multiple oligonucleotides formed after one or more repetitions of steps (d) to (f); The invention relates to a method of preparing an oligonucleotide-containing reaction mixture comprising: hybridizing one or more concatemer repeating units on the concatemer; iii) hybridizing the second extension template oligonucleotide with the second oligonucleotide primer; iv) extending the second oligonucleotide primer with a polymerase, thereby generating an extended nucleic acid chain complementary to the second extension template; v) heating the reaction mixture to separate the extended nucleic acid chain complementary to the second extension template from the second extension template; vi) cooling the heated product to allow the second extension template oligonucleotide to hybridize with the extended nucleic acid chain complementary to the second extension template generated in step (iv); vii) extending the extended nucleic acid chain complementary to the second extension template generated in step (iv) with a polymerase, thereby generating an extended nucleic acid chain comprising the concatemer repeating units of the sequence contained in the second oligonucleotide primer.

[0273] Embodiment 55: The method of Embodiment 54, further comprising repeating steps (v) to (vii) at least once.

[0274] Embodiment 56: The method described in embodiment 54 or 55, wherein step (i) includes providing a set of orthogonal second oligonucleotide primers and second extension template oligonucleotide pairs, so that steps (ii)-(vii) produce multiple sets of concatemers, each concatemer comprising concatemer repeat units of the corresponding orthogonal oligonucleotide primer sequence.

[0275] Embodiment 57: The method of any one of Embodiments 50-56, wherein the first oligonucleotide primer is coupled to the target binding ligand via its 5' end.

[0276] Embodiment 58: The method of Embodiment 57, wherein the target binding ligand is a polypeptide.

[0277] Embodiment 59: The method of Embodiment 58, wherein the polypeptide comprises an antibody or an antigen-binding fragment thereof.

[0278] Embodiment 60: The method of any one of Embodiments 57-59, further comprising contacting the target binding ligand with a preparation containing the target or cells or tissues for detecting the presence of the target.

[0279] Embodiment 61: The method of Embodiment 60, wherein the contacting is performed before concatemer formation steps (a)-(f) or (i)-(vii).

[0280] Embodiment 62: The method described in any one of Embodiments 50-61 further comprises contacting an extended nucleic acid chain comprising concatemer repeat units produced according to steps (a)-(f) and / or (i)-(vii) with a nucleic acid probe comprising a complementary sequence to the concatemer repeat sequence and a detectable label.

[0281] Embodiment 63: The method of any one of Embodiments 50-53, wherein the method produces a linear concatemer comprising a first oligonucleotide primer sequence.

[0282] Embodiment 64: The method of Embodiment 54, wherein the method produces a branched concatemer comprising a concatemer of a first oligonucleotide primer sequence combined with a concatemer of a second oligonucleotide primer sequence.

[0283] Embodiment 65: The method of any one of Embodiments 50-64, wherein the method is performed in contact with a preparation of cells or tissue.

[0284] Embodiment 66: The method of Embodiment 65, wherein the preparation of cells or tissue is fixed.

[0285] Embodiment 67: A method for labeling a target binding ligand, the method comprising: performing the method of any one of Embodiments 50-65, wherein the first oligonucleotide primer is coupled to the target binding ligand.

[0286] Embodiment 68: The method of Embodiment 67, wherein the target binding ligand comprises an antibody or an antigen-binding fragment thereof.

[0287] Embodiment 69: The method of Embodiment 67 or 68, wherein the first oligonucleotide primer is coupled to the target binding ligand in a manner that allows extension of the primer from its 3' end.

[0288] Embodiment 70: The method of any one of Embodiments 67-69, wherein the method is performed in contact with a preparation of cells or tissue.

[0289] Embodiment 71: The method of Embodiment 65, wherein the preparation of cells or tissue is fixed.

[0290] Embodiment 72: The method described in any one of Embodiments 67-71, further comprising contacting the concatemer produced in Embodiment 50 or 51 with a labeled nucleic acid probe, and detecting the labeled nucleic acid probe attached to the concatemer, wherein detection of the labeled nucleic acid probe indicates the presence and location of the target molecule.

[0291] Embodiment 73: The method of any one of Embodiments 1-72, wherein the nucleic acid polymerase is a thermostable nucleic acid polymerase.

[0292] Embodiment 74: A method for producing a nucleic acid molecule, the method comprising: (a) providing a first oligonucleotide primer; (b) contacting the first oligonucleotide primer with a first single-stranded extension template and a nucleic acid polymerase in a reaction mixture under conditions that allow the first oligonucleotide primer to hybridize and extend using the first single-stranded extension template, wherein the first single-stranded extension template comprises a first extension template element, a sequence complementary to the first oligonucleotide primer, and a 3' chain terminator in the 5' to 3' direction, so that the extension template is not extended by the polymerase; (c) heating the reaction mixture of step (b) to separate the first single-stranded extension template from the first single-stranded extension template and the nucleic acid polymerase; (d) contacting the extended first oligonucleotide primer with a second single-stranded extension template under conditions that allow hybridization and extension of the first extension oligonucleotide primer generated in step (b) using the second single-stranded extension template, wherein the second single-stranded extension template comprises, in a 5' to 3' direction, a second extension template element, a copy of the first extension template element, and a 3' chain terminator; wherein steps (a)-(d) produce a nucleic acid molecule comprising, in a 5' to 3' order, the first oligonucleotide primer sequence, the complement of the first extension template element, and the second extension template element.

[0293] Embodiment 75: The method of Embodiment 74, wherein the polymerase is a thermostable polymerase.

[0294] Embodiment 76: The method described in Embodiment 74 or Embodiment 75 further comprises repeating steps (b) and (c) at least once before step (d), wherein each repetition of steps (b) and (c) extends the generated nucleic acid molecule by one copy of the complementary sequence of the first extension template element.

[0295] Embodiment 77: The method described in any of Embodiments 74-76, further comprising at least one iteration of (e) heating the reaction mixture of step (d) to separate the second single-stranded extension template from the extended first oligonucleotide primer produced in step (d); and (f) repeating step (d), wherein each iteration of steps (e) and (f) extends the generated nucleic acid molecule by one copy of the complementary sequence of the second extension element.

[0296] Embodiment 78: The method described in any of Embodiments 74-77, further comprising heating the reaction mixture after step (d), and contacting the nucleic acid molecules produced in steps (a) to (d) with at least one additional single-stranded extension template under conditions that allow hybridization and extension of the produced nucleic acid molecules, wherein the additional single-stranded extension template comprises an additional extension template element, a copy of the extension template element from a previous round of extension, and a 3' chain terminator in the 5' to 3' direction, wherein the generated nucleic acid molecules are extended to include the complementary sequence of the additional extension template element.

