Methods, kits, systems and compositions for multiplex detection of targets

Abstract

The disclosure provides methods, composition, kits and systems for detecting, quantifying, and / or imaging target molecules, e.g., biomolecules, in a sample such as a preparation of cells or tissue.

Description

METHODS, KITS, SYSTEMS AND COMPOSITIONS FOR MULTIPLEXDETECTION OF TARGETSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 63 / 622,737, filed on January 19, 2024, and U.S. Provisional Application No. 63 / 555,668, filed on February 20, 2024, the content of all of which are incorporated herein by reference in their entireties.TECHNICAL FIELD

[0002] The technology described herein relates to methods, compositions, kits and systems for detecting, quantifying, and / or imaging target molecules, e.g., in a sample such as a preparation of cells or tissue.GOVERNMENT SUPPORT

[0003] This invention was made with government support under Grant No. 1R01CA283202- 01, and 5UH3CA255133-04 awarded by the National Institutes of Health. The government has certain rights in the invention.BACKGROUND

[0004] Spatial biology technologies have become valuable tools for investigating the spatial distribution of cell types within tissues. The utilization of DNA-based imaging has enabled a relatively high level of multiplexing and signal amplification. However, achieving specific signals requires DNA probes to be orthogonal to each other to prevent potential crosstalk. This limitation restricts the number of targets that can be labeled and imaged simultaneously. Furthermore, as multiplexing increases, the sequence similarity rises, leading to potential nonspecific binding during imaging. The present disclosure overcomes these issues.SUMMARY

[0005] The technology described herein relates, in general, to methods, compositions, kits, and systems for increasing multiplexity of assay for detecting targets, e.g., biomolecules either in situ or ex situ. This report highlights the use of nucleases to overcome constraints on the number of DNA probes and introduces a blocking method to reduce nonspecific binding during imaging.

[0006] Accordingly, in one aspect provided herein is a method of detecting a set of target molecule in a sample, e.g., in situ in a preparation of cells or tissue.

[0007] In some embodiments, the method comprises: (i) contacting a preparation of cells or tissue with a target-binding ligand conjugated to a nucleic acid tag, e.g., under conditions permitting specific binding of the target-binding ligand to a target molecule; (ii) contacting the nucleic acid tag with a labeled nucleic acid probe, wherein the labeled nucleic acid probe comprises a nucleotide sequence complementary to at least a portion of the nucleic acid; (iii) detecting the labeled nucleic acid probe bound to the nucleic acid tag, wherein detection of the labeled nucleic acid probe indicates the presence and location of the target molecule in the preparation of cells or tissue; (iv) cleaving or digesting the nucleic acid tag and / or the labeled nucleic acid probe, thereby removing the labeled nucleic acid probe from the target-binding ligand; (v) contacting the preparation of cells or tissue from step (iv) with a target-binding ligand conjugated to a nucleic acid tag, e.g., under conditions permitting specific binding of the target-binding ligand to a target molecule; vi) contacting the nucleic acid tag with a labeled nucleic acid probe, wherein the labeled nucleic acid probe comprises a nucleotide sequence complementary to at least a portion of the nucleic acid; (vii) detecting the labeled nucleic acid probe bound to the nucleic acid tag, wherein detection of the labeled nucleic acid probe indicates the presence and location of the target molecule in the preparation of cells or tissue; and (viii) optionally, repeating steps (iv)-(vii) one or more times, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times.

