Engineered dynamic DNA nanodevices to amplify signals

Abstract

The method, the composition and the kit provided by the invention provide a signal amplification method for detecting target biomolecules, and the sensitivity of detecting by using target binding ligand molecules is remarkably improved. These methods include adding nucleic acid repeat cycles 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, mass spectrometers / CyTOF and the like, and provide additional tools for research and diagnosis purposes.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 390,831, filed on July 20, 2022, and No. 63 / 390,946, filed on July 20, 2022, under 35 U.S. Patent Act, Section 119(e), the entire contents of both applications are incorporated herein by reference. Technical Field

[0003] The technology described herein relates to methods for amplifying signals when detecting, quantifying, and imaging biomolecules. Background Art

[0004] Methods for detecting, quantifying and / or imaging biological target molecules of interest are central to a wide range of research and diagnostic approaches. There is a need in the art for compositions and methods that allow for more sensitive detection of target biomolecules, as well as for example the detection of multiple such target biomolecules in the same assay - so-called multiplexed detection.

[0005] The recent emergence of single-cell technologies allows the investigation of the mechanisms of cancer development and progression from a completely new perspective. However, current epitope-based single-cell analyses cannot include the necessary number of targets of interest in a single analysis, and single-cell proteomics approaches are limited by sensitivity and throughput. For example, flow cytometry (e.g., BD Fortessa) is limited by fluorescence spectral overlap and cellular autofluorescence. Sequencing-based epitope quantification methods, such as CITE-seq and REAP-seq, suffer from high technical variation and nonspecific binding introduced by long oligonucleotide barcodes, so their quantification is lower than flow cytometry and mass cytometry methods. Lab-on-a-chip technologies, such as single-cell barcode chips (SCBC) and single-cell Western blots (scWestern), are more sensitive than cytometry methods and can detect low-abundance proteins. However, the multiplexing and throughput of such methods are both low. SCoPE MS and SCoPE2 are emerging single-cell proteomics analyses that allow proteome-scale characterization at the single-cell level. However, before such methods can be used to quantify changes in protein abundance at the single-cell level, sensitivity, throughput, and reproducibility must be improved.

[0006] Mass spectrometry is a recently established 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, allowing analysis of complex cellular behaviors in highly heterogeneous samples. In mass spectrometry, metal isotope-labeled antibodies are applied to label proteins or protein modifications in cells. During sample collection, each stained single cell is evaporated, atomized, and ionized. The metals in the resulting ion cloud are quantified by the mass spectrometer to obtain a high-dimensional single-cell proteome readout. However, mass spectrometry faces sensitivity limitations, requiring the accumulation of approximately 300 metal-labeled antibodies per epitope to generate a detectable signal. This inherent limitation has hampered the application of mass spectrometry in the analysis of low-abundance proteomes, including in the analysis of many transcription factors, surface receptor proteins, and intracellular phosphorylation sites that are critical for health and disease. Analysis of small-volume cells, such as immune cells and microorganisms, on a mass spectrometer is even more challenging. Systematic analysis of signaling networks in T lymphocytes (T cells are on average 23 times smaller than HeLa cells) at single-cell resolution has not yet been achieved. The abundance of many key phosphorylated proteins in human T lymphocytes does not reach the detection limit of the mass spectrometer. Summary of the invention

[0007] The technology described herein generally relates to amplifying signals associated with the binding of a target binding ligand to its target in or on a cell. Such signal amplification can provide readouts with sensitivity and resolution as high as single molecule detection. In various embodiments, the technology relates to generating nucleic acid sequence concatemers on a target ligand binding molecule, wherein the repeating sequence provides hybridization sites for multiple labeled nucleic acid probes for each target binding ligand molecule, thereby amplifying signals associated with the binding of a target molecule in a cell sample bound to the target ligand. The methods, compositions, and kits described herein have applications in a variety of situations, but have particular relevance in mass spectrometry cytometry and fluorescence activated cell sorting or flow cytometry, which are further described herein.

[0008] In one aspect, a method for detecting and / or quantifying a target molecule at a single cell level is described herein. The methods described herein are well suited for use in mass spectrometry and flow cytometry, and can also be used for techniques including, but not limited to, imaging mass spectrometry, multiplexed ion beam imaging (MIBI), and multiplexed ion beam imaging time of flight (MIBI-TOF). While applicable to these and other single cell methods, in one illustrative embodiment, described herein is a method for detecting a target molecule in a mass spectrometer, the method comprising: a) contacting a target binding ligand molecule comprising a coupled oligonucleotide primer with a cell or tissue sample under conditions that allow the target binding ligand molecule to bind to its target in the cell or tissue sample; b) extending the oligonucleotide primer to include a concatemer of a sequence included in the oligonucleotide primer; c) contacting the oligonucleotide primer extended in step (b) with a labeled probe nucleic acid, the labeled probe nucleic acid comprising a sequence complementary to a sequence repeated in the concatemer, such that a plurality of labeled probe nucleic acid molecules hybridize to a plurality of repeated sequences in the concatemer; d) cross-linking the hybridized probe nucleic acid molecules to the extended oligonucleotide primer; and e) detecting the labeled probe using mass cytometry.

[0009] In this aspect as described herein and in one embodiment of any other aspect, the probe nucleic acid of labeling comprises metal ion labeling.In one embodiment, metal is the stable isotope of lanthanide metal.Example comprises the isotope of lanthanum, cerium, praseodymium, neodymium, promethium, europium, gadolinium, terbium, dysprosium, holmium, erbium, thulium, ytterbium and lutetium.In for example mass spectrometer or related method, other metal / metal ions that may be used as distinguishable labeling can comprise for example the isotope of cadmium, palladium, indium, platinum, bismuth, selenium, tellurium, gold, silver, tantalum, yttrium, ruthenium, rhodium, iodine, osmium and iridium etc.The mass spectrum of lanthanide metal is easy to distinguish, makes it very suitable for multiple applications in mass spectrometer.

[0010] In another embodiment of this and any other aspect described herein, the labeled probe comprises a fluorophore. In another embodiment, the labeled probe may comprise a fluorophore label and a metal ion label.

[0011] In another embodiment of this and any other aspect described herein, extending the oligonucleotide primer comprises thermal cycling with a single-stranded extension template and a template-dependent nucleic acid polymerase.

[0012] In another embodiment of this and any other aspect described herein, the polymerase is thermostable.

[0013] In another embodiment of this and any other aspect described herein, the single-stranded extension template comprises a concatemer of at least two copies of a sequence complementary to the 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.

[0014] In another embodiment of this and any other aspect described herein, the extending step (b) comprises multiple cycles of extension, hot strand separation, and cooling to allow annealing of single-stranded extenders and extension by the polymerase, whereby successive cycles add successive multiple repeats to the oligonucleotide primer.

[0015] In another embodiment of this and any other aspect described herein, the method of detecting a target molecule in a mass spectrometer is multiplexed with a plurality of different target binding ligand molecules, orthogonal sets of single strand extension templates, and distinguishably labeled orthogonal probe nucleic acids.

[0016] In another embodiment of this and any other aspect described herein, the concatemer includes a branched concatemer formed on or bound to an extended oligonucleotide bound to a target binding ligand. Various branching methods are described below and can further increase the number of sites to which a nucleic acid probe hybridizes on a target binding ligand.

[0017] In another embodiment of this and any other aspect described herein, the method of detecting a target molecule in mass cytometry further comprises the step of imaging a fluorophore associated with the cell or tissue sample prior to the detecting step (e).

[0018] In another embodiment of this and any other aspects described herein, the hybridized probe nucleic acid molecules are cross-linked with the extended oligonucleotide primers and include photocrosslinking. In another embodiment, the probe nucleic acid molecules include a photocrosslinker. In another embodiment, the photocrosslinker includes 3-cyanovinylcarbazole phosphoramidite (CNVK) and crosslinking includes ultraviolet irradiation.

[0019] In another embodiment of this and any other aspect described herein, the target binding ligand comprises an antibody or antigen-binding fragment thereof.

[0020] In another aspect, the present invention describes a method for fluorescence-activated cell sorting, which comprises: a) providing a sample comprising fixed cells; b) contacting a target binding ligand comprising a coupled oligonucleotide primer with cells in the sample under conditions that allow the target binding ligand molecule to bind to its target in or on the cell; c) extending the oligonucleotide primer to include a multimer of the sequence included in the oligonucleotide primer; d) contacting the oligonucleotide primer extended in step (c) with a fluorescently labeled probe nucleic acid, wherein the probe nucleic acid includes a sequence complementary to a repeated sequence in the multimer, so that multiple labeled probe nucleic acid molecules hybridize to multiple repeated sequences in the multimer; e) cross-linking the hybridized probe nucleic acid molecules to the extended oligonucleotide primer; and f) sorting the cells by fluorescence-activated cell sorting.

[0021] In one embodiment of this and any other aspect described herein, the label probe further comprises a metal ion.

[0022] In another embodiment of this and any other aspect described herein, extending the oligonucleotide primer comprises thermal cycling with a single-stranded extension template and a template-dependent nucleic acid polymerase.

[0023] In another embodiment of this and any other aspect described herein, the polymerase is thermostable.

[0024] In another embodiment of this and any other aspect described herein, the single-stranded extension template comprises a concatemer of at least two copies of a sequence complementary to the 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.

[0025] In another embodiment of this and any other aspect described herein, the extending step (c) comprises multiple cycles of extension, hot strand separation, and cooling to allow the single-stranded extensions to anneal and be extended by the polymerase, whereby successive cycles add successive multiple repeat units to the oligonucleotide primer.

[0026] In another embodiment of this and any other aspect described herein, the method is multiplexed with a plurality of different target binding ligand molecules, orthogonal sets of single-stranded extension templates, and distinguishably labeled orthogonal probe nucleic acids.

[0027] In another embodiment of this and any other aspect described herein, the concatemer comprises a branched concatemer formed on the target binding ligand by thermal cycling.

[0028] In another embodiment of this and any other aspect described herein, the method further comprises, after the sorting step (f), a step of detecting the metal ion labeled probes by mass spectrometry.

[0029] In another embodiment of this aspect and any other aspect described herein, the hybridized probe nucleic acid molecule is cross-linked with the extended oligonucleotide primer, including photocrosslinking. In another embodiment, the probe nucleic acid molecule comprises a photocrosslinking portion. In another embodiment, the photocrosslinking portion comprises 3-cyanovinylcarbazole phosphoramidite (CNVK), and the crosslinking comprises ultraviolet irradiation. Other crosslinking agents include, but are not limited to, psoralen (ultraviolet activation), bromine-modified ultraviolet crosslinking (e.g., 5-bromodeoxyuridine-mediated crosslinking), carmustine crosslinking, and click chemistry-mediated crosslinking.

[0030] In another embodiment of this and any other aspect described herein, the target binding ligand comprises an antibody or antigen binding fragment thereof. In another embodiment, the target binding ligand comprises a DNA or RNA molecule, and may include, for example, modified bases or linkages.

[0031] In another aspect, described herein is a composition comprising a target binding ligand and an oligonucleotide that binds to the target binding ligand.

[0032] In another embodiment of this and any other aspect described herein, the concatenated oligonucleotide further comprises one or more branched concatenated oligonucleotides.

[0033] In another embodiment of this and any other aspect described herein, the composition further comprises a plurality of nucleic acid probe molecules hybridized to the concatenated repeats of the concatenated oligonucleotide.

[0034] In another embodiment of this and any other aspects described herein, the nucleic acid probe molecule comprises a cross-linking moiety. In one embodiment, the cross-linking moiety is a photocross-linking moiety. In another embodiment, the cross-linking moiety comprises 3-cyanovinylcarbazole phosphoramidite (CNVK).

[0035] In another embodiment of this and any other aspect described herein, the nucleic acid probe molecules are cross-linked to the concatenated repeats.