[0297] Embodiment 79: The method of any one of Embodiments 74-78, wherein the first oligonucleotide is coupled to a target binding ligand.

[0298] Embodiment 80: The method of any one of Embodiments 74-79, which is performed by contacting with a cell or tissue sample.

[0299] Embodiment 81: The method of any one of Embodiments 74-80, further comprising contacting the generated nucleic acid molecules with a labeled nucleic acid probe comprising one or more extension template element sequences.

[0300] Embodiment 82: A kit for performing one or more methods described herein, the kit comprising: A) a first oligonucleotide primer or a first oligonucleotide primer set; b) a single-stranded extension template or a single-stranded extension template set; and c) a sequential extension or nucleic acid molecule.

[0301] Embodiment 83: The kit of Embodiment 82, wherein the first oligonucleotide primer or the set of first oligonucleotide primers and the single-stranded extension template or the set of single-stranded extension templates are orthogonal sets.

[0302] Embodiment 84: The kit of Embodiment 83, wherein the orthogonal set is optimized to avoid primer / extender cross-talk, primer dimer formation, and off-target hybridization.

[0303] Embodiment 85: The kit of Embodiment 82, wherein the single-stranded extension template or the set of single-stranded extension templates is suitable for performing a concatemer generation method.

[0304] Embodiment 86: The kit described in Embodiment 82 further includes packaging materials for various components and instructions for use.

[0305] Embodiment 87: The kit of Embodiment 82 further comprises one or more target binding ligand molecules or reagents for coupling the first oligonucleotide to the target binding ligand.

[0306] Embodiment 88: The kit described in Embodiment 82 also includes a heat-resistant polymerase, nucleotides, reaction buffer components and reagents for labeling nucleic acid probe molecules.

[0307] Embodiment 89: The kit described in Embodiment 88 further includes a probe molecule complementary to the multi-repetitive element.

[0308] Embodiment 90: The kit of Embodiment 88, wherein the probe molecule is complementary to one or more elements in the generated ordered extension products.

[0309] The specific elements of any disclosed embodiment may be combined or replace elements in other embodiments. In addition, although the advantages associated with certain embodiments of the present disclosure are described in the context of these embodiments, other embodiments may also exhibit these advantages, and not all embodiments must exhibit these advantages to fall within the scope of the present disclosure.

[0310] Example

[0311] The following examples illustrate some embodiments and aspects of the present invention. It will be appreciated by those skilled in the relevant art that various modifications, additions, substitutions, etc. may be made without changing the spirit or scope of the present invention, and such modifications and variations are included within the scope of the present invention as defined in the following claims. The following examples do not limit the present invention in any way.

[0312] Example 1: Methods and applications of highly multiplexed, thermally controllable DNA extension.

[0313] TCE (Thermal Controlled DNA Extension) Technology Figures 1A-1C ) achieves highly multiplexed, uniform and controllable DNA amplification through high and low temperature cycling and extension mediated by non-extendable single-stranded extenders (ssEXTers) and DNA polymerase.

[0314] TCE can be used to generate concatemers with repeat sequences (Figure 1A). For example, if one wishes to generate concatemers with multiple repeats of a, one can mix primer a with a ssEXTer containing two repeats of a* (the reverse complement of a), a DNA polymerase, a buffer, and dNTPs. The reaction is started at a low temperature so that primer a will bind to the a*a*ssEXTer. The DNA polymerase will extend primer a to form an aa sequence that binds to the ssEXTer. The temperature can be increased so that the aa sequence melts from the ssEXTer. Once the temperature is lowered, the aa sequence can bind to the ssEXTer again and be extended by the DNA polymerase to form an aaa sequence. The ssEXTers have a terminator at the 3' end, so they are not extendable. Temperature cycling can be performed multiple times to control the extension length on the primers. Different amplification cycles were tested for the two 9nt primers ( Figure 3 ). The results show that amplification is strictly controlled by the number of thermal cycles. Since extension is an on-off reaction controlled by thermal cycling, the amplification of different sequences should be highly uniform. The amplification uniformity of 50 different 9nt sequences was tested ( Figure 4 ). Little sequence dependence of the extension efficiency was observed.

[0315] If multiple orthogonal sequences are designed as a to n, TCE can be used to achieve multiplex concatemer generation (Figure 1B). A low temperature can be set and all single repeats a to n will bind to their designated ssEXTer. A high temperature can be set and all double repeats aa to nn will dissociate from their designated ssEXTer. If the sequences are a to n and their ssEXTers are a*a* to n*n*, thermal cycling can be performed between these two temperatures in the same reaction to achieve multiplex amplification. Since the sequences are orthogonal to each other, there is little crosstalk extension. The total number of multiplexes corresponds to the length of the single repeat sequence ( Figure 2 ). With a single repeat length of 15 nt, 3855 different sequences can be amplified. To test this hypothesis, 50 orthogonal sequences of 9 nt in length were designed. The 50 multiplexes were amplified in a test tube and next generation sequencing was performed to sequence the generated concatemers ( Figure 5 ). The results showed that 45 ssEXTers had a specificity of >99.5% and 48 ssEXTers had a specificity of >99%.

[0316] TCE can also be used in primer exchange reactions (Figure 1C) to generate concatemers containing or composed of different sequences. For example, to generate concatemers containing orthogonal sequences a1 to an, multiple ssEXTers can be designed: a1*a2*, a2*a3*, ..., and an-1*an*, and primer a1 can be extended with these ssEXTers by TCE. After multiple extension cycles, concatemers containing sequences a1 to an should be obtained.

[0317] TCE chemistry has multiple application scenarios, including but not limited to imaging (see scenario 1), mass spectrometry (see scenario 2), flow cytometry (see scenario 3), and immunoblotting (see scenario 4).

[0318] Example 2: Scenario 1: Highly multiplexed signal amplification multi-omics imaging via in situ thermal cycling DNA extension

[0319] ISE imaging (in situ extension imaging) takes advantage of the small size and high orthogonality of DNA molecules to achieve high signal-to-noise ratio and highly multiplexed multi-omics imaging. It uses polymerase-mediated iterative in situ DNA extension controlled by thermal cycling to achieve highly multiplexed amplification.