[0008] In another embodiment, the method comprises:(i) contacting a preparation of cells or tissue with a set of target-binding ligand molecules, e.g., under conditions permitting specific binding of the target-binding ligand molecules to respective first target molecules present in the preparation of cells or tissue, wherein members of the set of target-binding ligand molecules are conjugated to respective nucleic acid tag, wherein the nucleic acid tag of each targetbinding ligand has a different sequence from other nucleic acid tags in the set, and is conjugated to a different member of a set of target-binding ligand molecules; (ii) contacting the nucleic acid tags with a set of distinguishably-labeled nucleic acid probes, wherein each labeled nucleic acid probe in the set comprises a nucleotide sequence complementary to at least a portion of a respective nucleic acid tag; (iii) detecting the labeled nucleic acid probes, wherein detection of the distinguishably-labeled nucleic acid probes indicate the presence and location of the first target molecules in the preparation of cells or tissue; (iv) cleaving or digesting the nucleic acid tags and / or the labeled nucleic acid probes, thereby removing the labeled nucleic acid probes from the target-binding ligands; (v) contacting the preparation of cells or tissue from step (iv) with a set of target-binding ligand molecules, e.g., under conditions permitting specific binding of the target-binding ligand molecules to respective second target molecules present in thepreparation of cells or tissue, wherein members of the set of target-binding ligand molecules are conjugated to respective nucleic acid tag, wherein the nucleic acid tag of each targetbinding ligand has a different sequence from other nucleic acid tags in the set, and is conjugated to a different member of the set of target-binding ligand molecules; (vi) contacting the nucleic acid tags with a second set of distinguishably-labeled nucleic acid probes, wherein each labeled nucleic acid probe in the set comprises a nucleotide sequence complementary to at least a portion of a respective second nucleic acid tag; (vii) detecting the labeled nucleic acid probes, wherein detection of the distinguishably-labeled nucleic acid probes indicate the presence and location of the second target molecules in the preparation of cells or tissue; and (viii) optionally, repeating steps (iv)-(vii) one or more times, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times.

[0009] In yet another embodiment, the method comprises: (i) contacting the preparation of cells or tissue with a set of target-binding ligand molecules under conditions permitting specific binding of the target-binding ligand molecules to target molecules present in the preparation of cells or tissue, wherein members of the set of target-binding ligand molecules are conjugated to respective nucleic acid tag, wherein the nucleic acid tag of each target-binding ligand has a different sequence from other first nucleic acid tags in the set; (ii) contacting the nucleic acid tags with a first set of probes, wherein the first set of probes comprises a set of blocking-nucleic acid probes and a first labeled-nucleic acid probe, wherein each blocking-nucleic acid probe has a different sequence from other blocking probes and said sequence is complementary to at least a portion of the nucleic acid tag of a different member of the set of target-binding ligand molecules, wherein the first labeled-nucleic acid probe comprises a sequence complementary to at least a portion of the nucleic acid tag conjugated to a first target-binding ligand in the set of target-binding ligand molecules and the sequence of the first labeled-nucleic acid probe is identical to the sequence of a first blocking-nucleic acid probe in the set of blocking-nucleic acid probes, wherein a concentration of the blocking-nucleic acid probes is lower than a concentration of the first labeled nucleic acid probe in the first set of blocking-nucleic acid probes; (iii) detecting the first labeled nucleic acid probe, wherein detection of the first labeled nucleic acid probe indicates the presence and location of a first target molecule in the preparation of cells or tissue; (iv) contacting the nucleic acid tags with a second set of probes, wherein the second set of probes comprises a set of blocking-nucleic acid probes and a second labeled-nucleic acid probe, wherein each blocking-nucleic acid probe has a different sequence from other blocking probes and said sequence is complementary to at least a portion of the nucleic acid tag of a different member of the set of target-binding ligand molecules, wherein the second labeled-nucleic acid probe comprises a sequence complementary to at least a portionof the nucleic acid tag conjugated to a second target-binding ligand in the set of target-binding ligand molecules and the sequence of the second labeled-nucleic acid probe is identical to the sequence of a second blocking-nucleic acid probe in the set of blocking-nucleic acid probes, wherein a concentration of the blocking-nucleic acid probes is lower than a concentration of the second labeled nucleic acid probe in the second set of blocking-nucleic acid probes; (v) detecting the second labeled nucleic acid probe, wherein detection of the second labeled nucleic acid probe indicates the presence and location of a second target molecule in the preparation of cells or tissue; and (vi) optionally, repeating steps (iv)-(v) one or more times, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times.