[0036] In another embodiment of this and any other aspect described herein, the nucleic acid probe molecule comprises a fluorescent label or a metal ion label.

[0037] In another embodiment of this and any other aspect described herein, the nucleic acid probe molecule comprises a fluorescent label and a metal ion label.

[0038] In another embodiment of this and any other aspect described herein, the target binding ligand comprises an antibody or antigen-binding fragment thereof.

[0039] In another aspect, described herein is a cell or tissue sample comprising a composition described herein, wherein the target binding ligand binds to a target molecule in or on the cell or tissue sample.

[0040] In another aspect, the present invention describes a kit for implementing one or more methods described herein, the kit comprising: a) a target binding ligand coupled to an oligonucleotide primer; or a set of target binding ligands coupled to an orthogonal oligonucleotide primer; b) a single-stranded extension template or a set of orthogonal single-stranded extension templates; c) a labeled probe nucleic acid or an orthogonal labeled probe nucleic acid set, wherein the labeled probe nucleic acid or the orthogonal labeled probe nucleic acid set comprises a cross-linking portion.

[0041] In one embodiment of this and any other aspect described herein, the single-stranded extension template comprises a concatemer of at least two copies of a sequence complementary to the 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.

[0042] In another embodiment of this and any other aspect described herein, the kit further comprises a template-dependent nucleic acid polymerase. In one embodiment, the polymerase is thermostable.

[0043] In another embodiment of this and any other aspect described herein, the target binding ligand comprises an antibody or antigen-binding fragment thereof.

[0044] In another embodiment of this and any other aspect described herein, orthogonal nucleotide primers, single-stranded extension templates, and nucleic acid probes are optimized to avoid primer / extension body cross-talk, primer-dimer formation, and off-target hybridization.

[0045] In another embodiment of this and any other aspect described herein, the single-stranded extension template or set of single-stranded extension templates is suitable for performing a concatemer generation method.

[0046] In another embodiment of this and any other aspect described herein, the kit further comprises packaging materials for the various components and instructions for use.

[0047] In another embodiment of this and any other aspect described herein, the kit further comprises one or more target binding ligand molecules or reagents for coupling the first oligonucleotide to the target binding ligand.

[0048] In another embodiment of this and any other aspect described herein, the kit further comprises a thermostable polymerase, nucleotides, reaction buffer components, and reagents for labeling the nucleic acid probe molecules.

[0049] In another embodiment of this and any other aspect described herein, the probe molecule may be complementary to a concatemeric repeat element.

[0050] In another embodiment of this and any other aspect described herein, the crosslinking moiety comprises a photocrosslinking moiety. In one embodiment, the crosslinking moiety comprises 3-cyanovinylcarbazole phosphoramidite (CNVK).

[0051] In another embodiment of this and any other aspect described herein, the labeled probe nucleic acid or orthogonally labeled probe nucleotide set comprises a fluorescent label or a metal ion label.

[0052] In another embodiment of this and any other aspect described herein, the labeled nucleic acid probe molecules include a fluorescent label and a metal ion label.

[0053] Definitions 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 multiple 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.

[0054] As used herein, the term "specific binding" refers to a physical interaction between two molecules, compounds, cells and / or particles, wherein the binding of the first entity to the second target entity has greater specificity and affinity than the binding to the third entity of the non-target. A reagent specific for 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 on an antigen-binding protein (such as an antibody or its antigen-binding fragment): 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 is said to be "specific" or "binds specifically" or "selectively binds" to the first target or antigen as compared to a second target or antigen. In general, a molecule that "specifically binds", "selectively binds" or "has specificity for a given target" will bind to the 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. -5 M (10000 nM) or lower KD binding, for example 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12M 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 use any suitable method, such as titrating the polypeptide agent in a suitable cell binding assay, to determine suitable conditions for the polypeptide agent described herein to selectively bind to the target.

[0055] In this context, it should be understood that, where an antibody or antigen-binding fragment thereof is involved, specific binding is mediated by the CDRs of the antibody polypeptide, rather than by 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 No. WO2005 / 012359); Forte Bio-Octe™ analysis, enzyme-linked immunosorbent assay (ELISA); and competition assays (e.g., RIA).

[0056] As used herein, the term "coupling" refers to, for example, the connection of an oligonucleotide to a target binding ligand in a stable manner through 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.

[0057] 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 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 repeat unit length of the concatemers as described herein can determine the multiplexing degree that the orthogonal repeat group of a given length can achieve. The length of the repeat unit 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.

[0058] As used herein, the term "conditions that allow hybridization and extension" for a given molecule (e.g., 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 extension by a given polymerase 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 one skilled in the art. Thus, in a single 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-generally, the annealing temperature in the cyclic reaction is about 5°C lower than the Tm at which the repeat sequence hybridizes to its complement at the optimal salt and reaction component concentrations for the selected enzyme. When performed in multiplexing, the length of the repeat unit becomes more important because the Tm of the various repeat sequences will vary. Primer / single-stranded extension template design can take into account sequence variation to design primers and single-stranded extension templates with relatively close Tm values, typically within a range of 5-7°C for all members of the primer / extension template sequence set. In these cases, a single annealing or hybridization temperature in the cycled reaction can allow for efficient hybridization and extension of the members of the set in the multiplex. In various embodiments of the method that include a step of contacting a nucleic acid molecule with a single-stranded extension template under conditions that allow hybridization and extension of the nucleic acid molecules, the reaction can be incubated at the annealing temperature for a period of time (typically from 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-stranded extension template under conditions that allow hybridization and extension of the nucleic acid molecules can include such annealing / extension temperature variation. In some embodiments, annealing or hybridization occurs effectively at a temperature at which the polymerase is sufficiently active. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figure 1A-1B Figure 1A is a schematic diagram of epitope detection using conventional mass spectrometer antibodies. Figure 1B is a schematic diagram of epitope detection using ACED (Amplification by Cyclic Extension of DNA Oligonucleotides) amplification method.

[0060] Figure 2 We show that CNvK-based photocrosslinking allows hybridized detection strands to remain intact during mass spectrometer sample acquisition.

[0061] Figures 3A-3C.Figure 3A uses transient transfection to generate an expression gradient of GFP in a cell group subsequently targeted by an anti-GFP antibody coupled to an ACED oligonucleotide. By 1-500 thermal cycles, the signal amplification of ACED (y axis) is compared with the counterstaining using an anti-rat secondary antibody to show the specificity of ACED. Figure 3B examines the data in Figure 3A divided into 10 boxes of equal width according to the GFP expression level displayed by the secondary antibody. Box 1 represents untransfected cells as an internal control. Box 10 shows the cells with the highest GFP expression level. Figure 3C shows the box median and signal-to-noise ratio (i.e., the median of boxes 1-9 compared to the median in box 1) in 1-500 thermal cycles, shown in the two figures on the left, indicating that the amplification intensity is 13 times and the signal-to-noise ratio is enhanced by 6 times. The right side plots the multiple changes of each box in 1-500 cycles in the form of a bar graph.

[0062] Figure 4 Depicted is the comparison of signal amplification with one or two rounds of branching to linear amplification without branching. A 17-fold amplification was observed with each branching round.

[0063] Figure 5 By performing pairwise analysis on six extenders and metal chains, we investigate how ACED exhibits a high degree of orthogonality.

[0064] Figure 6 Py2T cells undergoing EMT were analyzed by ACED mass spectrometry. 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.

[0065] Fig. 7A -7C. Thermally controllable DNA extension protocol. Fig. 7A It is shown that thermal cycle extension (TCE) can be used to generate concatemers with repetitive sequences. Figure 7B A TCE is shown that enables multiplex concatemer generation. Figure 7C shows that TCE can also be used for primer exchange reactions.

[0066] Figures 8A-8BSignal amplification enhanced by branched ACE amplification is shown. Figure 8A examines that after linear extension (step 1), the primary detection chain can be used directly for mass spectrometry analysis (step 2). When branching is required to further amplify the signal, the number of detection chain binding sites on each antibody can be expanded by applying a primary branched chain containing CNvK (step 3), followed by UV cross-linking and thermal ring primer extension (step 4). Then, a secondary detection chain (step 5) can be applied before mass spectrometry analysis. Based on the same principle, secondary branching (steps 6-8) can be achieved compared to the unamplified signal, ultimately achieving a signal enhancement of more than 500 times. Figure 8B examines the GFP ion counts generated by ACE after one or two rounds of branching in an ERK2-GFP transient expression experiment using ultra-low amounts of GFP antibodies (10ng / ml) compared to the counts generated by linear amplification without branches. Cells expressing ERK2-GFP were stained with oligo-coupled rat anti-GFP antibodies, linear or branched ACE amplification, and then 141Pr-labeled detection chain hybridization. Conventionally labeled 172Yb anti-ERK2 antibody was used to detect ERK2-GFP fusion protein in the same cells and subsequently analyzed by mass spectrometry. We observed that primary branch amplification with 50 thermal cycled branch extension steps within 1 hour produced a 9-fold signal enhancement compared to linear amplification. In addition, a secondary branching approach could be applied, resulting in a 5-fold increase in signal, which increased the initial unamplified signal by more than 500-fold.

[0067] Figures 9A-9B Depicts how ACE exhibits high orthogonality with an overall 1.02% crosstalk signal in a 33-plex multiplex evaluation. Figure 9A shows that HEK293T cells expressing GFP were individually stained with anti-GFPACE antibodies coupled to 33 starting sequences before being barcoded, pooled, and processed through the ACE protocol in the same tube and then analyzed on a mass spectrometer. Figure 9B shows that the data was then barcoded and decoded to allow for pairwise analysis of potential ACE crosstalk. Before calculating any mismatched initiator-detector pairs (e.g., Ab Initiator 1-Detector 2'- 142 Nd) and the real signal (e.g., Ab-initiator 1-detector 1'- 141 Before calculating the ratio between the Pr), the ion counts generated by the detection chain under all conditions (per column) were normalized to 0-1. On average, ACE had a crosstalk signal of 1.02%. Four pairs of probes were detected with crosstalk exceeding 10% (initiator 2-detector 3', initiator 4-detector 5', initiator 7-detector 8', initiator 20-detector 21').

[0068] Fig.10GFP+ and GFP- E. coli cells stained with DNA barcode-conjugated anti-GFP antibodies are shown.With two rounds of branching, protein abundance in microorganisms can be measured using mass spectrometry.

[0069] Figure 11A depicts that in primary human CD4 T cells, the p-ERK signal was only slightly above the mass cytometry detection limit using conventional conjugated antibodies. Using ACE, the p-ERK signal increased dramatically, showing the expected trend in the TCR stimulation time course experiment. Figure 11B analyzes primary human T cells harvested during a 1-hour TCR stimulation time course, and ACE reveals differential signal responses on key TCR signal mediators, including p-CD3ζ, p-CD28, p-ZAP70 / SYK, p-LAT, p-SLP76, p-PLCγ1, p-BTK / ITK, p-MEK1 / 2, p-ERK1 / 2, p-p90RSK, and p-S6.

[0070] Figure 12A shows that high abundance proteins can be captured to generate detectable signals in IMC analysis. Due to this limitation, recent IMC-based studies of human breast cancer have been designed to focus on cell surface epitopes, cytokeratins, and some cell function indicators. Figure 12B depicts that by applying ACE to amplify the IMC signal, multiple low abundance targets can be detected on the IMC instrument. Figure 12C depicts that by applying two rounds of branching, ACE is expected to increase the average metal ion count by nearly 4000 times. Therefore, compared with the currently used 1μm 2 Compared with laser beam, the size of laser ablation craters can be reduced to a sub-diffraction limit level of 100 nm in diameter.