[0320] Specifically, the target sample is stained with an antibody coupled to an orthogonal short starting oligonucleotide, and / or stained with a DNA probe hybridized to an orthogonal short starting sequence of RNA ( Figure 6 ). Multiple 3' sealed single-stranded extensions (ssEXTers) containing two repeats of the initiation sequence are then added to the sample. Each ssEXTer will bind and extend a specific initiator through DNA polymerization at low temperature. The sample is then heated to a high temperature to melt the ssEXTer (the melting temperature of all ssEXTers in the reaction buffer is below 56°C). Multiple rounds of temperature cycling will be performed to allow the ssEXTer to repeatedly anneal and extend, thereby generating long concatemers in situ. Orthogonal fluorescent labeled imagers that hybridize to the concatemers can then be added for signal amplification. Subsequently, rapid imager exchange rounds can be performed to achieve high multiplexing.

[0321] Signal amplification is highly tunable by varying the number of thermal cycling rounds. Using β-tubulin in an immortalized cell line (Hela) as an example of a protein target (Figure 7A, Fig.24 ). The cells will first be fixed with paraformaldehyde. Then, the initiator-conjugated primary antibody will be stained onto the sample. The sample will be transferred to a thermal cycler. The DNA initiator on the β-tubulin antibody will be linearly extended to the DNA concatemer through the thermal cycling reaction. A fluorescently labeled DNA imager is provided and hybridized to the concatemer. A fluorescence microscope is then used to acquire the amplified signal of the target molecule. AXE achieved more than 14-fold amplification in 4 hours of thermal cycling (about 130 cycles) and reached a plateau at 8 hours of thermal cycling (about 260 cycles), reaching a 16.5-fold amplification (Figure 7A, Fig.24 ). Similarly, the RNA target (APC gene) could be amplified in Hela cells (Figure 7B, Fig.24 ) After 16 hours of thermal cycling, an approximately 8-fold amplification was achieved.

[0322] Signal amplification for ISE imaging can be further enhanced by branching the DNA concatemers. After the initial linear extension of the concatemers, probes containing secondary initiators are added to hybridize to the primary linear concatemers. This creates more imager binding sites and enables additional amplification. Alternatively, secondary long concatemers can also be generated separately in an ex situ reaction and added to the sample to achieve a total amplification of >100-fold ( Figure 8 , Fig.25 ).

[0323] Multiplexed ISE imaging can be achieved by co-staining proteins in the sample. Fig. 9 , Fig.26 ).

[0324] For example, ISE imaging successfully showed the changes in the expression levels of marker proteins in Py2T model cells during the epithelial-mesenchymal transition (EMT) process ( Fig. 9 ). In addition, joint imaging of RNA and protein can be achieved by ISE multiplex imaging. RNA probe hybridization and antibody staining are performed, followed by ISE extension in tissue (Figure 10).

[0325] The ISE scheme uses short coupled oligonucleotides and short ssEXT for amplification, which in principle leads to high penetration of the probe in thick tissues. Therefore, ISE imaging enables cell phenotyping in thick samples. Combined with traditional tissue clearing methods (SHIELD), ISE imaging achieved labeling and signal amplification of neuronal protein markers in the 0.5mm mouse cerebral cortex and hippocampus ( Fig.11 ).

[0326] ISE amplifies signals with a high signal-to-noise ratio. Many previous amplification methods rely on the addition of an ectopically synthesized preamplifier on the target of interest. Any nonspecific binding of the preamplifier may result in nonspecific signal amplification, thereby reducing the signal-to-noise ratio. In the ISE protocol, no preamplifier is added and the signal is only amplified in situ from the initiator. In addition, only short 11nt initiators are used for antibody coupling, thereby minimizing nonspecific binding of antibodies. To demonstrate the minimal nonspecific signal of the ISE protocol, Hela cells were transfected with a tubulin-GFP fusion gene. Then, the anti-GFP primary antibody was diluted to 100pg / mL and signal amplification was performed by the ISE protocol. Most of the signal spots in the cells were observed to colocalize with areas of high tubulin-GFP expression (Figure 12A). In contrast, signal amplification of the fluorophore-labeled secondary antibody resulted in high nonspecific background signals in areas lacking tubulin-GFP expression (Figure 12B). Fig.29 Additional imaging using ISE is shown, demonstrating that ISE is compatible with various types of clearing protocols for thick tissue imaging in mice. Humans can also be used for ISE protein imaging ( Fig.30). The samples were photobleached by LED to reduce autofluorescence.

[0327] Example 3: Scenario 2: Designing dynamic DNA nanodevices to amplify signals in mass cytometry analysis

[0328] Introduction. Mass cytometry is a state-of-the-art method based on inductively coupled plasma time-of-flight mass spectrometry and a single-cell sample introduction system that can simultaneously quantify >50 proteins or protein modifications at single-cell resolution, enabling analysis of complex cellular behaviors in highly heterogeneous samples. In the mass spectrometer, metal isotope-labeled antibodies are used to label proteins or protein modifications in cells. During sample collection, each stained single cell is vaporized, atomized, and ionized. The mass spectrometer quantifies the metals in the formed ion cloud to obtain a high-dimensional single-cell proteome readout. Previous studies have introduced mass cytometry as a general method to assess the signaling network status of more than 30 phosphorylation sites in millions of single cells. The relationship between all measured phosphorylation site pairs can be calculated to infer network responses to stimuli or track network remodeling through phenotypic transitions. Combined with high-throughput screening assays, these types of experiments have revealed new signaling mechanisms involved in cancer progression and drug resistance.

[0329] However, single-cell signaling network analysis has not yet been translated to study patient-derived tumor samples in clinical cancer research. Similar to many other types of single-cell methods, mass cytometry faces sensitivity limitations, and approximately 300 metal-labeled antibodies targeting the same epitope must be present in a cell to produce a detectable signal. For single-cell signaling network mapping in cancer samples, sensitivity limitations have a higher impact because the basal level of phosphorylation sites (i.e., in the absence of additional stimulation) often does not reach the detection limit of mass cytometry, especially in cells with smaller size, such as infiltrating T cells in tumor samples. In addition, phosphorylated residues are difficult to fully preserve in tissue samples. Factors such as temperature fluctuations and fixation protocols may further reduce phosphorylation levels before analyzing samples. In the imaging mode of mass cytometry analysis (IMC), imaging resolution must be compromised to compensate for the low sensitivity that makes it difficult to identify spatial cues that determine signal outcomes.

[0330] A DNA nanodevice has recently been invented that performs repeated in situ ligation under thermocycling conditions. Combining this device with a newly developed photocrosslinking strategy based on 3-cyanovinylcarbazole phosphoramidite (CNvK) modification, the approach has been successfully used to amplify mass spectrometer signals to address its sensitivity bottleneck. This allows us to understand cell states and predict cell fate in biological or clinical samples.