[0010] In still another embodiment, the method comprises: (i) contacting the preparation of cells or tissue with a set of target-binding ligand molecules under conditions permitting specific binding of the target-binding ligand molecules to target molecules present in the preparation of cells or tissue, wherein members of the set of target-binding ligand molecules are conjugated to respective nucleic acid tag, wherein the nucleic acid tag of each target-binding ligand has a different sequence from other first nucleic acid tags in the set; (ii) contacting the nucleic acid tags with a first set of probes, wherein the first set of probes comprises a set of blocking-nucleic acid probes and a first labeled-nucleic acid probe, wherein each blocking-nucleic acid probe has a different sequence from other blocking-nucleic acid probes and said sequence is complementary to at least a portion of the nucleic acid tag of a different member of the set of target-binding ligand molecules, wherein the first labeled-nucleic acid probe comprises a sequence complementary to at least a portion of the nucleic acid tag conjugated to a first targetbinding ligand in the set of target-binding ligand molecules, and wherein the set of blocking- nucleic acid probes does not comprise a blocking-nucleic acid probe having a sequence complementary to the nucleic acid tag conjugated to the first target-binding ligand; (iii) detecting the first labeled nucleic acid probe, wherein detection of the first labeled nucleic acid probe indicates the presence and location of a first target molecule in the preparation of cells or tissue; (iv) contacting the nucleic acid tags with a second set of probes, wherein the second set of probes comprises a set of blocking-nucleic acid probes and a second labeled-nucleic acid probe, wherein each blocking-nucleic acid probe has a different sequence from other blocking- nucleic acid probes and said sequence is complementary to at least a portion of the nucleic acid tag of a different member of the set of target-binding ligand molecules, wherein the second labeled-nucleic acid probe comprises a sequence complementary to at least a portion of the nucleic acid tag conjugated to a second target-binding ligand in the set of target-binding ligand molecules, and wherein the set of blocking-nucleic acid probes does not comprise a blocking-nucleic acid probe having a sequence complementary to the nucleic acid tag conjugated to the second target-binding ligand; (v) detecting the second labeled nucleic acid probe, wherein detection of the second labeled nucleic acid probe indicates the presence and location of a second target molecule in the preparation of cells or tissue; and (vi) optionally, repeating steps (iv)-(v) one or more times, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times. In some further embodiments of this, a concentration of the blocking probes is higher than a concentration of the labeled-nucleic acid probes.

[0011] In some embodiments, the step of contacting the first nucleic acid tags with the first set of distinguishably-labeled nucleic acid probes comprises contacting the first nucleic acid tags with a set of probe, wherein the first set of probes comprises a first set of blocking-nucleic acid probes and the first set of distinguishably-labeled nucleic acid probes, wherein each blocking- nucleic acid probe has a different sequence from other blocking-nucleic acid probes and said sequence is complementary to at least a portion of the nucleic acid tag of a different member of the set of target-binding ligand molecules, and wherein a concentration of the blocking- nucleic acid probes in the first set of blocking-nucleic acid probes is lower than a concentration of the labeled nucleic acid probes in the first set of blocking-nucleic acid probes.

[0012] In some embodiments, the step of contacting the first nucleic acid tags with the first set of distinguishably-labeled nucleic acid probes comprises contacting the first nucleic acid tags with a set of probe, wherein the first set of probes comprises a first set of blocking-nucleic acid probes and the first set of distinguishably-labeled nucleic acid probes, wherein each blocking-nucleic acid probe has a different sequence from other blocking-nucleic acid probes and said sequence is complementary to at least a portion of the nucleic acid tag of a different member of the set of target-binding ligand molecules, and wherein the set of blocking-nucleic acid probes does not comprise a blocking-nucleic acid probe having a sequence complementary to the nucleic acid tag having a sequence complementary to the distinguishably-labeled nucleic acid probes.