[0071] Figure 13A shows a human kidney section with 20 ACE oligo-conjugated antibodies at the same time. After post-staining fixation, before analyzing the slide on IMC, these markers are amplified with linear or branched ACE. Figure 13B shows that 18 ACE-amplified kidney markers are displayed in three overlapping images, wherein each marker is represented by a specific color. The position of these markers in kidney tissue is consistent with expectations. Figure 13C shows that single cell segmentation has been performed, and the segmented cells are embedded in a two-dimensional UMAP map. Figure 13D shows that the detected cell types show unique protein expression profiles, which can only be studied with multiple spatial proteomics analysis supported by ACE. DETAILED DESCRIPTION

[0072] The technology described herein relates to improvements in the ability to detect target molecules with high sensitivity at the single cell level, and optionally in a multiplexed manner. Certain embodiments of the technology described herein relate to the generation of binding nucleic acids on target binding molecules, which can be bound in situ, such as to target molecules in or on cells. By providing binding sites or "landing pads" for multiple copies of probes complementary to multi-linked repeat elements, the technology labels target binding molecules in a manner that allows detection of even low-abundance target molecules, which in some embodiments can even include single-molecule sensitivity. These methods are applicable to a range of different scenarios, but are particularly useful for, for example, mass spectrometry cytometry and flow cytometry.

[0073] The following describes various considerations involved in practicing the techniques disclosed herein.

[0074] Preparation of cell samples:

[0075] Mass spectrometry, flow cytometry and similar methods are best suited for analyzing single cell suspensions. Non-limiting examples can include cells in suspension culture, cells in adherent culture placed in single cell suspension (e.g., by trypsinization), cells in blood samples, and cells separated from tissues (healthy or diseased, including but not limited to tumor tissue) by physical and / or enzymatic methods, etc. Cell samples can be fixed to avoid deterioration in 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, various concentrations of paraformaldehyde (PFA) (typically between 1% and 5%, such as 1%, 1.2%, 1.4%, 1.6%, 1.8%, 2.0%, 2.2%, 2.4%, 2.6%, 2.8%, 3.0%, 3.2%, 3.4%, 3.6%, 3.8%, 4%, 4.2%, 4.4%, 4.6%, 4.8%, or 5.0%), various concentrations of formaldehyde (e.g., 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, or 5% formaldehyde), 10% neutral buffered formalin, Bonn's solution, methanol, acetone, glutaraldehyde, and the like. Cells or tissue samples may be treated or processed to minimize nucleic acid degradation, e.g., RNA is a target molecule. One of ordinary skill in the art will be able to determine the most appropriate fixative for a cell or tissue sample and the technique to be performed. In some embodiments, the fixative that may be used is 1.6% paraformaldehyde.

[0076] In more detail, for cultured cell lines, paraformaldehyde (PFA, from Electron Microscopy Sciences) can be added to the cell suspension to a final percentage of 1.6%, and the cells can be incubated at room temperature for 10 minutes. The cross-linked cells are washed twice with cell staining medium (CSM, PBS containing 0.5% BSA, PBS with 0.02% NaN3), and after centrifugation, the cells are resuspended using ice-cold methanol and then permeabilized on ice for 10 minutes or stored at -80°C for long term.

[0077] For tumor cell samples, fresh tissue samples can be immediately transferred to pre-cooled MACS tissue storage solution (Miltenyi Biotec) after surgical resection and shipped at 4°C. Tissue processing is best completed within 24 hours of collection. For dissociation, the tissue was minced using a scalpel and further dissociated using a human tumor dissociation kit (Miltenyi Biotech) and gentle MACS dissociation solution (Miltenyi Biotech) according to the manufacturer's instructions. The resulting single cell suspension was filtered through sterile 70μm and 40μm cell strainers in sequence. The cell suspension was stained for viability with 25μM cisplatin (Enzo Life Sciences) for 1 minute pulses before quenching with 10% FBS. The cells were then fixed with 1.6% paraformaldehyde (PFA, Electron Microscopy Sciences) for 10 minutes at room temperature and stored at -80°C.

[0078] For human monocytes, monocytes were isolated by density gradient centrifugation in tissue aqueous solution (Sigma-Aldrich) followed by MACS purification using a pan-monocyte isolation kit (Miltenyi Biotech) according to the manufacturer's instructions. 2 Monocytes were differentiated into immature macrophages after 5 days of culture in tissue culture dishes. Cells were then polarized for 24 hours. For mass spectrometry analysis, cells were stained for viability with 10 μM cisplatin (Enzo Life Sciences) in a 1-minute pulse before quenching with 10% FBS, as previously described (Fienberg et al., 2012). Cells were then fixed with 1.6% paraformaldehyde (Electron Microscopy Sciences) for 10 minutes at room temperature and stored at -80°C.

[0079] Target Binding Ligand:

[0080] In some embodiments of any of the aspects, the oligonucleotide primer strand is attached to the target binding ligand molecule.

[0081] As used herein, a "target binding ligand" is a molecule or portion 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, members of affinity binding pairs (e.g., avidin / streptavidin), oligonucleotides, aptamers, and receptors.

[0082] In some embodiments of any aspect, the target binding ligand binds, i.e., the target molecule is a non-limiting molecule selected from lipids, sugars, oligosaccharides or polysaccharides, amino acids, peptides or polypeptides, nucleosides, nucleotides, oligonucleotides or polynucleotides, hormones, vitamins, small molecules, miRNA, metabolites, and any combination thereof.

[0083] In some embodiments of any of the aspects, the target binding ligand binds to a barcoded DNA or RNA molecule.

[0084] 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 may include an immunoglobulin heavy chain (H) variable region (abbreviated herein as V H ) and immunoglobulin light chain (L) variable region (abbreviated herein as V L ). In another example, the antibody includes two heavy chain (H) variable regions and two light chain (L) variable regions. The antibody may include structural features of IgA, IgG, IgE, IgD, IgM (and their subtypes). The term antibody as used herein includes any of a variety of different constructs that use one or more antigen binding domains or fragments of an antibody to mediate binding to a target molecule. Therefore, 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 skeletons to support multiple target binding domains can also be used as target binding ligands.

[0085] As used herein, "antigen-binding fragment" refers to the portion 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 complementary 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 V H and V L domains, which may be linked by a portion of the constant domains of the antibody heavy and light chains, or by a linker, such as a peptide linker. Unconventional antibodies, such as camelid and short antibodies, have only heavy chain sequences, such as V HH These can be used in a similar manner to those containing V H / V L The term antigen-binding fragment is used as an antigen-binding fragment. Non-limiting examples of antibody fragments encompassed by the term antigen-binding fragment include: (i) L , C L 、V H and C H 1 domain; (ii) Fab' fragment, which is a H 1 domain has one or more cysteine ​​residues at the C-terminus; (iii) a Fab fragment having a V H and C H 1 domain; (iv) an Fd fragment having V H and C H 1 domain and C H 1 domain C-terminal one or more cysteine ​​residues Fd 'fragment; (v) having an antibody single arm V L and V H Fv fragment of the structural domain; (vi) V H (vii) F(ab')2 fragments, bivalent fragments, comprising two Fab' fragments linked by a disulfide bond at the hinge region; (viii) single-chain antibody molecules (e.g., single-chain Fv; scFv) (Bird et al., Science 242:423-426 (1988); Huston et al., PNAS (USA) 85:5879-5883 (1988)); (ix) "diabodies" with two antigen-binding sites, which comprise a light chain variable domain (V L ) connected to the heavy chain variable region (V H ) (see, e.g., EP 404097; WO 93 / 11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); and comprising a pair of tandem Fd fragments (VH -C H 1-V H -C H 1) "linear antibody", which together with the complementary light chain polypeptide forms a pair of antigen binding regions (Zapata et al., Protein Eng. 8 (10): 1057-1062 (1995); and U.S. Patent No. 5,641,870). Fv, scFv or diabody molecules can be linked by binding to V H and V L The CH1 domain is stabilized by disulfide bonds. Minibodies are also available that contain scFv fragments linked to a CH3 domain. 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.

[0086] Oligonucleotides:

[0087] 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 connected 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.

[0088] 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 nucleosides 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 "stopper" portion) that can block or exclude template-dependent extension. Such modifications may also be referred to as chain blockers, for example, including dideoxynucleosides, 3'-CPR IICPG, 3'-phosphoCPG, 5'-Ome dT CE phosphoramidites, 5'-amino dT CE phosphoramidites, 2'3'-ddACE phosphoramidines, 2'3-ddC CE phosphoramidites, 2'3-ddG-CE phosphoramidites, 2'3'-ddT CE phosphoramidites, 3'-dACPG, 3'-ddC CPG, 5'-dC CPG, 3' spacer C3CPG, etc. In some embodiments, the oligonucleotide may also include, for example, a nucleoside modified to include a crosslinking moiety. The inclusion 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 in 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 diazepine (DB-dUMP, psoralen), 4-thio-dT (S4dT), and the like.

[0089] The oligonucleotides used in the methods, compositions and kits described herein are generally at least 9 nucleotides in length, but can vary in length from 8 to 100 or more nucleotides. Thus, the length of the oligonucleotides used in the methods, compositions or kits described herein can be, for example, between 9-100 nucleotides, between 9-95 nucleotides, between 9-90 nucleotides, between 9-85 nucleotides, between 9-80 nucleotides, between 9-75 nucleotides, between 9-70 nucleotides, between 9-65 nucleotides, between 9-60 nucleotides, between 9-55 nucleotides, between 9-50 nucleotides, between 9-45 nucleotides, between 9-40 nucleotides, between 9-35 nucleotides, between 9-30 nucleotides, between 9-25 nucleotides, between 9-2 ... , 9-15 nucleotides, 10-100 nucleotides, 10-90 nucleotides, 10-80 nucleotides, 10-70 nucleotides, 10-60 nucleotides, 10-50 nucleotides, 10-40 nucleotides, 10-30 nucleotides, 10-20 nucleotides, 11-100 nucleotides, 11-90 nucleotides, 11-80 nucleotides, 11-70 nucleotides, 11-60 nucleotides, 11-50 nucleotides, 11-40 nucleotides, 11-30 nucleotides, 11-2 0 nucleotides, 12-100 nucleotides, 12-90 nucleotides, 12-80 nucleotides, 12-70 nucleotides, 12-60 nucleotides, 12-50 nucleotides, 12-40 nucleotides, 12-30 nucleotides, 12-20 nucleotides, 13-100 nucleotides, 13-90 nucleotides, 13-80 nucleotides, 13-70 nucleotides, 13-60 nucleotides, 13-50 nucleotides, 13-40 nucleotides, 13-30 nucleotides, 13-20 nucleotides nucleotides, 14-100 nucleotides, 14-90 nucleotides, 14-80 nucleotides, 14-70 nucleotides, 14-60 nucleotides, 14-50 nucleotides, 14-40 nucleotides, 14-30 nucleotides, 14-20 nucleotides, 15-100 nucleotides, 15-90 nucleotides, 15-80 nucleotides, 15-70 nucleotides, 15-60 nucleotides, 15-50 nucleotides, 15-40 nucleotides, 15-30 nucleotides, or 15-20 nucleotides.

[0090] Methods for coupling oligonucleotides to 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 via a connecting portion connected to or near the 5' end of an oligonucleotide primer.

[0091] The oligonucleotides described herein include nucleic acid probe molecules. In general, a nucleic acid probe molecule is an oligonucleotide that includes a label that is directly or indirectly detectable to provide a signal. Non-limiting examples of labels can include fluorescent labels or metals, such as lanthanide elements or other metals that allow detection in a mass spectrometer. Other examples of detectable labels include, for example, isotopes (such as 32P, 35S, etc.), quantum dots, organic dyes, polymer nanoparticles, metal nanoparticles, and Raman dots. Methods for preparing labeled oligonucleotide probes with any of a variety of different detectable parts are known to those skilled in the art. Multiple copies of nucleic acid probes that bind to the repeated sequences in the concatemers produced as described herein can be combined with such concatemers, so that each target molecule bound by the target binding ligand described herein has multiple label parts associated with it, greatly amplifying the signal, thereby being able to determine the presence, position, and / or amount of a given target, achieving single molecule sensitivity.