[0331] Methods. In conventional mass cytometry methods, metal isotopes are first chelated to maleimide-modified diethylenetriaminepentaacetic acid (DTPA) polymers and subsequently conjugated to reactive cysteine ​​residues located in the hinge region of partially reduced antibodies 1 . With this approach, only a few modification sites on each antibody can be conjugated, carrying a limited number of metal ions ( Figure 13A ). To improve the sensitivity of mass cytometry analysis, the main innovation of this new method, called cyclic extension amplification by DNA oligonucleotides (ACED), is the design of DNA nanodevices to generate infinitely repeated metal probe hybridization sites in situ ( Figure 13B ). In ACED, antibodies targeting a protein of interest are conjugated to short DNA oligonucleotide initiators (TT-a, 11-mer) that maintain antibody binding affinity, maximize antibody diffusion within cells, and reduce nonspecific binding, which is common in methods that apply long oligonucleotide (>40mer) conjugates 14,15 ( Figure 13B , step 1). The coupled antibody is mixed in a staining solution and applied to a cell suspension for cell surface or intracellular marker staining (step 2). Next, an extension body (a'-T-a', 19-mer) with two complementary repeated initiator sequences is introduced into the stained cells. At low temperatures, the extension body and the initiator hybridize to allow the chain extension to which the BST polymerase is connected (forming TT-aAa, step 3). By increasing the reaction temperature, the extension body is removed to expose the single-stranded extension probe (step 4). The thermal cycle is then repeated with the required rounds to continuously extend the probe coupled to the antibody (step 5), generating hundreds of aA repeats at each antibody modification site (step 6). The DTPA polymer with chelated Ln3+ metal ions is coupled to a detection chain with a "-Ta" sequence through a maleimide-thiol reaction, which can then be hybridized with an extended DNA probe on the antibody (step 7), with each probe occupying a metal probe binding site (aAa). Brief (1 second) ultraviolet (UV) irradiation activates the 3-cyanovinylcarbazole phosphoramidite (CNVK) photocrosslinker on the metal probe strand, creating covalent bonds between the hybridized DNA molecules and allowing the DTPA polymer to attach to the antibody (step 8)( Fig.14 ).

[0332] In order to verify the specificity and quantify the amplification ability of ACED in mass spectrometry flow cytometry analysis, the system was applied to human embryonic kidney HEK293T cells transiently overexpressing green fluorescent protein GFP with a high expression gradient. As expected, the GFP antibody signal amplified by ACED was highly correlated with the second antibody signal targeting the first GFP antibody, confirming the specificity of the established DNA device in intracellular antibody signal amplification (Figure 15A). Through the time series analysis of 500 thermal cycles, the ACED method always shows the amplified mass spectrometry flow cytometry signal during the thermal cycle time (Figure 15A). Compared with the unamplified samples, the sample with 500 rounds of amplification was observed to be quantitatively analyzed according to the GFP expression level using binned data (Figure 15B), 13 times the amplification intensity and 6 times the signal-to-noise ratio enhancement (Figure 15C). In order to detect and quantify ultra-low abundance proteins, linear ACED amplification may be inefficient and time-consuming. For these proteins, a branch amplification strategy was developed, and the amplification ability can be increased exponentially through iterative cycles of ACED reactions. Quantification of branching results by mass cytometry analysis revealed a 17-fold magnification of each branching whorl ( FIG. 16 ).

[0333] Ultimately, unlimited signal amplification can be achieved through multiple branching reactions. In order to multiplex the ACED approach, a high degree of orthogonality between DNA probes targeting different antibodies is required. 50 orthogonal ACED barcode sequences were designed to cover the full capacity of the mass cytometer. In a proof-of-concept test, six initiator probes were cross-reacted with six extender probes to thoroughly evaluate their orthogonality ( Fig.17 In subsequent mass cytometry analysis, only the paired initiator and extender sequences produced detectable signals ( Fig.17 ). A 30-fold signal amplification was then performed on the mouse Py2T cell line undergoing epithelial-mesenchymal transition (EMT) over a 7-day time course. Using ACED, key signature molecules can be detected, differentiated, and quantified during the progression of the EMT process, including E-cadherin, vimentin, Smad2 / 3, Smad4, and most importantly, the transcription factors Snail / Slug, Zeb1( Fig.18 ). Dimensionality reduction analysis using unified manifold approximation and projection (UMAP) projected the trajectory of EMT progression over time, validating ACED as a powerful technology platform that can accurately amplify mass spectrometry flow cytometry signals, especially for low-abundance proteins ( Fig.18 ).

[0334] In addition to protein detection, in a recent analysis, it has been demonstrated that this method can distinguish RNA abundance at single-cell resolution. Vimentin mRNA levels increased by an average of 2.5-fold in Py2T cells treated with TGF-β compared to untreated (PBS) cells ( Fig.19 ).

[0335] Example 4: Scenario 3: TCE combined with flow cytometry and flow sorting

[0336] Flow cytometry and flow sorting involve suspending a cell sample in the fluid of a flow cytometer instrument to detect and measure the physical and chemical properties of the sample. Despite recent advances in flow cytometry and flow sorting, the method still suffers from low sensitivity and low multiplexity for detection and sorting.

[0337] Applying TCE to amplify signals and increase the multiplexity of flow-related techniques ( Fig. 20 ). Specifically, a fixative is used to fix single suspended cells. Antibodies targeting the protein of interest are then added to the sample. The antibodies are conjugated to different orthogonal sequences. TCE amplification is performed to extend the conjugated DNA in situ. Imagers with different fluorophores that can bind to different concatemers are then added to the sample. Flow cytometry is used to measure the intensity of the different fluorophores, revealing the abundance of their targets.

[0338] As a proof of concept, HEK293T cells were transfected with GFP mRNA and immunostained with anti-GFP antibody (Figure 21A). The signal was then amplified with 130 cycles or 260 cycles of TCE, or with secondary antibodies. Flow cytometry measurements were performed on single cell suspension samples (Figure 21B). The absolute signal intensity and the correlation of signal intensity with GFP fluorescence signal were compared. The results showed that TCE flow had higher signal amplification and signal-to-noise ratio compared to the secondary antibody used as the gold standard.

[0339] Example 5: Scenario 4: TCE combined with immunoblotting

[0340] The immunoblotting technique uses gel electrophoresis to separate proteins from a sample and then transfer them to a membrane. Antibodies specific to the target protein are bound to the membrane and detected with chemical or radioactive tags. It is used to detect specific proteins in a sample. A key limitation of immunoblotting is the lack of multiplexing due to the limited host species of secondary antibodies.