[0013] In some embodiments, the step of contacting the second nucleic acid tags with the second set of distinguishably-labeled nucleic acid probes comprises contacting the second nucleic acid tags with a set of probe, wherein the second set of probes comprises a second set of blocking-nucleic acid probes and the second set of distinguishably-labeled nucleic acid probes, wherein each blocking-nucleic acid probe has a different sequence from other blocking-nucleic acid probes and said sequence is complementary to at least a portion of the nucleic acid tag of a different member of the set of target-binding ligand molecules, and wherein a concentration of the blocking-nucleic acid probes in the second set of blocking-nucleic acid probes is lower than a concentration of the labeled nucleic acid probes in the second set of blocking-nucleic acid probes.

[0014] In some embodiments, the step of contacting the second nucleic acid tags with the second set of distinguishably-labeled nucleic acid probes comprises contacting the second nucleic acid tags with a set of probe, wherein the second set of probes comprises a second set of blocking-nucleic acid probes and the second set of distinguishably-labeled nucleic acid probes, wherein each blocking-nucleic acid probe has a different sequence from other blocking- nucleic acid probes and said sequence is complementary to at least a portion of the nucleic acid tag of a different member of the set of target-binding ligand molecules, and wherein the set of blocking-nucleic acid probes does not comprise a blocking-nucleic acid probe having a sequence complementary to the nucleic acid tag having a sequence complementary to the distinguishably-labeled nucleic acid probes.

[0015] In some embodiments, the first and second target molecules are different. In some other embodiments, the first and second target molecules are same.

[0016] In some embodiments, the first or second target-binding ligand comprises a protein.

[0017] In some embodiments, the first or second target-binding ligand comprises an antibody or antigen-binding fragment thereof.

[0018] In some embodiments, the first or second target-binding ligand comprises RNA.

[0019] In some embodiments, wherein the first or second nucleic acid tag is DNA.

[0020] In some embodiments, wherein the nuclease is a DNase.

[0021] In some embodiments, the first nucleic acid tag is DNA conjugated with a protein.

[0022] In some embodiments, the first nucleic acid tag is DNA conjugated with the antibody or antigen-binding fragment thereof.

[0023] In some embodiments, the first nucleic acid tag is DNA conjugated with an RNA.

[0024] In some embodiments, the first target-binding ligand is an RNA.

[0025] In some embodiments, the nuclease is an RNase.

[0026] In some embodiments, the first target-binding ligand is an RNA.

[0027] In some embodiments, the second target-binding ligand comprises protein.

[0028] In some embodiments, the second nucleic acid tag is DNA conjugated with protein.

[0029] In some embodiments, wherein the second nucleic acid tag is DNA conjugated with an antibody or antigen-binding fragment thereof.

[0030] In some embodiments, the second nucleic acid tag is DNA conjugated with an RNA.

[0031] In some embodiments, the first nucleic acid tag is a concatemer comprising repeats of a first oligonucleotide primer sequence.

[0032] In some embodiments, the step of contacting the preparation of cells or tissue with the first target-binding ligand comprises: (i) contacting the preparation of cells or tissue with a first target-binding ligand conjugated to a first oligonucleotide primer, under conditions permitting specific binding of the first target-binding ligand to the target molecule; (ii) adding to the preparation of cells or tissue a reaction mixture comprising a first single-stranded extender template and a nucleic acid polymerase enzyme under conditions permitting hybridization and extension of the first oligonucleotide primer using the first single-stranded extender template; (iii) heating the reaction mixture of step (ii) to separate the first singlestranded extender template from extended first oligonucleotide primer produced in step (ii); and (iv)repeating steps (ii) and (iii) at least once, thereby generating a concatemer comprising repeats of the first oligonucleotide primer sequence conjugated to the first target-binding ligand.

[0033] In some embodiments, the second nucleic acid tag is a concatemer comprising repeats of a second oligonucleotide primer sequence.