[0092] 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.

[0093] Exemplary fluorophores include, but are not limited to, 1,5-IAEDANS, 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), Alexa Fluor 350 TM Alexa Fluor 430 TM 、AlexaFluor 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, BlancophorFFG, 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, Bodipy R6G 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 , Cascade Yellow, 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、Cy7 TM , 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 、FM 4-46、Fura Red 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 shift (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, NylosanBrilliant Iavin E8G, Oregon Green TM , Oregon Green488-X, Oregon Green TM 488、OregonGreen 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, ProcionYellow, Propidium Iodide (PI), PyMPO, Pyrene, Pyronine, Pyronine B, PyrozalBrilliantFlavin 7GF, QSY 7, Quinacridone, Resorufin, RH 414, Rhod-2, Rhodamine, Rhodamine 110, Rhodamine 123, Rhodamine 5-GLD, Rhodamine 6G, Rhodamine B 540, Rhodamine B 200, Rhodamine B (Rhodamine Bextra), 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, SevronBrilliantRed 2B, SevronBrilliantRed4G, SevronBrilliant RedB, 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, thiodicarbocyanine (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, WW781, XL665, X-rhodamine, XRITC, xylenol orange, Y66F, Y66H, Y66W, yellow GFP, YFP, YO-PRO-1, YO-PRO-3, YOYO-1, and YOYO-3. Methods for fluorescent labeling of nucleic acids are known in the art. For multiplex analysis approaches, probes labeled with fluorophores having distinguishable excitation and / or emission spectra can be used.

[0094] Generate concatemers:

[0095] In various aspects, the basic method of labeling a target binding ligand comprises generating concatemers on the target binding ligand, wherein the concatemer repeating unit provides multiple binding sites for nucleic acid probes on a single target binding ligand molecule, thereby providing multiplex amplification of the signal from the target binding ligand. As described above, the methods, compositions and kits described herein allow for multiplex labeling and detection of different target binding ligands, which can be achieved by using orthogonal concatemer / probe sets and using distinguishable labels. In one aspect, a method of binding labeling includes, for example: (a) providing a conjugate of a target binding ligand and a first oligonucleotide primer; (b) in a reaction mixture, under conditions that allow hybridization and extension of the first oligonucleotide primer using a 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 generating a concatemer comprising repeating units of an oligonucleotide primer sequence, the concatemer being conjugated to a target binding ligand. In one embodiment of the methods and other methods described herein, the steps are performed in the order shown.

[0096] In various embodiments of the concatemer labeling method, 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.

[0097] In various embodiments, the concatemer associated with the target binding ligand may include branches that further expand the number of times the concatemer is repeated, thereby expanding the number of sites available for binding to the probe molecule, thereby further improving sensitivity. Therefore, in these embodiments, the binding labeling method also includes, for example, after at least one repetition of steps (b)-(d), 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 concatemer produced in steps (b)-(d), and allowing 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. Additional branching can be performed with appropriate primer / extender combinations to further expand the number of potential probe binding sites associated with a given target binding ligand molecule.

[0098] In an alternative to generating branched concatemer oligonucleotides, a method can be used in which a first oligonucleotide primer is extended to a target binding ligand that binds to its target to generate a first concatemer as described herein, and then the preformed branched concatemer is allowed to hybridize to multiple repeat elements on the first concatemer. In this alternative embodiment, the method further comprises, for example, after step (d): (i) adding a nucleic acid under conditions that allow hybridization, the nucleic acid comprising: (1) a second concatemer consisting of repeats 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 a sequence complementary to the first oligonucleotide primer. Additional branched concatemer additions can be performed to further increase the number of probe binding sites associated with a given target binding ligand.

[0099] The concatemer generation or concatemer labeling methods described herein can be performed on cells or tissue samples containing target ligands or detecting target ligands. This has the advantage that the migration, diffusion and binding properties of the target binding ligand will not be strongly affected by the labeling method because the target binding ligand can already bind to the cellular target - for example, 40 nucleotides or less oligonucleotides will not significantly change these properties of, for example, antibodies as target binding ligands. Therefore, although the first oligonucleotide primer can be longer or shorter depending on the situation, in some embodiments, the first oligonucleotide primer associated with the target binding ligand is on the order of 40 nucleotides or less, such as 35 nucleotides or less, 30 nucleotides or less, 25 nucleotides or less, 20 nucleotides or less or 15 nucleotides or less.

[0100] The labeling concatemer generation method can be multiplexed so that in the same set of reactions, multiple different target binding ligands are labeled with linear or branched concatemers. An example of a multiplex labeling method for a target ligand panel is listed below. In one method, the method includes: (a) providing an orthogonal first oligonucleotide primer and a first single-stranded extension template oligonucleotide pair, wherein each pair of first oligonucleotide primers has a sequence different from other first oligonucleotide primers in the group and is coupled to a different member of the target binding ligand group; (b) in a reaction mixture, under conditions that allow the use of the first single-stranded extension template of the orthogonal pair to hybridize and extend the first oligonucleotide primer, the coupled first oligonucleotide primers are contacted with the first single-stranded extension template of the orthogonal pair 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 target ligand group, wherein the concatemer comprises a repeating unit of the oligonucleotide primer sequence that binds to the target binding ligand.

[0101] In one embodiment, the first single-stranded extension template in each orthogonal first oligonucleotide primer / first single-stranded extension body 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 so that the extension template is not extended by a polymerase.

[0102] In another embodiment, the steps of the methods and other methods described herein are performed in the order shown.

[0103] The above method provides for the generation of multiple linear concatemers. As described above, branched concatemers can further amplify the signal of target binding ligands, and this method can be directly applied to multiplexing. Therefore, the method of introducing branched concatemers in a multiplex reaction also includes, for example, after step (d), the following steps: (i) adding a second set of second oligonucleotide primers and second single-stranded extension template oligonucleotides, wherein the second oligonucleotide primers in each pair contain a sequence complementary to a member of the first oligonucleotide primer set and a sequence complementary to a second single-stranded extension template of a second set of orthogonal oligonucleotide pairs in a 5' to 3' order, wherein each second single-stranded extension template contains 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 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 the corresponding second oligonucleotide primer repeating units on the corresponding members of the set of target binding ligands. This method provides branched concatemers in a multiplexed manner. Additional rounds of branching can be performed to further increase the signal for each target-binding ligand in the set.

[0104] Alternative methods for concatemer generation and multiple concatemer generation include the following: In one method, a method for generating a nucleic acid chain comprising concatemer repeats of a given sequence comprises: a) providing a first oligonucleotide primer and a first extension template oligonucleotide in a reaction mixture, the first extension template comprising a concatemer of at least two copies of the sequence linked head-to-tail, wherein the first extension template comprises a chain terminator at its 3' end, the sequence being complementary to the first oligonucleotide primer; b) incubating the reaction mixture under conditions that allow the first oligonucleotide primer to hybridize with the first extension template oligonucleotide; c) extending the hybridized first primer with a nucleic acid polymerase to produce 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 to produce an extended nucleic acid chain comprising concatemer repeats of the sequence contained in the first oligonucleotide primer. In one embodiment, the method further comprises repeating steps (d) to (f) at least once, preferably multiple times, such as 2 times, 3 times, 5 times, 10 times, 15 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, 100 times or more. In another embodiment, these steps are performed in the order presented. 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 concatenated nucleic acid by one concatenated repeat.

[0105] Where multiplexing is desired, step (a) may comprise providing an orthogonal first oligonucleotide primer and first extension template oligonucleotide pair set such that steps (b)-(f) produce a multiplexed set of concatemers, each concatemer comprising multiple repeats of the corresponding orthogonal oligonucleotide primer sequence.

[0106] Branching can also be applied in the context of this method in cases where branching can further increase the signal. Specifically, after one or more repetitions of steps (d) to (f), the improvement of the method further incorporating branching can also include: 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 at least two repeating sequences linked head-to-tail, and a chain terminator at its 3' end, the sequence being complementary to the second oligonucleotide primer; ii) aligning the second oligonucleotide primer with one of the concatemers formed after one or more repetitions of steps (d) to (f); The method further comprises the steps of: hybridizing one or more concatemer repeating units of the second oligonucleotide primer with the second extension template oligonucleotide; iii) hybridizing the second extension template oligonucleotide with the second oligonucleotide primer; iv) extending the second oligonucleotide primer with a polymerase to produce 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 produced in step (iv); vii) extending the extended nucleic acid chain complementary to the second extension template produced in step (iv) with a polymerase to produce an extended nucleic acid chain comprising multiple repeats of the sequence contained in the second oligonucleotide primer. In another embodiment, the method further comprises repeating steps (v) to (vii) at least once, preferably multiple times, for example at least 2 times, 3 times, 5 times, 10 times, 15 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, 100 times or more.

[0107] Where multiple labelling and branching is desired, step (i) may comprise providing an orthogonal set of second oligonucleotide primers and second extension template oligonucleotide pairs such that steps (ii)-(vii) produce a plurality of sets of concatemers, each concatemer comprising concatemer repeats of the corresponding orthogonal oligonucleotide primer sequence.

[0108] Several embodiments of the concatemer generation methods described herein are shown in FIG. 7 .

[0109] The single-stranded oligonucleotide primer will contain or consist of a unit sequence that will be repeated in the concatemers generated using the methods described herein. Fig. 7AIn practice, 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 that becomes the concatemer repeat sequence, but the 3' portion of the original oligonucleotide primer that is complementary to the sequence of the head-to-tail oriented repeats in the single-stranded extension template will determine the repeat sequence. For example, this can be determined by reference to Fig. 7A To clarify, Fig. 7A Oligonucleotide primer "a" and extension "a*a*" are shown, where a* is the complementary sequence of a. An additional sequence (not shown) that is not necessarily complementary to a* is optionally added to the 5' end of the primer, which essentially acts as a linker and will not be replicated in the concatemer based on the a* copy of the extension template. The single-stranded extension template will be discussed in more detail below and demonstrated in the embodiment.

[0110] As discussed further below, the advantage of the methods, compositions and kits described herein is that concatemer-labeled target binding ligands can be produced 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 multiplexing degree that can be achieved using orthogonal primers 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.

[0111] In one embodiment, the target molecule itself can be an RNA molecule, including but not limited to mRNA molecules. The hybridization of one or more single-stranded oligonucleotides provides a starting site for producing concatemers using single-stranded extension template oligonucleotides as described herein, wherein the single-stranded oligonucleotide comprises a 5' region complementary to the sequence on the target mRNA and a 3' region comprising at least one sequence extended as 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. When the target molecule is RNA, the single-stranded target binding ligand oligonucleotide can be hybridized with its RNA target in the cell, 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 as 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.

[0112] 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 one sequence extended as 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. When the target molecule is a DNA molecule of a given sequence, the single-stranded target binding ligand oligonucleotide can be hybridized with the DNA target in the cell by the target complementary sequence at the 5' end of the 3' sequence, extended as a concatemer repeat element, and then (after removing unhybridized oligonucleotides) cross-linked, such as known in the art or described herein, so as to maintain its binding to the target DNA by subsequent concatemer generation steps.

[0113] As previously mentioned, an important advantage of the methods, compositions and kits described herein is the ability to perform the concatemer generation step in multiplex, such that multiple different target binding ligands have different concatemer probe landing pads generated in multiplex from target binding ligands associated with their cellular targets. Figure 7B As shown, an orthogonal primer having a repeating sequence "a", "b" ... "n" is extended using a single-stranded extension template having at least two repeats or copies of a complementary sequence "a*", "b*", ... "n*". Once generated, the different concatemers provide landing pads for orthogonal complementary probes that can also multiplex to detect various target ligands. Various methods for detecting and distinguishing probes in multiplexing are known in the art and / or discussed elsewhere herein.