[0341] To overcome this limitation, TCE was applied to immunoblotting to achieve higher multiplexity ( Fig. 22). Specifically, orthogonal docking chains are coupled to the primary antibody. After the target is transferred to the membrane after gel electrophoresis, we add the antibody to the membrane to enable the antibody to bind to the target. TCE is used to generate long concatemers in a test tube, which can each bind to a different docking chain. The concatemers are then added to the membrane. After the concatemers are bound, imagers coupled to fluorophores targeting different concatemers are added to the membrane, allowing the target to be visualized by fluorescence imaging. Imager swapping is performed to quickly visualize other targets on the same membrane.

[0342] As a proof of concept, GFP protein immunoblotting was performed using ectopically generated concatemers and a fluorophore-coupled imager ( Fig.23 ). The GFP target band is visible on the membrane, with no nonspecific bands.

[0343] All identified patents and other publications are expressly incorporated herein by reference for the purpose of describing and disclosing methods such as those described in such publications that may be relevant to the present invention. These publications are provided solely for disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventor is not entitled to prior disclosure by reason of prior invention or for any other reason. All statements regarding the dates or contents of these documents are based on the information available to the applicant and do not constitute any admission as to the correctness of the dates or contents of these documents.

Claims

1. A method for labeling a target binding ligand, the method comprising: (a) providing a conjugate of a target binding ligand and a first oligonucleotide primer; (b) contacting the coupled first oligonucleotide primer with the first single-stranded extension template and a nucleic acid polymerase in a reaction mixture under conditions that allow hybridization and extension of the first oligonucleotide primer using the first single-stranded extension template; (c) heating the reaction mixture of step (b) to separate the first single-stranded extension template from the extended first oligonucleotide primer produced in step (b); (d) repeating steps (b) and (c) at least once, thereby generating a concatemer comprising repeating units of the oligonucleotide primer sequence coupled to a target binding ligand.

2. The method of claim 1, wherein the first single-stranded extension template comprises a concatemer of at least two copies of a sequence linked head-to-tail and complementary to the first oligonucleotide primer, and a 3' chain terminator that prevents the extension template from being extended by a polymerase.

3. The method according to claim 1 or 2, further comprising the following steps after at least one repetition of steps (b)-(d): (i) adding a second oligonucleotide primer and a second single-strand extension template oligonucleotide, wherein the second oligonucleotide primer comprises, in 5′ to 3′ order, a sequence complementary to the first oligonucleotide primer and a sequence complementary to the second single-strand extension template, wherein the second single-strand extension template comprises a 3′ chain terminator; (ii) heating the reaction mixture to separate the nucleic acid strands and cooling to allow the second oligonucleotide primer to hybridize to the concatemers produced in steps (b)-(d) and to allow the second single-stranded extension template to hybridize to the second oligonucleotide primer; (iii) extending the second oligonucleotide primer using the second single-stranded extension template oligonucleotide as a template; and (iv) repeating steps (ii) and (iii) at least once, thereby generating concatemers comprising repeat units of the second oligonucleotide primer.

4. The method according to claim 1 or 2, further comprising after step (d): (i) adding a nucleic acid comprising: (1) a second concatemer consisting of repeating units of a second oligonucleotide sequence; and (2) a sequence complementary to the first oligonucleotide primer under conditions that allow hybridization, such that multiple molecules of the nucleic acid comprising the second concatemer hybridize to the concatemer formed in step (d) through the sequence complementary to the first oligonucleotide primer.

5. The method according to any one of claims 1 to 4, wherein the method is performed in situ on a cell or tissue sample comprising a target ligand or for assaying a target ligand. The method according to claim 5 , wherein the cell or tissue sample is fixed.

7. The method of claim 5 or 6, wherein the tissue sample is paraffin embedded.

8. The method according to any one of claims 1 to 7, further comprising contacting the concatemers produced in the previous step with a labeled nucleic acid probe.

9. The method of claim 8, wherein the nucleic acid probe comprises a sequence complementary to a concatemer repeat unit.

10. The method of claim 8 or 9, wherein the nucleic acid probe comprises a sequence complementary to the first oligonucleotide primer.

11. The method according to any one of claims 3 or 5-7, further comprising contacting the concatemer consisting of the second oligonucleotide primer repeating unit with a labeled nucleic acid probe, wherein the nucleic acid probe comprises a sequence complementary to the second oligonucleotide primer.

12. The method according to any one of claims 4 or 5-7, further comprising contacting the concatemer consisting of the repeating units of the second oligonucleotide sequence with a labeled nucleic acid probe, wherein the nucleic acid probe comprises a sequence complementary to the second oligonucleotide sequence.

13. The method according to any one of claims 8 to 12, wherein a plurality of labeled nucleic acid probe molecules hybridize to a plurality of concatenated repeat units.

14. The method of any one of claims 1-8, wherein the target binding ligand comprises an antibody or an antigen-binding fragment thereof.

15. The method according to any one of claims 1 to 14, wherein a plurality of different target binding ligands are multiplex labeled by different orthogonal oligonucleotide primer / single strand extension template pairs.

16. A method for in situ detection of a target molecule in a cell or tissue preparation, the method comprising: (a) contacting a preparation of cells or tissue with a target binding ligand coupled to a first oligonucleotide primer under conditions that allow the target binding ligand to specifically bind to a target molecule; (b) adding a reaction mixture comprising a first single-stranded extension template and a nucleic acid polymerase to the preparation of cells or tissue under conditions that allow hybridization and extension of the first oligonucleotide primer using the first single-stranded extension template; (c) heating the reaction mixture of step (b) to separate the first single-stranded extension template from the extended first oligonucleotide primer produced in step (b); (d) repeating steps (b) and (c) at least once, thereby generating a concatemer comprising repeating units of the first oligonucleotide primer sequence coupled to a target binding ligand; (e) contacting the concatemers produced in step (d) with a labeled nucleic acid probe; as well as (f) detecting the labeled nucleic acid probe, wherein detection of the labeled nucleic acid probe indicates the presence and location of the target molecule in the preparation of cells or tissue.

17. The method of claim 16, wherein the first single-stranded extension template comprises a concatemer of at least two copies of a sequence oriented head-to-tail and linked, and a 3' chain terminator such that the extension template is not extended by the polymerase, the sequence being complementary to the first oligonucleotide primer.