[0034] In some embodiments, the step of contacting the preparation of cells or tissue with the second target-binding ligand comprises: (i) contacting the preparation of cells or tissue with a second target-binding ligand conjugated to a second oligonucleotide primer, under conditions permitting specific binding of the second target-binding ligand to the target molecule; (ii) adding to the preparation of cells or tissue a reaction mixture comprising a second singlestranded extender template and a nucleic acid polymerase enzyme under conditions permitting hybridization and extension of the second oligonucleotide primer using the second singlestranded extender template; (iii) heating the reaction mixture of step (ii) to separate the second single-stranded extender template from extended second oligonucleotide primer produced in step (ii); and (iv) repeating steps (ii) and (iii) at least once, thereby generating a concatemer comprising repeats of the second oligonucleotide primer sequence conjugated to the second target-binding ligand.

[0035] In some embodiments, the first nucleic acid tag is a branched nucleic acid, optionally, the first nucleic acid tag is a branched DNA.

[0036] In some embodiments, a plurality of labeled nucleic acid probe molecules hybridizes to first or second nucleic acid tag.

[0037] In some embodiments, a single labeled nucleic acid probe molecules hybridizes to first or second nucleic acid tag.

[0038] In some embodiments, a concentration of the blocking probes is higher than a concentration of the labeled-nucleic acid probes. In some other embodiments, a concentration of the blocking probes is lower than a concentration of the labeled-nucleic acid probes.

[0039] In some embodiments, at least one, e.g., each nucleic acid tag is a concatemer comprising repeats of a respective first oligonucleotide primer sequence.

[0040] In some embodiments, the step of contacting the preparation of cells or tissue with a set of target-binding ligand molecules comprises: (i) contacting the preparation of cells or tissue with a set of target-binding ligand molecules under conditions permitting specific binding of the target-binding ligand molecules to target molecules present in the preparation of cells or tissue, wherein members of the set of target-binding ligand molecules are conjugated to respective first oligonucleotide primer members of a set of orthogonal first oligonucleotide primer and first single-stranded extender template oligonucleotide pairs, wherein the first oligonucleotide primer of each pair has a different sequence from other first oligonucleotide primers in the set, and is conjugated to a different member of the set of target-binding ligand molecules; (ii) contacting the preparation of cells or tissue with the set of target-binding ligand molecules under conditions permitting specific binding of the target-binding ligand molecules to target molecules present in the preparation of cells or tissue; (iii) adding to the preparation of cells or tissue a reaction mixture comprising first single-stranded extender templates of the set of orthogonal first oligonucleotide primer and first single-stranded extender template oligonucleotide pairs, and a nucleic acid polymerase, under conditions permitting hybridization and extension of the first oligonucleotide primers using the respective first single-stranded extender templates; (iv) heating the reaction mixture of step (iii) to separate the first singlestranded extender templates from extended first oligonucleotide primers produced in step (iii); and (v) repeating steps (iii) and (iv) at least once, thereby generating, on respective members of the set of target-binding ligand molecules, a concatemer comprising repeats of the respective first oligonucleotide primer sequence, conjugated to the target-binding ligand molecule.

[0041] In some embodiments, at least one, e.g., each nucleic acid tag is a branched nucleic acid, optionally, the branched nucleic acid is DNA.

[0042] In some embodiments, at least one, e.g., each target-binding ligand comprises a protein.

[0043] In some embodiments, at least one, e.g., each target-binding ligand comprises an antibody or antigen-binding fragment thereof.

[0044] In some embodiments, at least one, e.g., each target-binding ligand comprises RNA.

[0045] In some embodiments, a plurality of labeled nucleic acid probe molecules hybridizes to a plurality of concatemer repeats conjugated to the target-binding ligands.

[0046] In some embodiments, the preparation of cells or tissue is fixed.

[0047] In some embodiments, the preparation of tissue is paraffin embedded.