[0114] As described herein, the target binding ligand with oligonucleotide primers can be contacted with a 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 immune tissue to detect a given target ligand. Therefore, the method for contacting a cell or tissue sample with a target binding ligand can be parallel to the method for, for example, a fluorescently labeled antibody or antibody fragment. When performed in a multiplex manner, contact or staining can include adding a target binding ligand group, 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 a cell or tissue preparation with a 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.

[0115] Generation of concatemers:

[0116] 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, for example, can be performed in a cell sample with a target binding ligand bound to its target molecule. These methods generally use an oligonucleotide bound to a target binding ligand and a single strand extension template, and repeat cycles of annealing, polymerase extension, and hot strand separation, wherein each cycle adds another concatemer repeat 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, wherein the concatemer is conjugated to the target binding ligand.

[0117] 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.

[0118] 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 and include, for example, OligoPerfect TM Primer Designer, etc.

[0119] Therefore, in a given concatemer generation scheme, an oligonucleotide primer that binds to a target binding ligand, such as an oligonucleotide that binds to its cellular target, is contacted with a single-stranded extension template oligonucleotide at a temperature determined, for example, based on the Tm of the primer-template hybrid double strand in a reaction mixture suitable for the polymerase used. This allows the single-stranded extension template to be annealed to the primer, and when incubated at the extension temperature of the polymerase, the annealed primer is extended by the selected polymerase. Heating is performed to separate the extended chain from the single-stranded extension template, cooling is performed to allow the single-stranded extension template to anneal, and incubating at the extension temperature of the polymerase, thereby generating a concatemer of oligonucleotide primer sequence repeating units defined by the single-stranded extension template. When performed in a multiplex manner, a combination of different orthogonal repeats on different target binding ligands that bind to respective cellular targets is generated. Subsequent detection based on distinguishable labeled probes hybridized to concatemers allows detection, quantification and / or imaging of target molecules in or on cells.

[0120] Single strand extension template:

[0121] The use of single-stranded extension templates is the core of the methods, compositions and kits described herein. The single-stranded extension templates that can be used for the methods, compositions and kits described herein include a concatemer of at least two copies of a sequence that is oriented head-to-tail and connected, and the sequence is complementary to an oligonucleotide primer that binds a target ligand. 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 a polymerase extension of the concatemer. The design of the single-stranded extension template provides a method in which a copy of the concatemer repeat sequence complement is hybridized with an oligonucleotide that binds 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 the oligonucleotide that is bound to the target ligand. As a representative illustration, please refer again to Figure 1A (extension body a*a*, with a 3' "blocker" 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 sequence is determined by parameters that allow multiple target detection while minimizing primer interactions and off-target hybridization.

[0122] In a multiplex experiment, different sets of single-stranded extension templates can be used, orthogonal to the oligonucleotide primer sets, which are coupled to corresponding members of different target binding ligand sets. As with the single-stranded extension templates used alone, each single-stranded extension template in the set comprises a concatemer of at least two copy sequences connected in a head-to-tail orientation, which are complementary to different oligonucleotide primers that bind to the target binding ligand, for example, as described herein. Each single-stranded extension template in the set also includes a 3' blocking or chain termination modification that prevents polymerase extension of the extension template. For example, as shown in Figure 1B, this multiplexing method (extension bodies composed of a*a*, b*b* and n*n*, each of which has a 3' termination position) produces concatemers with different repeat unit sequences on each member of the target binding ligand set.

[0123] 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.

[0124] Branched chain concatemers:

[0125] One option for further increasing the number of probe binding sites on a target binding ligand is to introduce branches so that the initial linear concatemer of the target binding ligand provides sites for generating additional concatemers that branch from 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 repeats, thereby increasing the number of potential probe binding sites on a given target binding ligand. In some embodiments, branches are generated 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.

[0126] In one embodiment, to generate branched concatemers 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 as described herein. The considerations for designing the second oligonucleotide primer for single and multiple branched concatemer generation are the same as those for designing the first oligonucleotide primer 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.

[0127] 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 molecules of the second oligonucleotide primer are allowed to anneal to at least one multi-repeat unit (preferably a plurality of multi-repeat units) in the first extension multi-linked oligonucleotide through their sequence complementary to the first oligonucleotide primer.

[0128] 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.

[0129] For concatemer generation, the second single-stranded extension template anneals to a complementary sequence on the second oligonucleotide primer, and the second oligonucleotide primer is extended, thereby adding new copies of the second concatemer repeats to new branches of the first concatemer. Repeated cycles of hot strand separation and second single-stranded template-directed primer extension produce concatemers containing repeat sequences, including the sequence in the second oligonucleotide primer. When multiple second oligonucleotide primers hybridize to multiple concatemer repeats on the first extended concatemer oligonucleotide, the first extended oligonucleotide effectively provides a backbone with many branches, each branch including multiple repeats of the second concatemer repeat sequence. Additional branching using a third, fourth, fifth, or more oligonucleotide primers and orthogonal single-stranded extension templates that are designed similarly to the second oligonucleotide primer / second single-stranded extension template can further expand the number of probe binding sites associated with a given target binding ligand.

[0130] Another branching method produces concatemers of a second repeating sequence that contains a sequence at its 5' end that is complementary to the first concatemer repeats. These second concatemer molecules are then allowed to hybridize with the repeating sequence in the first target binding ligand-associated concatemer through the complementary sequence to provide branched concatemers. The sequence that is complementary to the first concatemer repeats may include a (photo) crosslinking portion that allows the second concatemer to be crosslinked to the first concatemer. It is expected that the second component molecule itself may include branches with a third, fourth, fifth or more repeating elements to further amplify the number of probe binding sites associated with a given target binding ligand.

[0131] Any of the alternative branching methods described above can be used in the multiplex reactions described herein.

[0132] Detection of target molecules:

[0133] Among other uses, compositions, methods and kits described herein can be used to detect target molecules in biological samples. Once a multiple sequence is produced on a target binding partner that is bound to its target in or on a biological or cell sample, a labeled nucleic acid probe (or an orthogonal probe set when performing multiple detection) can contact the multiple repeat sequences of the target binding partner and allow it to hybridize. Labeled probe molecules as described herein can include a cross-linking portion that allows the probe to be cross-linked with its complement. The labeled probe molecules are optionally cross-linked to their constituent sequences by ultraviolet activation of the cross-linking portion, and can then be detected in a manner suitable for a given label, including but not limited to fluorescence-activated flow cytometry or cell sorting, and a mass spectrometer.

[0134] Therefore, in various embodiments, detection includes contacting the concatemers produced in the previous step with labeled nucleic acid probes. In some embodiments, the nucleic acid probe comprises a sequence that is complementary to the concatemer repeats. In some embodiments, the nucleic acid probe comprises a sequence that is complementary to the oligonucleotide primer associated with the target binding ligand. When branched concatemers are involved, the repetitive sequences on the branched concatemers can be contacted with nucleic acid probes that are complementary to these repetitive sequences. The hybridization of multiple nucleic acid probe molecules with multiple repetitive sequences associated with the target binding ligand provides sensitive target detection, and when used in the multiplexing allowed by the disclosed method, provides simultaneous determination of 5 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, or up to 60 or more expression profiles of different biomolecular targets.

[0135] 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 envisioned that repeated detection of different fluorophores with non-overlapping excitation spectra could be performed using different wavelengths. Similarly, detecting non-overlapping emission spectra of different fluorophores can be used to detect multiple different fluorophores associated with multiple different targets. It is also envisioned that a first round of fluorescently labeled probes could be applied and detected, followed by photobleaching of the fluorophores, before a second round of different probes labeled with the same fluorophore or fluorophores as the first round is applied.

[0136] When mass cytometry is used for detection (see below and in the Examples herein), each different metal ion label present in or on the cell can be distinguished by its mass-to-charge ratio, so that orthogonal probes with different metal ion labels can be used to multiplex different target molecules in the same assay.

[0137] Given that the multiple repeat sequences on the 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.

[0138] Mass cytometry:

[0139] Mass cytometry, also known as time-of-flight cytometry Provides a tool for high-dimensional and high-throughput single-cell analysis. Mass spectrometers were first introduced in 2009 (Bandura et al., Anal. Chem. 81: 6813-6822 (2009)) and have been widely used in the analysis of immune cell function / activation and other processes due to their high parameter capabilities. By using metal ion labels instead of fluorescent labels commonly used in fluorescence-based flow cytometry, mass spectrometers overcome the problem of overlapping emission spectra, thereby reducing the number of different targets or parameters that can be analyzed in a single assay.

[0140] For example, Iyer et al., Front.Immunol.13, Article 815828 (2022) review the methods, applications, and considerations for performing mass spectrometry cytometry. In brief, in a mass spectrometer, cells are typically incubated with a mixture of antibodies labeled with unique non-radioactive heavy metal (most commonly lanthanide elements) 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, which atomizes and ionizes the sample. This process converts each cell into a cloud containing ions of the elements 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, the ions are separated by mass-to-charge ratio. Upon encountering a detection agent, the ion counts are amplified and converted into electrical signals. 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 spectrometer panel. When adapted for use with the concatemer-based labeling / signal amplification approach described herein, highly multiplexed, highly sensitive detection of a large number of even low-abundance targets is possible.

[0141] As described above, the concatemer-based signal amplification methods described herein can be readily applied to other biomolecular target detection or analysis methods. Non-limiting examples include imaging mass spectrometry cytometry (see, e.g., Giesen et al., Nat. Methods 11:417-422 (2014), see also Hyperion+ TM Imaging system (Fluidigm, Inc.; see the World Wide Web at Fluidigm.com / products-services / technologies / imageg-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.

[0142] It is noteworthy that the concatemer-based methods and compositions described herein, as well as the cross-linking of probes to target binding ligand-related nucleic acids, 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 signals in (see Wang, H. et al. (2015) Multiplex Fluorescent RNA In Situ Hybridization Via RNAscope. In: Hauptmann, G. (eds) In SituHybridization Methods. Neuromethods, vol 99 (2015). Humana Press, New York City, NY). Another option is applicable to branched DNA FISH or InSituPlexTM (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 associated with target molecules or target binding ligand molecules. In this method, the probes described herein (e.g., using CNVK or other crosslinking agents described herein in conjunction with the probes) are crosslinked to nucleic acids associated with molecules that hybridize or bind to a given target RNA species, such as in RNAScope. TM Or in branched DNA FISH, it can be used to maintain the binding of the probe to the nucleotide associated with the molecule.

[0143] 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, e.g., 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 the nucleic acid probe 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).

[0144] 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 adapted in a similar manner using CNVK cross-linking chemistry. For example, long repeat sequences generated according to the SABER method can be hybridized to oligonucleotides on a target binding ligand (e.g., an antibody or other target binding ligand) and cross-linked to CNVK, and nucleic acid probe molecules can be hybridized to these repeat sequences and similarly cross-linked to CNVK to provide signal amplification (e.g., in a manner such that the repeat sequence and the probe do not dissociate from the target in subsequent steps). In the case of generating repeat sequences, the same approach can be applied, for example, by hybridization chain reaction (HCR; see, e.g., Hybridization chain reaction: a versatile molecular tool for biosensing, bioimaging, and biomedicine. Chem. Soc. Rev. 46: 4281-4298 (2017).

[0145] In a further modification, the concatemer approach as described above and demonstrated in the embodiments can be adapted to use click chemistry, rather than CNVK cross-linking, to covalently bind, for example, nucleic acids containing repeats 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)).

[0146] Ordered extension and nucleic acid generation

[0147] 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 body elements can be used, each extension element comprising a sequence complementary to the 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.