18. The method of claim 16 or 17, wherein the target binding ligand comprises an antibody or an antigen binding fragment thereof.

19. The method of any one of claims 16-18, wherein the labeled nucleic acid probe comprises a sequence complementary to the first oligonucleotide primer.

20. The method of any one of claims 16-19, wherein the labeled nucleic acid probe comprises a sequence complementary to a concatemer repeat unit.

21. The method of any one of claims 16-20, wherein a plurality of labeled nucleic acid probe molecules are hybridized to a plurality of concatemer repeat units coupled to a target binding ligand.

22. The method according to any one of claims 16 to 18, further comprising the following steps after step (d) and before step (e): (i) adding a second oligonucleotide primer and a second single-stranded extension template oligonucleotide, wherein the second oligonucleotide primer comprises, in 5′ to 3′ order, a sequence complementary to the first oligonucleotide primer and a sequence complementary to the second single-stranded template, wherein the second single-stranded extension template comprises a 3′ chain terminator; (ii) heating the reaction mixture to separate the nucleic acid strands and cooling to allow the second oligonucleotide primer to hybridize to the concatemer produced in step (d) and to allow the second single-stranded extension template to hybridize to the second oligonucleotide primer; (iii) extending the second oligonucleotide primer using the second single-stranded extension template oligonucleotide as a template; and (iv) repeating steps (ii) and (iii) at least once, thereby generating concatemers comprising repeat units of the second oligonucleotide primer.

23. The method of claim 22, wherein the labeled nucleic acid probe comprises a sequence complementary to the second oligonucleotide primer.

24. The method of claim 22 or 23, wherein a plurality of labeled nucleic acid probe molecules are hybridized to a plurality of concatameric repeat units coupled to the target binding ligand.

25. The method according to any one of claims 16 to 18, further comprising after step (d) and before step (e): (i) adding a nucleic acid comprising: (1) a second concatemer consisting of repeating units of a second oligonucleotide sequence; and (2) a sequence complementary to the first oligonucleotide primer under conditions that allow hybridization, such that multiple molecules of the nucleic acid comprising the second concatemer hybridize to the concatemer comprising repeats of the first oligonucleotide primer sequence formed in step (d) through the sequence complementary to the first oligonucleotide primer.

26. The method of claim 25, wherein the labeled nucleic acid probe comprises a sequence complementary to the second oligonucleotide sequence.

27. The method of claim 25 or 26, wherein a plurality of labeled nucleic acid probe molecules hybridize to a plurality of the second concatemer repeat units.

28. The method of any one of claims 16-27, wherein the preparation of cells or tissue is fixed.

29. The method of claim 28, wherein the tissue preparation is paraffin embedded.

30. A method for labeling a set of target binding ligands, the method comprising: (a) providing a set of orthogonal first oligonucleotide primer and first single-stranded extension template oligonucleotide pairs, wherein the first oligonucleotide primer of each pair has a sequence different from the other first oligonucleotide primers in the set and is coupled to a different member of the set of target binding ligands; (b) contacting the coupled first oligonucleotide primer with the first single-stranded extension template of the orthogonal pair and a nucleic acid polymerase in a reaction mixture under conditions that allow hybridization and extension of the first single-stranded extension template with the first oligonucleotide primer using the orthogonal pair; (c) heating the reaction mixture of step (b) to separate the first single-stranded extension template from the extended first oligonucleotide primer produced in step (b); (d) repeating steps (b) and (c) at least once, thereby generating a concatemer on each member of the target ligand set, the concatemer comprising a repeating unit of the oligonucleotide primer sequence coupled to the target binding ligand.

31. A method according to claim 30, wherein the first single-stranded extension template in each orthogonal first oligonucleotide primer / first single-stranded extension pair comprises a concatemer of at least two copies of a sequence that is complementary to the first oligonucleotide primer and a 3' chain terminator in a head-to-tail orientation so that the extension template is not extended by a polymerase.

32. The method according to claim 30 or 31, further comprising the following steps after step (d): (i) adding a second set of orthogonal second oligonucleotide primers, second single-stranded extension template oligonucleotide pairs, wherein the second oligonucleotide primer in each pair comprises, in 5′ to 3′ order, a sequence complementary to one member of the first oligonucleotide primer set, and a sequence complementary to the second single-stranded extension template of the second set of orthogonal oligonucleotide pairs; (ii) heating the reaction mixture to separate the nucleic acid strands and cooling to allow the second oligonucleotide primer to hybridize to the concatemers produced in steps (b)-(d) and to allow the second single-stranded extension template to hybridize to the second oligonucleotide primer; (iii) extending the second oligonucleotide primer using the second single-stranded extension template oligonucleotide as a template; and (iv) repeating steps (ii) and (iii) at least once, thereby generating concatemers comprising corresponding second oligonucleotide primer repeat units on corresponding members of the set of target binding ligands.

33. The method of claim 30 or 31 further comprises contacting the concatemer produced in the previous step with a plurality of labeled nucleic acid probes, wherein each different probe is labeled with a distinguishable detectable label portion, and wherein members of the plurality of labeled nucleic acid probes comprise sequences complementary to corresponding members of the first oligonucleotide primer set.

34. The method of claim 32, further comprising contacting the concatemer produced in step (iv) with a plurality of labeled nucleic acid probes, wherein each different probe is labeled with a distinguishable detectable label portion, and wherein members of the plurality of labeled nucleic acid probes comprise sequences that are complementary to corresponding members of the second oligonucleotide primer set.

35. The method according to claim 33 or 34, wherein: For each member of the set of target binding ligands, a plurality of labeled nucleic acid probe molecules are hybridized to a plurality of concatenated repeat units.

36. The method of any one of claims 30-35, wherein the method is performed in situ on a cell or tissue sample comprising or for assaying a target ligand.

37. The method of claim 36, wherein the preparation of cells or tissue is fixed.

38. The method of claim 37, wherein the tissue preparation is paraffin embedded.

39. The method of any one of claims 30-38, wherein the target binding ligand comprises an antibody or an antigen-binding fragment thereof.