[0048] The method described herein lend itself well to multiplex labeling, detection and / or imaging. As discussed in further detail below, one multiplex approach uses a set of orthogonal target ligand-binding ligands, each specific for a different target of interest, and each conjugated to a different nucleic acid tag. The respective nucleic acid tags provide different, highly selective binding site(s) for a set of different, distinguishably-labeled probes that permit multiplex detection, quantitation and / or imaging of the members of a set of target molecules in a sample at each detection step.

[0049] In another aspect, provided herein is a kit comprising at least one, e.g., 1, 2, 3, 4, 5 or more of the components, e.g., target-binding ligand conjugated to a nucleic acid tag, targetbinding ligand conjugated to an oligonucleotide primer, labeled-nucleic acid probe, nuclease, single-stranded extender template, and polymerase, described herein. In some embodiments, the kit comprises: (i) a target-binding ligand conjugated with a nucleic acid tag; (ii) a labeled- nucleic acid probe; and (iii) at least one of: (a) a nuclease; and (b) a set of blocking probes.

[0050] In some embodiments, the kit comprises: (i) a target-binding ligand conjugated with an oligonucleotide primer; (ii) a single-stranded extender template; (iii) a polymerase; (iv) a labeled-nucleic acid probe; and (v) at least one of: (a) a nuclease; and (b) a set of blocking probes.

[0051] In another aspect, provided herein is a system for detecting targets in a sample, the system comprising at least one, e.g., 1, 2, 3, 4, 5 or more of the components, e.g., target-binding ligand conjugated to a nucleic acid tag, target-binding ligand conjugated to an oligonucleotide primer, labeled-nucleic acid probe, nuclease, single-stranded extender template, and polymerase, described herein. In some embodiments, the system comprises: (i) a targetbinding ligand conjugated with a nucleic acid tag; (ii) a labeled-nucleic acid probe; and (iii) at least one of: (a) a nuclease; and (b) a set of blocking probes.

[0052] In some embodiments, the system comprises: (i) a target-binding ligand conjugated with an oligonucleotide primer; (ii) a single-stranded extender template; (iii) a polymerase; (iv) a labeled-nucleic acid probe; and (v) at least one of: (a) a nuclease; and (b) a set of blocking probes.

[0053] In another aspect, provided herein is a reaction mixture comprising at least one, e.g., 1, 2, 3, 4, 5 or more of the components, e.g., target-binding ligand conjugated to a nucleic acidtag, target-binding ligand conjugated to an oligonucleotide primer, labeled-nucleic acid probe, nuclease, single-stranded extender template, and polymerase, described herein. In some embodiments, the reaction mixture comprises: (i) a target-binding ligand conjugated with a nucleic acid tag; (ii) a labeled-nucleic acid probe; and (iii) at least one of: (a) a nuclease; and (b) a set of blocking probes.

[0054] In some embodiments, the reaction mixture comprises: (i) a target-binding ligand conjugated with an oligonucleotide primer; (ii) a single-stranded extender template; (iii) a polymerase; (iv) a labeled-nucleic acid probe; and (v) at least one of: (a) a nuclease; and (b) a set of blocking probes.

[0055] In still another aspect, provided herein is a composition comprising at least one, e.g., 1, 2, 3, 4, 5 or more of the components, e.g., target-binding ligand conjugated to a nucleic acid tag, target-binding ligand conjugated to an oligonucleotide primer, labeled-nucleic acid probe, nuclease, single-stranded extender template, and polymerase, described herein. In some embodiments, the composition comprises: (i) a target-binding ligand conjugated with a nucleic acid tag; (ii) a labeled-nucleic acid probe and (iii) at least one of: (a) a nuclease; and (b) a set of blocking probes.

[0056] In some embodiments, the composition comprises: (i) a target-binding ligand conjugated with an oligonucleotide primer; (ii) a single-stranded extender template; (iii) a polymerase; (iv) a labeled-nucleic acid probe; and (v) at least one of: (a) a nuclease; and (b) a set of blocking probes, optionally, the set of blocking probes does not comprise a blocking probe having a sequence complementary to the nucleic acid tag conjugated to the target-binding ligand.