[0148] In one embodiment, as shown in FIG. 7C , the ordered extension method for producing a desired sequence nucleic acid molecule includes providing a first oligonucleotide primer, and contacting the first oligonucleotide primer with a first single-stranded extension template in a reaction mixture and in a reaction mixture, contacting the first oligonucleotide primer with the first single-stranded extension template and a nucleic acid polymerase under conditions that allow the first oligonucleotide primer to hybridize and extend using the 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 the 5' to 3' direction. This step extends the first oligonucleotide primer by the complementary sequence of the first extension template element. It should be noted that this ordered extension method can be performed in the following manner: adding continuous single-stranded extension templates in continuous steps, or, as an alternative, a complete set of single-stranded extension templates can be added at one time, and an ordered extension is constructed in a single reaction system based on the specific hybridization of the appropriate single-stranded extension template with each continuously added extension product through continuous thermal cycles.

[0149] 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 hybridization and extension of 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 copy of the second extension template element, 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. Several consecutive rounds of strand separation and contact with the continuous single-stranded extension template can be performed, each round including 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, which 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, among other uses, a surface, a target binding ligand, or other moiety to which the first oligonucleotide is coupled.

[0150] 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 repetitions added in this way can provide signal amplification as described herein.

[0151] The ordered extension method can also be performed in a multiplex. Primer design considerations are similar to those described herein for designing primers for the multiplex concatemer generation protocol.

[0152] The ordered extension method can be carried out in solution with the first oligonucleotide primer, or on the surface of the nucleic acid molecule to be constructed, or by coupling the first oligonucleotide primer to the surface. The method can also be carried out in the case where the oligonucleotide primer is coupled to a target binding ligand as described herein. The method can also be carried out in contact with a cell or tissue sample, so that the nucleic acid molecule is generated in situ in the cell or tissue sample, for example, in association with a target molecule bound by a target binding ligand in the cell or tissue sample.

[0153] 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. Detecting the label associated with the probe may provide information on the presence, amount and / or location of the target associated with the first oligonucleotide primer.

[0154] In another approach, a method is also considered to add long DNA chains that are extended only once, rather than short DNA chains through multiple rounds of thermal cycling. The long DNA chain extended in one step is a variation of the schematic of Figure 1C - considering a2* as a repeat of imaging probe binding. 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, another option is to incorporate uracil bases into the long DNA chain and digest it using uracil-DNA glycosylase (e.g., uracil-DNA-glycosylase; M0280S, New England Biolabs) to fragment the long DNA chain after the one-step extension to allow imager binding and CNVQ cross-linking.

[0155] Reagent test kit:

[0156] In various embodiments, a kit for performing one or more methods described herein is provided. Such kits may include, for example, a first oligonucleotide primer or a first oligonucleotide primer set as described herein, and reagents sufficient to couple the oligonucleotide primer to one or more target binding partners. The kit may include a target binding partner coupled to an oligonucleotide primer, or a different target binding partner group coupled to an orthogonal oligonucleotide primer set. The target binding partner may include, for example, a peptide, a polypeptide, a nucleic acid, an aptamer, a receptor and / or its multiple ligands, a member of an affinity binding pair (including but not limited to biotin / streptavidin), and a small molecule agent that specifically binds to a target molecule as defined herein. Antibodies and antigen binding fragments or their constructs represent a class of target binding partners that can be used for 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 partners that can be used for methods, compositions, and kits described herein.

[0157] The kit can include a single-stranded extension template, or a set of single-stranded extension templates suitable for performing the concatemer generation methods described herein.

[0158] The kit can include, for example, an orthogonal set of oligonucleotide primers (bound to a target binding ligand or prepared as such coupling) and a single-stranded extension template, allowing multiple uses. 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 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 concatemer repeat element, or complementary to one or more elements in an ordered extension product generated as described herein. The labeled nucleic acid probe molecule can include a crosslinking portion. The crosslinking portion can be a UV crosslinking portion, and can include, for example, 3-cyano vinyl carbazole phosphoramidite (CNvK). The label can include any label described herein or known in the art, including but not limited to fluorescent labels, metal ion labels, etc. The metal ion label can be, for example, a lanthanide metal ion, or can include, for example, cadmium, palladium, indium, platinum, bismuth, selenium, tellurium, gold, silver and tantalum, yttrium, ruthenium, rhodium, iodine, osmium and iridium, etc. In some embodiments, the labeled nucleic acid probe can include a metal ion label and a fluorescent label.

[0159] The kit may include packaging materials for the various components, such as instructions for use.

[0160] The technology may be as described in any one of the following numbered embodiments:

[0161] Embodiment 1: A method for detecting a target molecule in mass spectrometry cytometry, the method comprising: a) contacting a target binding ligand molecule comprising a coupled oligonucleotide primer with a cell or tissue sample under conditions that allow the target binding ligand molecule to bind to its target in the cell or tissue sample; b) extending the oligonucleotide primer to include a multimer of the sequence included in the oligonucleotide primer; c) contacting the extended oligonucleotide primer of step (b) with a labeled probe nucleic acid, the labeled probe nucleotide comprising a sequence complementary to a repeated sequence in the multimer, so that multiple labeled probe DNA molecules hybridize with multiple sequence repeating units in the multimer; d) cross-linking the hybridized probe nucleic acid molecules to the extended oligonucleotide primer; e) detecting the labeled probe using mass spectrometry cytometry.

[0162] Embodiment 2: The method according to embodiment 1, wherein the labeled probe nucleic acid includes a metal ion label.

[0163] Embodiment 3: The method according to embodiment 1 or embodiment 2, wherein the labeled probe comprises a fluorophore.

[0164] Embodiment 4: The method according to any one of embodiments 1-3, wherein extending the oligonucleotide primer comprises thermal cycling with a single-stranded extension template and a template-dependent nucleic acid polymerase.

[0165] Embodiment 5: The method according to embodiment 4, wherein the polymerase is thermostable.

[0166] Embodiment 6: The method according to embodiment 4 or embodiment 5, wherein the single-stranded extension template comprises a concatemer of at least two copies of the sequence connected in a head-to-tail direction and a 3' chain terminator so that the extension template is not extended by the polymerase, and the sequence is complementary to the oligonucleotide primer.

[0167] Embodiment 7: A method according to any one of embodiments 1-6, wherein the extension step (b) includes multiple extension, hot chain separation and cooling cycles to allow annealing of single-stranded extension bodies and extension by the polymerase, whereby consecutive cycles add consecutive multi-linked repeat units to the oligonucleotide primer.

[0168] Embodiment 8: The method according to any one of embodiments 1-7 is multiplexed with a plurality of different target binding ligand molecules, orthogonal sets of single-stranded extension templates, and distinguishably labeled orthogonal probe nucleic acids.

[0169] Embodiment 9: A method according to any one of Embodiments 1-8, wherein the concatemer comprises a branched concatemer formed on the target binding ligand by thermal cycling.

[0170] Embodiment 10: The method according to Embodiment 3 further comprises the step of imaging the fluorophores associated with the cell or tissue sample before the detection step (e).

[0171] Embodiment 11: The method according to any one of embodiments 1-10, wherein cross-linking the hybridized probe nucleic acid molecule to the extended oligonucleotide primer comprises photocross-linking.

[0172] Embodiment 12: A method according to embodiment 11, wherein the probe nucleic acid molecule comprises a photocrosslinker.

[0173] Embodiment 13: The method according to Embodiment 12, wherein the photocrosslinking agent comprises 3-cyanovinylcarbazole phosphoramidite (CNVK) and the crosslinking comprises ultraviolet irradiation.

[0174] Embodiment 14: A method according to any one of Embodiments 1-13, wherein the target binding ligand comprises an antibody or an antigen-binding fragment thereof.

[0175] Embodiment 15: A fluorescence-activated cell sorting method, the method comprising: a) providing a sample comprising fixed cells; b) contacting a target binding ligand molecule comprising a coupled oligonucleotide primer with cells in the sample under conditions that allow the target binding ligand molecule to bind to its target in or on the cell; c) extending the oligonucleotide primer to include a multimer of the sequence contained in the oligonucleotide primer; d) contacting the extended oligonucleotide primer of step (c) with a fluorescently labeled probe nucleic acid, the probe nucleic acid comprising a sequence complementary to a repeated sequence in the multimer, so that multiple labeled probe nucleic acid molecules repeatedly hybridize with multiple sequences in the multimer; e) cross-linking the hybridized probe nucleic acid molecules to the extended oligonucleotide primer; and f) sorting the cells by fluorescence-activated cell sorting.

[0176] Embodiment 16: The method according to Embodiment 15, wherein the labeled probe further comprises a metal ion.

[0177] Embodiment 17: The method according to embodiment 15 or 16, wherein extending the oligonucleotide primer comprises thermal cycling with a single-stranded extension template and a template-dependent nucleic acid polymerase.

[0178] Embodiment 18: The method of embodiment 17, wherein the polymerase is thermostable.

[0179] Embodiment 19: A method according to embodiment 17 or embodiment 18, wherein the 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 oligonucleotide primer.

[0180] Embodiment 20: A method according to any one of embodiments 15-19, wherein the extension step (c) comprises multiple extension, hot chain separation and cooling cycles to allow annealing of single-stranded extension bodies and extension by the polymerase, whereby consecutive cycles add consecutive multi-linked repeat units to the oligonucleotide primer.

[0181] Embodiment 21: The method according to any one of embodiments 15-20 is multiplexed with a plurality of different target binding ligand molecules, orthogonal sets of single-stranded extension templates, and distinguishably labeled orthogonal probe nucleic acids.

[0182] Embodiment 22: A method according to any one of Embodiments 15-21, wherein the concatemer comprises a branched concatemer formed on the target binding ligand by thermal cycling.

[0183] Embodiment 23: The method according to any one of Embodiments 16-22, further comprising a step of detecting the metal ion labeled probes by a mass spectrometer after the sorting step (f).

[0184] Embodiment 24: The method of any one of Embodiments 15-23, wherein cross-linking the hybridized probe nucleic acid molecule to the extended oligonucleotide primer comprises photo-cross-linking.

[0185] Embodiment 25: A method according to any one of embodiments 15-24, wherein the probe nucleic acid molecule comprises a photo-crosslinking portion.

[0186] Embodiment 26: A method according to Embodiment 25, wherein the photocrosslinking moiety comprises 3-cyanovinylcarbazole phosphoramidite (CNVK) and the crosslinking comprises ultraviolet irradiation.

[0187] Embodiment 27: A method according to any one of Embodiments 15-26, wherein the target binding ligand comprises an antibody or an antigen-binding fragment thereof.

[0188] Embodiment 28: A composition comprising a target binding ligand and an oligonucleotide that binds to the target binding ligand.

[0189] Embodiment 29: The composition of Embodiment 28, wherein the concatenated oligonucleotide further comprises one or more branched concatenated oligonucleotides.

[0190] Embodiment 30: The composition according to embodiment 28, further comprising a plurality of nucleic acid probe molecules hybridized to the concatenated repeating units of the concatenated oligonucleotide.

[0191] Embodiment 31: A composition according to embodiment 30, wherein the nucleic acid probe molecule comprises a cross-linking portion.

[0192] Embodiment 32: The composition of Embodiment 31, wherein the crosslinking moiety comprises 3-cyanovinylcarbazole phosphoramidite (CNVK).

[0193] Embodiment 33: A composition according to embodiment 31 or 32, wherein the nucleic acid probe molecules are cross-linked to the multiple repeat sequences.

[0194] Embodiment 34: A composition according to any one of Embodiments 30-33, wherein the nucleic acid probe molecule comprises a fluorescent label or a metal ion label.

[0195] Embodiment 35: A composition according to any one of Embodiments 30-34, wherein the nucleic acid probe molecule comprises a fluorescent label and a metal ion label.

[0196] Embodiment 36: A composition according to any one of Embodiments 30-35, wherein the target binding ligand comprises an antibody or an antigen-binding fragment thereof.