40. A method for in situ detection of a set of target molecules in a preparation of cells or tissues, the method comprising: (a) providing a set of target binding ligand molecules, wherein members of the set of target binding ligand molecules are coupled to corresponding first oligonucleotide primer members of the set of orthogonal first oligonucleotide primer and first single-stranded extension template oligonucleotide pairs, wherein each pair of first oligonucleotide primers has a sequence different from other first oligonucleotide primers in the set and is coupled to a different member of the set of target binding ligand molecules; (b) contacting the preparation of cells or tissue with the set of target binding ligands under conditions that allow the target binding ligand molecules to specifically bind to target molecules present in the preparation of cells or tissue; (c) adding a reaction mixture comprising the first single-stranded extension template of the orthogonal first oligonucleotide primer and the first single-stranded extension template oligonucleotide pair, and a nucleic acid polymerase to the preparation of cells or tissues under conditions that allow hybridization and extension of the first oligonucleotide primer using the corresponding first single-stranded extension template; (d) heating the reaction mixture of step (c) to separate the first single-stranded extension template from the extended first oligonucleotide primer produced in step (c); (e) repeating steps (c) and (d) at least once, thereby generating concatemers on corresponding members of the set of target binding ligand molecules, the concatemers comprising corresponding first oligonucleotide primer sequence repeating units coupled to the target binding ligand molecules; (f) contacting the concatemers produced in step (e) with a plurality of distinguishably labeled nucleic acid probes; as well as (g) detecting the labeled nucleic acid probe, wherein detection of the labeled nucleic acid probe indicates the presence and location of the target molecule in the preparation of cells or tissue.

41. The method of claim 40, wherein the first single-stranded extension template comprises a concatemer of at least two copies of a sequence complementary to the first oligonucleotide primer and a 3' chain terminator in a head-to-tail orientation, wherein the 3' chain terminator prevents the extension template from being extended by a polymerase.

42. The method according to claim 40 or 41, further comprising the following steps after step (e) and before step (f): (i) adding a second set of orthogonal second oligonucleotide primers, second single-stranded extension template oligonucleotide pairs, wherein the second oligonucleotide primer in each pair comprises, in 5′ to 3′ order, a sequence complementary to a member of the first oligonucleotide primer set, and a sequence complementary to a second single-stranded extension template of the second set of orthogonal oligonucleotide pairs; (ii) heating the reaction mixture to separate the nucleic acid strands and cooling to allow the second oligonucleotide primer to hybridize to the concatemers produced in steps (c)-(e) and to allow the second single-stranded extension template to hybridize to the second oligonucleotide primer; (iii) extending the second oligonucleotide primer using the second single-stranded extension template oligonucleotide as a template; and (iv) repeating steps (ii) and (iii) at least once, thereby generating concatemers comprising corresponding second oligonucleotide primer repeat units on corresponding members of the set of target binding ligands.

43. The method of any one of claims 40-42, wherein the plurality of distinguishably labeled nucleic acid probes comprise sequences complementary to corresponding first oligonucleotide primer members of the orthogonal first oligonucleotide primer and first single-stranded extension template oligonucleotide pair set.

44. The method of claim 42, wherein the plurality of distinguishably labeled nucleic acid probes comprise sequences complementary to corresponding second oligonucleotide primer members of the second set of orthogonal second oligonucleotide primers, second single-stranded extension template oligonucleotide pairs.

45. The method of any one of claims 40-44, wherein the target binding ligand comprises an antibody or antigen-binding fragment thereof.

46. ​​The method of any one of claims 40-45, wherein a plurality of labeled nucleic acid probe molecules are hybridized to a plurality of concatemer repeat units coupled to a target binding ligand.

47. A kit comprising reagents for performing the method of any one of claims 1-46.

48. A target binding ligand comprising a concatemer produced by the method of any one of claims 1-15.

49. The target binding ligand of claim 48, wherein the target binding ligand comprises an antibody or an antigen binding fragment thereof.

50. A method for producing a nucleic acid chain comprising concatemeric repeat units of a given sequence, the method comprising: a) providing a reaction mixture comprising a first oligonucleotide primer and a first extension template oligonucleotide, the first extension template comprising a concatemer of at least two copies of a sequence complementary to the first oligonucleotide primer in a head-to-tail orientation, wherein the first extension template comprises a chain terminator at its 3' end; b) incubating the reaction mixture under conditions that allow hybridization of the first oligonucleotide primer to the first extension template oligonucleotide; c) extending the hybridized first primer with a nucleic acid polymerase, thereby generating an extended nucleic acid strand complementary to the first extension template; d) heating the reaction mixture to separate the extended nucleic acid strand complementary to the first extension template from the first extension template; e) cooling the heated product to allow the first extension template oligonucleotide to hybridize with the extended nucleic acid strand complementary to the first extension template produced in step (c); f) extending the extended nucleic acid chain complementary to the first extension template generated in step (c) using a polymerase, thereby generating an extended nucleic acid chain comprising multiple repeating units of the sequence contained in the first oligonucleotide primer.

51. The method of claim 50, further comprising repeating steps (d) to (f) at least once.

52. The method of claim 51, comprising repeating steps (d) to (f) at least n times, wherein each iteration of steps (d) to (f) increases the length of the concatemer nucleic acid by one concatemer repeat unit.

53. A method according to any one of claims 50-52, wherein step (a) comprises providing a set of orthogonal first oligonucleotide primers and first extension template oligonucleotide pairs, so that steps (b)-(f) produce a set of concatemers, each concatemer comprising concatemer repeat units of corresponding orthogonal oligonucleotide primer sequences.

54. The method of any one of claims 51-53, further comprising after one or more repetitions of steps (d) to (f): i) adding a second oligonucleotide primer and a second extension template oligonucleotide to the reaction, wherein the second oligonucleotide primer comprises a 5' proximal sequence complementary to the first oligonucleotide primer sequence and a 3' proximal sequence complementary to one of the at least two repeat portions of the second extension template oligonucleotide, wherein, The second extension template oligonucleotide comprises a sequence of at least two repeating units linked in a head-to-tail orientation and a chain terminator at its 3' end, wherein the sequence is complementary to the second oligonucleotide primer; ii) hybridizing the second oligonucleotide primer to one or more concatemer repeat units on the concatemer formed after one or more repetitions of steps (d) to (f); iii) hybridizing the second extension template oligonucleotide to the second oligonucleotide primer; iv) extending the second oligonucleotide primer with a polymerase, thereby generating an extended nucleic acid strand complementary to the second extension template; v) heating the reaction mixture to separate the extended nucleic acid strand complementary to the second extension template from the second extension template; vi) cooling the heated product to allow the second extension template oligonucleotide to hybridize with the extended nucleic acid strand complementary to the second extension template produced in step (iv); vii) extending the extended nucleic acid chain complementary to the second extension template generated in step (iv) using a polymerase, thereby generating an extended nucleic acid chain comprising multiple repeating units of the sequence contained in the second oligonucleotide primer.