[0057] The kit described herein lend itself well to multiplex labeling, detection and / or imaging. Accordingly, in some embodiments, the kit comprises: (i) a set of orthogonal target ligandbinding ligands, each specific for a different target of interest, and each conjugated to a different nucleic acid tag; (ii) a set of different, distinguishably-labeled probes, where the different nucleic acid tags provide different, highly selective binding site(s) for the set of different, distinguishably-labeled probes; and (iii) and at least one of: (a) a nuclease; and (b) a set of blocking probes.BRIEF DESCRIPTION OF THE FIGURES

[0058] FIGS. 1A and IB are schematic representations of some exemplary embodiments of the disclosure illustrating how nuclease treatment can enhance the multiplexity of DNA-based imaging. In FIG. 1A, cells or tissue samples are labeled with a first set of RNA probes carryingDNA barcode labels. The addition of imagers and optional rounds of imager exchanges allows for RNA imaging. The subsequent digestion with RNase releases the probes bound to RNA, making them easy to be washed off. The sample can then be labeled with protein targets using DNA-barcoded antibodies, enabling a second round of imaging. In this embodiment, the DNA barcode on the antibodies can match that of the RNA probes, resulting in 2x multiplexity. In FIG. IB, DNase, instead of RNase, is used to digest all the DNA in the sample. This removes the DNA barcodes on the samples allowing for multiple rounds of labeling. Compared with the embodiment of FIG. 1A, this embodiment does not have the constrain of imaging RNA first then antibodies. Each imaging round can involve DNA-barcoded antibodies and / or DNA- barcoded RNA probes.

[0059] FIGS. 2A-2C are schematic representation of some exemplary embodiments of the disclosure illustrating how the blocking method can reduce background in DNA-based imaging. In FIG. 2A, imagers (labeled-nucleic acid probes) added without blocking strands can bind not only to DNA (nucleic acid tags) on the intended targets (illustrated as antibodies) but also to DNA on other targets (illustrated as RNA) due to sequence complementarity. This can lead to incorrect labeling and data interpretation. In FIG. 2B, imagers are added with blocking strands to all targets. The blocking strands have higher sequence complementarity to DNAs on other targets than the imagers. The blocking strands are added with a concentration that is a fraction of the imager concentration. Nonspecific bindings are displaced by the blocking strands, allowing imagers to bind only to the intended targets. This protocol is convenient, requiring the creation of a master mix of blocker strands to which imagers are added. In FIG. 2C, imagers are added with blocking strands to all other targets except the target to which the imagers bind. The blocking strands can be added at a high concentration, and displace nonspecific bindings, ensuring imagers only bind to the intended targets. This strategy is useful when there is high sequence complementarity between imagers and nonspecific DNA. This strategy is also useful when only a single or a few binding sites are available for the imagers to bind.

[0060] FIGS. 3A and 3B show the effect of using RNase H (FIG. 3A) and RNaseH + RNase I (FIG. 3B) to increase the multiplexity of imaging. RNA targets are imaged first followed by imaging of protein targets. In FIG. 3A, the RNA target molecules are imaged first (RNA), after digestion with RNase H (After RnaseH DIG), after reamplifying the digested RNA targets (Reamplification for digested targets), and imaging of protein targets (New targets). In FIG. 3B, the RNA target molecules are imaged first (RNA), after digestion with RNase H + RNase I (After RnaseH+Rnasel DIG), after reamplifying the digested RNA targets (Reamplificationfor digested targets), and imaging of protein targets (New targets). In FIGS. 3A and 3B, a good signal is seen before digestion. After digestion, the signal is gone even after the digested targets are reamplified. As seen, a signal is observed from new protein targets after addition of target-binding ligands to the new targets. This shows digestion with RNase efficiently removes the signal from the previous round of RNA imaging and allows for iterative imaging of new protein targets.