[0197] Embodiment 37: A cell or tissue sample comprising the composition of any one of embodiments 30-36, wherein the target binding ligand binds to a target molecule in or on the cell or tissue sample.

[0198] Embodiment 38: A kit for implementing one or more methods described herein, the kit comprising: a) a target binding ligand coupled to an oligonucleotide primer; or a target binding ligand group coupled to an orthogonal oligonucleotide primer; b) a single-stranded extension template or an orthogonal single-stranded extension template group; c) a labeled probe nucleic acid or an orthogonal labeled probe nucleic acid group, wherein the labeled probe nucleic acid or the orthogonal labeled probe nucleic acid group comprises a cross-linking portion.

[0199] Embodiment 39: A kit according to embodiment 38, wherein the single-stranded extension template comprises a concatemer of at least two copies of a 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 oligonucleotide primer.

[0200] Embodiment 40: The kit according to Embodiment 38 further comprises a template-dependent nucleic acid polymerase.

[0201] Embodiment 41: A kit according to Embodiment 40, wherein the polymerase is thermostable.

[0202] Embodiment 42: A kit according to any one of Embodiments 38-41, wherein the target binding ligand comprises an antibody or an antigen-binding fragment thereof.

[0203] Embodiment 43: A kit according to any one of embodiments 38-42, wherein the orthogonal nucleotide primers, single-stranded extension template and nucleic acid probe are optimized to avoid primer / extension body cross-talk, primer dimer formation and off-target hybridization.

[0204] Embodiment 44: The kit according to any one of Embodiments 38-43, wherein the single-stranded extension template or the set of single-stranded extension templates is suitable for performing a concatemer generation method.

[0205] Embodiment 45: The kit according to any one of embodiments 38-44, further comprising packaging materials and instructions for use of the various components.

[0206] Embodiment 46: The kit according to any one of Embodiments 38-45, further comprising one or more target binding ligand molecules or reagents for coupling the first oligonucleotide to the target binding ligand.

[0207] Embodiment 47: The kit according to any one of Embodiments 38-46, further comprising a thermostable polymerase, nucleotides, reaction buffer components and reagents for labeling nucleic acid probe molecules.

[0208] Embodiment 48: A kit according to any one of embodiments 38-47, wherein the probe molecule can be complementary to the concatemeric repeat element.

[0209] Embodiment 49: A kit according to any one of Embodiments 38-48, wherein the cross-linking moiety comprises a photo-cross-linking moiety.

[0210] Embodiment 50: The kit of any one of Embodiments 38-49, wherein the cross-linking moiety comprises 3-cyanovinylcarbazole phosphoramidite (CNVK).

[0211] Embodiment 51: A kit according to any one of Embodiments 38-50, wherein the labeled probe nucleic acid or the set of orthogonally labeled probe nucleic acids comprises a fluorescent label or a metal ion label.

[0212] Embodiment 52: The kit described in any of Embodiments 38-51, wherein the labeled nucleic acid probe molecules include fluorescent labels and metal ion labels.

[0213] The specific elements of any disclosed embodiment may be combined or substituted for 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.

[0214] Example

[0215] 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 that these modifications and variations are included within the scope of the present invention defined in the following claims. The following examples do not limit the present invention in any way.

[0216] Example 1

[0217] Mass spectrometry is a state-of-the-art approach 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 researchers have introduced mass spectrometry cytometry as a versatile 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 analysis, these types of experiments have revealed new signaling mechanisms involved in cancer progression and drug resistance.

[0218] However, single-cell signaling network analysis has not yet been translated to studying patient-derived tumor samples in cancer clinical research. Similar to many other types of single-cell methods, mass spectrometers face 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 levels of phosphorylation sites (i.e., in the absence of additional stimulation) often do not reach the detection limit of the mass spectrometer, especially in cells with smaller volumes, 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 prior to analyzing samples. In the imaging mode of mass spectrometry cytometry (IMC), imaging resolution must be compromised to compensate for the low sensitivity that makes it difficult to identify spatial cues that determine signal outcomes.

[0219] A DNA nanodevice has recently been created that performs repeated in situ ligation under thermal cycling conditions. Combining this device with a newly developed photocrosslinking strategy based on 3-cyanovinylcarbazole phosphoramidite (CNvK) modification, the described 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.

[0220] Methods. In conventional mass spectrometry cytometry approaches, 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. With this approach, only a few modification sites on each antibody can be bound, carrying a limited number of metal ions (Figure 1A). To improve the sensitivity of mass spectrometry analysis, the main innovation of my new approach, termed cyclic extension amplification by DNA oligonucleotides (ACED), is the design of a DNA nanodevice to generate infinitely repeated metal probe hybridization sites in situ (Figure 1B). In ACED, antibodies targeting proteins of interest are conjugated to short DNA oligomer triggers (TT-a, 11-mer), which maintain antibody binding affinity, maximize the ability of the antibody to diffuse within the cell, and reduce nonspecific binding, which is common in approaches that apply long oligomer (>40mer) conjugates. 14,15 (Figure 1B, step 1). The bound antibody is mixed in a staining solution and applied to a cell suspension for staining of cell surface or intracellular markers (step 2). Next, an extension body (a'-T-a', 19-mer) with a starting sequence of two complementary repeat units is introduced into the stained cells. At low temperatures, the extension body and the initiator hybridize to allow BST polymerase drug chain extension (forming TT-aAa, step 3). By increasing the reaction temperature, the extension body is removed to expose the single-stranded extended probe (step 4). The thermal cycle is then repeated with the desired rounds to continuously extend the antibody-bound probe (step 5), generating hundreds of aA repeats at each antibody-modified site (step 6). + The DTPA polymer of the metal ion is coupled to the detection strand with the sequence a'-T-a' through a maleimide-thiol reaction, which can then be hybridized with the extended DNA probes on the antibody (step 7), with each probe occupying one metal probe binding site (aAa). Brief (1 second) ultraviolet (UV) irradiation activates the 3-cyanovinylcarbazole phosphoramidite (CNVK) photocrosslinker on the metal probe strand, creating a covalent bond between the hybridized DNA molecules and allowing the DTPA polymer to be attached to the antibody (step 8)( Figure 2 ).

[0221] To verify the specificity and quantify the amplification capacity of ACED in mass spectrometry analysis, the system was applied to human embryonic kidney HEK293T cells transiently overexpressing green fluorescent protein GFP in a high expression gradient. As expected, the GFP antibody signal amplified by ACED was highly correlated with the secondary antibody signal targeting the first GFP antibody, confirming the specificity of the established DNA device in intracellular antibody signal amplification (Figure 3A). Through time series analysis of 500 thermal cycles, we found that the ACED method continuously amplified the mass spectrometry cytometer signal during the thermal cycling time (Figure 3A). Compared with the unamplified samples (Figure 3C), 13-fold amplification intensity and 6-fold signal-to-noise ratio enhancement were observed in samples amplified for 500 rounds according to GFP expression levels (Figure 3B). In order to detect and quantify ultra-low abundance proteins, linear ACED amplification can be inefficient and time-consuming. For these proteins, we developed a branch amplification strategy in which the amplification capacity can be increased exponentially through iterative cycles of the ACED reaction. Quantification of the branch results by mass spectrometry cytometer analysis observed a 17-fold amplification for each branch round ( Figure 4 ). Ultimately, unlimited signal amplification can be achieved through multiple branching reactions. In order to multiplex the ACED approach, a high degree of orthogonality between the DNA probes targeting different antibodies is required. 50 orthogonal ACED barcode sequences were designed to cover the full capacity of the mass spectrometer. In a proof-of-concept test, six initiator probes were cross-reacted with six extender probes to thoroughly evaluate their orthogonality ( Figure 5 In subsequent mass spectrometry analysis, only the paired initiator and extender sequences produced detectable signals ( Figure 5 ). Then, a 30-fold signal amplification was performed on the mouse Py2T cell line that underwent epithelial-mesenchymal transition (EMT) over a 7-day period. 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 ( Figure 6 Dimensionality reduction analysis using Uniform Manifold Approximation and Projection (UMAP)16 projected the trajectory of EMT progression over time, validating ACED as a powerful technology platform that can accurately amplify mass spectrometer signals, especially for low-abundance proteins ( Figure 6 ).

[0222] References

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[0224] 2.Bendall,S.C.,Simonds,E.F.,Qiu,P.,Amir,E.D.,Krutzik,P.O.,Finck,R.,Bruggner,R.V,Melamed,R.,Trejo,A.,Ornatsky,O.I.,Balderas,R.S.,Plevritis,S.K.,Sachs,K.,Pe’er,D.,Tanner,S.D.,&Nolan,G.P.Single-cell mass cytometryofdifferential immune and drug responses across a human hematopoieticcontinuum.Science 332,687-96(2011)PMID:21551058.

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[0227] 5.Levine,J.H.,Simonds,E.F.,Bendall,S.C.,Davis,K.L.,Amir,E.D.,Tadmor,M.D.,Litvin,O.,Fienberg,H.G.,Jager,A.,Zunder,E.R.,Finck,R.,Gedman,A.L.,Radtke,I.,Downing,J.R.,Pe’er,D.,&Nolan,G.P.Data-Driven Phenotypic DissectionofAML Reveals Progenitor-like Cells that Correlate with Prognosis.Cell 162,184-197(2015)PMID:26095251.

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[0234] 12.Jackson,H.W.,Fischer,J.R.,Zanotelli,V.R.T.,Ali,H.R.,Mechera,R.,Soysal,S.D.,Moch,H.,Muenst,S.,Varga,Z.,Weber,W.P.,&Bodenmiller,B.The single-cell pathology landscape of breast cancer.Nature578,615-620(2020)PMID:31959985.

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[0239] Example 2: Single prokaryotic cell analysis of ACE on a mass spectrometer

[0240] To evaluate the ability of ACE to detect changes in protein abundance in small cells such as bacteria, GFP+ and GFP- Escherichia coli cells were stained with DNA barcode-conjugated anti-GFP antibodies ( Fig.10 ). With two rounds of branching, protein abundance in microorganisms can be measured using mass spectrometry. A seven-fold difference in signal intensity was observed between the two populations, demonstrating that the signal amplification method can even quantitatively analyze single bacterial cells using mass spectrometry.

[0241] Example 3: Primary T cell signaling analysis using ACE

[0242] Systematic analysis of TCR signaling networks at single-cell resolution is technically challenging due to the limited abundance and small size of phosphorylated proteins in T lymphocytes. ACE achieved an average of 17-fold and up to 41-fold signal amplification in a 30-fold TCR signal analysis panel. In primary human CD4 T cells, the p-ERK signal was only slightly above the mass spectrometer detection limit using conventional conjugated antibodies (Figure 11A). Using ACE, the p-ERK signal increased dramatically, showing the expected trend in TCR stimulation time course experiments. By analyzing primary human T cells harvested during a 1-hour TCR stimulation time course, ACE revealed differential signal responses on key TCR signal mediators, including p-CD3ζ, p-CD28, p-ZAP70 / SYK, p-LAT, p-SLP76, p-PLCγ1, p-BTK / ITK, p-MEK1 / 2, p-ERK1 / 2, p-p90RSK, and p-S6 (Figure 11B).