55. The method of claim 54, further comprising repeating steps (v) to (vii) at least once.

56. A method according to claim 54 or 55, wherein step (i) includes providing a set of orthogonal second oligonucleotide primers and second extension template oligonucleotide pairs, so that steps (ii)-(vii) produce multiple sets of concatemers, each concatemer comprising concatemer repeat units of the corresponding orthogonal oligonucleotide primer sequence.

57. The method of any one of claims 50-56, wherein the first oligonucleotide primer is coupled to a target binding ligand via its 5' end.

58. The method of claim 57, wherein the target binding ligand is a polypeptide.

59. The method of claim 58, wherein the polypeptide comprises an antibody or an antigen-binding fragment thereof.

60. The method of any one of claims 57-59, further comprising contacting the target binding ligand with a preparation of cells or tissues containing the target or for detecting the presence of the target.

61. The method of claim 60, wherein the contacting is performed prior to steps (a)-(f) or (i)-(vii) of producing concatemers.

62. The method according to any one of claims 50-61 further comprises contacting the extended nucleic acid chain comprising the concatemer repeat units produced according to steps (a)-(f) and / or (i)-(vii) with a nucleic acid probe comprising a complementary sequence to the concatemer repeat units and a detectable label.

63. The method of any one of claims 50-53, wherein the method produces a linear concatemer comprising the first oligonucleotide primer sequence.

64. The method of claim 54, wherein the method produces branched concatemers comprising concatemers of the first oligonucleotide primer sequence bound to concatemers of the second oligonucleotide primer sequence.

65. The method of any one of claims 50-64, wherein the method is performed in contact with a preparation of cells or tissue.

66. The method of claim 65, wherein the preparation of cells or tissue is fixed.

67. A method for labeling a target binding ligand, the method comprising: Perform the method of any one of claims 50-65, wherein the first oligonucleotide primer is coupled to the target binding ligand.

68. The method of claim 67, wherein the target binding ligand comprises an antibody or an antigen binding fragment thereof.

69. The method of claim 67 or 68, wherein the first oligonucleotide primer is coupled to the target binding ligand in a manner that allows extension of the primer from its 3' end.

70. The method of any one of claims 67-69, wherein the method is performed in contact with a preparation of cells or tissue.

71. The method of claim 65, wherein the preparation of cells or tissue is fixed.

72. The method of any one of claims 67-71, further comprising contacting the concatemer produced in claim 50 or 51 with a labeled nucleic acid probe, and detecting the labeled nucleic acid probe attached to the concatemer, wherein detection of the labeled nucleic acid probe indicates the presence and location of the target molecule.

73. The method of any one of claims 1-72, wherein the nucleic acid polymerase is a thermostable nucleic acid polymerase.

74. A method for producing a nucleic acid molecule, the method comprising: (a) providing a first oligonucleotide primer; (b) contacting the first oligonucleotide primer with the first single-stranded extension template and a nucleic acid polymerase in a reaction mixture under conditions that allow hybridization and extension of the first oligonucleotide primer using the first single-stranded extension template, wherein the first single-stranded extension template comprises, in the 5′ to 3′ direction, a first extension template element, a sequence complementary to the first oligonucleotide primer, and a 3′ chain terminator, such that the extension template is not extended by the polymerase; (c) heating the reaction mixture of step (b) to separate the first single-stranded extension template from the extended first oligonucleotide primer produced in step (b); (d) contacting the extended first oligonucleotide primer with a second single-stranded extension template under conditions that allow hybridization and extension of the first extension oligonucleotide primer generated in step (b) using the second single-stranded extension template, wherein the second single-stranded extension template comprises, in the 5' to 3' direction, the second extension template element, a copy of the first extension template element, and a 3' chain terminator; wherein steps (a)-(d) produce a nucleic acid molecule comprising, in 5' to 3' order, a first oligonucleotide primer sequence, a complementary sequence to a first extension template element, and a complementary sequence to a second extension template element.

75. The method of claim 74, wherein the polymerase is a thermostable polymerase.

76. The method of claim 74 or 75, further comprising repeating steps (b) and (c) at least once prior to step (d), wherein each repetition of steps (b) and (c) results in the resulting nucleic acid molecule being extended by one copy of the complementary sequence of the first extension template element.

77. The method of any one of claims 74-76, further comprising at least one iteration of the following steps: (e) heating the reaction mixture of step (d) to separate the second single-stranded extension template from the extended first oligonucleotide primer produced in step (d); and (f) repeating step (d), Each iteration of steps (e) and (f) extends the resulting nucleic acid molecule by one copy of the complement of the second extension element.

78. The method of any one of claims 74-77, further comprising heating the reaction mixture after step (d) and contacting the nucleic acids produced in steps (a) to (d) with at least one additional single-stranded extension template under conditions that allow hybridization and extension of the produced nucleic acid molecules, wherein the additional single-stranded extension template comprises an additional extension template element, a copy of the extension template element from a previous round of extension, and a 3' chain terminator in the 5' to 3' direction, wherein the produced nucleic acid molecules are extended to include the complementary sequence of the additional extension template element.

79. The method of any one of claims 74-78, wherein the first oligonucleotide is coupled to a target binding ligand.

80. The method of any one of claims 74-79, performed in contact with a cell or tissue sample.

81. The method of any one of claims 74-80, further comprising contacting the generated nucleic acid molecules with a labeled nucleic acid probe comprising one or more extension template element sequences.

82. A kit for practicing one or more methods described herein, comprising: a) a first oligonucleotide primer or a first oligonucleotide primer set; b) a single-stranded extension template or a set of single-stranded extension templates; and c) Ordered stretches or nucleic acid molecules.

83. The kit of claim 82, wherein the first oligonucleotide primer or the first oligonucleotide primer set and the single-stranded extension template or the single-stranded extension template set are orthogonal sets.

84. The kit of claim 83, wherein the orthogonal set is optimized to avoid primer / extension crosstalk, primer dimer formation, and off-target hybridization.

85. The kit of claim 82, wherein the single-stranded extension template or the set of single-stranded extension templates is suitable for performing a method of concatemer generation.

86. The kit of claim 82, further comprising packaging materials and instructions for use for the various components.

87. The kit of claim 82, further comprising one or more target binding ligand molecules or reagents for coupling the first oligonucleotide to the target binding ligand.

88. The kit according to claim 82 further comprises a thermostable polymerase, nucleotides, reaction buffer components and reagents for labeling nucleic acid probe molecules.

89. The kit of claim 88, further comprising a probe molecule complementary to the concatemeric repeat element.

90. The kit of claim 88, wherein the probe molecule is complementary to one or more elements in the generated ordered extension products.

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