[0243] Example 4: Design of ACE to improve sensitivity and resolution of IMC

[0244] In imaging mass cytometry (IMC), a UV laser is used to ablate antibody-stained samples point by point. A mixed flow of argon and helium then delivers the ablated material to the mass cytometer. Using the preserved subcellular level (1 μm2 ) Spatial information simultaneously quantifies more than 50 proteins (Figure 12A) 1,2 IMC is increasingly being applied to analyze microenvironmental features, including cell types, functions, and cell-cell interactions in tumors and metabolic diseases. 3,4 However, since every 1μm 2 Very little biological material is present in the ablated pixel, so only highly abundant proteins can be captured to generate detectable signals in IMC analysis. Due to this limitation, recent IMC-based studies of human breast cancer have been designed to focus on cell surface epitopes, cytokeratins, and a few cell function indicators. 3,5 (Figure 12A). These proteins are often highly abundant, especially in the tissue samples analyzed. This type of spatial and multiplexed epitope analysis plays an important role in deep classification of cell types and subsequent analysis of tumor-stroma and immune cell interactions, but the lack of sensitivity to study key phosphorylation sites or transcription factors that control or indicate growth, proliferation, survival, and migration limits the role of IMC in deciphering cell fate and functional determinants in cancer cells. By applying ACE to amplify the IMC signal, multiple low-abundance targets can be detected on the IMC instrument (Figure 12B).

[0245] Using ACE, it is also expected that the spatial resolution of IMC analysis will be improved. Improving IMC resolution was previously considered difficult because reducing the laser ablation crater size will exponentially reduce the number of ablated ions produced per laser pulse (i.e., one pixel in the reconstructed image). A high-intensity signal amplification method that can multiply the number of coupled ions on each antibody will be key to achieving potential high-resolution IMC analysis. Applying two rounds of branching, ACE is expected to increase the average metal ion count by nearly 4000 times (Figure 12C). Thus, compared with the currently used 1 μm 2 Compared to laser beams, the size of laser ablation craters can be reduced to sub-diffraction-limited levels of 100 nm in diameter. After the resolution upgrade, the ion counts per laser shot will be collected from a crater 100 times smaller and are therefore expected to be reduced by a factor of 100. However, due to compensation by the ultra-high amplification power of ACED, about 40 times brighter signals can still be generated compared to the ion counts of conventional 1μm resolution IMCs.

[0246] Example 5: 20-fold ACE-IMC analysis of human kidney samples

[0247] To verify the role of ACE in imaging mass spectrometer signal amplification, human kidney sections were stained simultaneously with 20 ACE oligoconjugate antibodies (Figure 13A). After staining and fixation, these markers were amplified with linear or branched ACE before analyzing the slides on IMC. 18 ACE-amplified kidney markers are shown in three overlapping images, each of which is represented by a specific color (Figure 13B). The locations of these markers in kidney tissue are consistent with expectations. Different tissue compartments and functional units can be identified in multiplexed images. Clear signals of low-abundance markers, including transcription factor nestin and phosphorylated protein p-S6, were observed. Single cell segmentation was performed, and the segmented cells were embedded in a two-dimensional UMAP map (Figure 13C). 18 different cell clusters were identified using a phenograph color-coded on a UMAP map. Each cluster represents a different cell type detected using ACE-assisted IMC analysis. The cell types detected in the analysis show unique protein expression profiles that can only be studied by multiple spatial proteomics analysis supported by ACE (Figure 13D).

[0248] 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 their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate disclosure by virtue 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 applicants and do not constitute any admission as to the correctness of the dates or contents of these documents.

Claims

1. A method for detecting a target molecule in mass spectrometry cytometry, the method comprising: a) contacting a target binding ligand molecule comprising a coupled oligonucleotide primer with a cell or tissue sample under conditions that allow the target binding ligand molecule to bind to its target in the cell or tissue sample; b) extending the oligonucleotide primer to include concatemers of the sequence included in the oligonucleotide primer; c) contacting the extended oligonucleotide primer of step (b) with a labeled probe nucleic acid, wherein the labeled probe nucleic acid comprises a sequence complementary to a sequence repeated in the concatemer, such that a plurality of labeled probe DNA molecules hybridize to a plurality of sequence repeating units in the concatemer; d) cross-linking the hybridized probe nucleic acid molecule to the extended oligonucleotide primer; and e) Detection of labeled probes using mass cytometry.

2. The method of claim 1, wherein the labeled probe nucleic acid comprises a metal ion label.

3. The method of claim 1 or 2, wherein the label probe comprises a fluorophore.

4. The method according to any one of claims 1 to 3, wherein extending the oligonucleotide primer comprises thermal cycling using a single-stranded extension template and a template-dependent nucleic acid polymerase. The method of claim 4 , wherein the polymerase is thermostable.

6. The method according to claim 4 or 5, wherein the single-stranded extension template comprises a concatemer of at least two copies of a sequence linked head-to-tail and a 3' chain terminator so that the extension template is not extended by a polymerase, the sequence being complementary to an oligonucleotide primer.

7. The method according to any one of claims 1 to 6, wherein the extension step (b) comprises multiple cycles of extension, hot strand separation and cooling to allow annealing of single-stranded extension bodies and extension by the polymerase, whereby successive cycles add consecutive multiple repeat units to the oligonucleotide primer.

8. The method of any one of claims 1-7, which is performed in multiplex with a plurality of different target binding ligand molecules, orthogonal sets of single-stranded extension templates, and distinguishably labeled orthogonal probe nucleic acids.

9. The method of any one of claims 1-8, wherein the concatemer comprises a branched concatemer formed on the target binding ligand by thermal cycling.

10. The method of claim 3, further comprising the step of imaging a fluorophore associated with the cell or tissue sample prior to the detecting step (e).

11. The method of any one of claims 1-10, wherein cross-linking the hybridized probe nucleic acid molecules to the extended oligonucleotide primers comprises photocross-linking.

12. The method of claim 11, wherein the probe nucleic acid molecule comprises a photocrosslinker.

13. The method of claim 12, wherein the photocrosslinker comprises 3-cyanovinylcarbazole phosphoramidite (CNVK) and crosslinking comprises ultraviolet irradiation.

14. The method of any one of claims 1-13, wherein the target binding ligand comprises an antibody or an antigen-binding fragment thereof.

15. A method for fluorescence activated cell sorting, comprising: a) providing a sample comprising fixed cells; b) contacting the target binding ligand molecule comprising the coupled oligonucleotide primer with the cells in the sample under conditions that allow the target binding ligand molecule to bind to its target in or on the cell; c) extending the oligonucleotide primer to include concatemers of the sequence included in the oligonucleotide primer; d) contacting the extended oligonucleotide primer of step (c) with a fluorescently labeled probe nucleic acid comprising a sequence complementary to a sequence repeated in the concatemer, such that a plurality of labeled probe nucleic acid molecules repeatedly hybridize to a plurality of sequences in the concatemer; e) cross-linking the hybridized probe nucleic acid molecule to the extended oligonucleotide primer; and f) Sorting cells by fluorescence activated cell sorting. The method according to claim 15 , wherein the labeled probe further comprises a metal ion.

17. The method of claim 15 or 16, wherein extending the oligonucleotide primer comprises thermal cycling using a single-stranded extension template and a template-dependent nucleic acid polymerase.

18. The method of claim 17, wherein the polymerase is thermostable.

19. The method according to claim 17 or 18, wherein the single-stranded extension template comprises a concatemer of at least two copies of a sequence linked head-to-tail and a 3' chain terminator so that the extension template is not extended by the polymerase, the sequence being complementary to the oligonucleotide primer.

20. The method of any one of claims 15-19, wherein the extension step (c) comprises multiple cycles of extension, hot strand separation and cooling to allow annealing of single-stranded extension bodies and extension by the polymerase, whereby successive cycles add consecutive concatenated repeat units to the oligonucleotide primer.

21. The method of any one of claims 15-20, which is performed in multiplex with a plurality of different target binding ligand molecules, orthogonal sets of single-stranded extension templates, and distinguishably labeled orthogonal probe nucleic acids.

22. The method of any one of claims 15-21, wherein the concatemer comprises a branched concatemer formed on the target binding ligand by thermal cycling.

23. The method according to any one of claims 16 to 22, further comprising a step of detecting the metal ion labeled probes by a mass spectrometer after the sorting step (f).

24. The method of any one of claims 15-23, wherein cross-linking the hybridized probe nucleic acid molecules to the extended oligonucleotide primers comprises photocross-linking.

25. The method of any one of claims 15-24, wherein the probe nucleic acid molecule comprises a photo-crosslinkable moiety.

26. The method of claim 25, wherein the photocrosslinking moiety comprises 3-cyanovinylcarbazole phosphoramidite (CNVK) and crosslinking comprises ultraviolet irradiation.

27. The method of any one of claims 15-26, wherein the target binding ligand comprises an antibody or an antigen-binding fragment thereof.

28. A composition comprising a target binding ligand and an oligonucleotide coupled to the target binding ligand.

29. The composition of claim 28, wherein the concatenated oligonucleotide further comprises one or more branched concatenated oligonucleotides.

30. The composition of claim 28, further comprising a plurality of nucleic acid probe molecules hybridized to the concatenated repeating units of the concatenated oligonucleotide.

31. The composition of claim 30, wherein the nucleic acid probe molecule comprises a cross-linking moiety.

32. The composition of claim 31, wherein the crosslinking moiety comprises 3-cyanovinylcarbazole phosphoramidite (CNVK).

33. The composition of claim 31 or 32, wherein the nucleic acid probe molecules are cross-linked to the multiple repeat sequences.

34. The composition of any one of claims 30-33, wherein the nucleic acid probe molecule comprises a fluorescent label or a metal ion label.

35. The composition of any one of claims 30-34, wherein the nucleic acid probe molecule comprises a fluorescent label and a metal ion label.

36. The composition of any one of claims 30-35, wherein the target binding ligand comprises an antibody or an antigen-binding fragment thereof.

37. A cell or tissue sample comprising the composition of any one of claims 30-36, wherein the target binding ligand binds to a target molecule in or on the cell or tissue sample.

38. A kit for carrying out one or more of the methods described herein, the kit comprising: a) a target binding ligand coupled to an oligonucleotide primer; or a set of target binding ligands coupled to an orthogonal oligonucleotide primer; b) a single-stranded extension template or a set of orthogonal single-stranded extension templates; c) a label probe nucleic acid or a set of orthogonal label probe nucleic acids, wherein the label probe nucleic acid or the set of orthogonal label probe nucleic acids comprises a cross-linking moiety.

39. The kit of claim 38, wherein the single-stranded extension template comprises a concatemer of at least two copies of a sequence complementary to an 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.

40. The kit of claim 38, further comprising a template-dependent nucleic acid polymerase.

41. The kit of claim 40, wherein the polymerase is thermostable.

42. The kit of any one of claims 38-41, wherein the target binding ligand comprises an antibody or an antigen-binding fragment thereof.

43. The kit of any one of claims 38-42, wherein the orthogonal nucleotide primers, single-stranded extension template, and nucleic acid probe are optimized to avoid primer / extension body cross-talk, primer dimer formation, and off-target hybridization.

44. The kit of any one of claims 38 to 43, wherein the single-stranded extension template or the set of single-stranded extension templates is suitable for performing a concatemer generation method.

45. The kit according to any one of claims 38-44, further comprising packaging materials and instructions for use for the various components.

46. ​​The kit of any one of claims 38-45, further comprising one or more target binding ligand molecules or reagents for coupling the first oligonucleotide to the target binding ligand.

47. The kit according to any one of claims 38 to 46, further comprising a thermostable polymerase, nucleotides, reaction buffer components and reagents for labeling nucleic acid probe molecules.

48. The kit according to any one of claims 38 to 47, wherein the probe molecule is complementary to a concatemer repeat element.

49. The kit of any one of claims 38-48, wherein the cross-linking moiety comprises a photo-cross-linking moiety.

50. The kit of any one of claims 38-49, wherein the cross-linking moiety comprises 3-cyanovinylcarbazole phosphoramidite (CNVK).

51. The kit according to any one of claims 38-50, wherein the labeled probe nucleic acid or the set of orthogonally labeled probe nucleic acids comprises a fluorescent label or a metal ion label.

52. The kit according to any one of claims 38-51, wherein the labeled nucleic acid probe molecules include fluorescent labels and metal ion labels.

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