[0061] FIG. 4 shows the effect of using DNase to increase the multiplexity of imaging. A first set of target molecules are imaged using DNA barcode-labeled target binding ligand, i.e., nucleic acid tag is DNA (Before DIG signal), after digestion with DNase (After DIG), after reamplifying the digested targets (Reamplification for digested targets), and imaging of new targets with new DNA barcode-labeled target binding ligand (New targets). As seen, a signal is observed from new protein targets after addition of target-binding ligands to the new targets. This shows digestion with DNase efficiently removes the signal from the previous round of target imaging and allows for iterative imaging of new targets.

[0062] FIG. 5 shows results of protein imaging using blocking probes to address labeled- nucleic acid probe crosstalk according to an embodiment of the disclosure. 2ndAb shows the ground truth. Without the blocking probes, the DNA channel (DNA flour (mTOR)) showed Lamin signal which is incorrect. With the blocking probes, the DNA channel showed the correct mTOR signal. The blocking probes also did not significantly reduce the true signal intensity.

[0063] FIG. 6 shows results of target imaging using blocking probes to address labeled-nucleic acid probe crosstalk according to an embodiment of the disclosure. Without the blocking probe blocker, the labeled-nucleic acid probe 2 (imager 2) showed nonspecific signal as it could bind to the nucleic acid tag for labeled-nucleic acid probe 1 (imager 1). With the blocking probes, which bind to the nucleic acid tag for labeled-nucleic acid probe 1, the labeled-nucleic acid probe 2 channel showed no crosstalk signal. The blocking probes also did not significantly reduce the true signal intensity.DETAILED DESCRIPTION

[0064] It should be understood that this invention is not limited to the particular methodology, protocols, and reagents, etc., described herein and as such can vary. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention, which is defined solely by the claims.

[0065] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. All documents, or portions of documents, cited in this application, including, but not limited to, patents, patent applications, articles, books, and treatises, are hereby expressly incorporated by reference in their entirety for any purpose.

[0066] Exemplary strategies for enhancing the multiplexity of imaging / detecting targets according to various aspects described herein are depicted in FIGS. 1A-2C.

[0067] Some embodiments of the methods described herein are based, in part, on removing nucleic acid tags or nucleic acid bar codes conjugated to target binding ligands, e.g., with nuclease treatment. See, for example FIGS. 1A and IB. Generally, a first target molecule is imaged by contacting a sample, e.g., a preparation of cells or a tissue containing the target molecule with a first target-binding ligand. The first target-binding molecule is capable of binding or interacting, e.g., specifically with the first target molecule. The first target-binding molecule is conjugated with a nucleic acid tag. After the first target-binding ligand binds, e.g., specifically with the first target molecule in the sample, the sample is contacted with a labeled- nucleic acid probe. The labeled-nucleic acid probe comprises a nucleotide sequence complementary to at least a portion of a nucleotide sequence of the nucleic acid tag conjugated to the first target-binding ligand. Generally, the complementary sequence is of sufficient length to allow the labeled-nucleic acid probe to bind, interact or hybridize with the nucleic acid tag conjugated to the first target-binding ligand. The labeled-nucleic acid probe comprises a detectable label and the labeled-nucleic acid probe bound to the nucleic acid tag conjugated to the first target-binding ligand is detected via the detectable label. Detection of the labeled- nucleic acid probe indicates the presence and / or location of the first target molecule in the sample. After detection of the labeled-nucleic acid probe, the nucleic acid tag conjugated to the first target-binding ligand is removed. For example, the sample is contacted with a nuclease under conditions permitting digestion of the nucleic acid tag conjugated to the first targetbinding ligand so that the labeled-nucleic acid probe no longer binds or hybridizes with the nucleic acid tag conjugated to the first target-binding ligand. After digestion, the sample is contacted with a second target-binding molecule that is capable of binding or interacting, e.g., specifically with a second target molecule, which second target molecule is different from the first target molecule. The second target-binding molecule is conjugated with a nucleic acid tag. After the second target-binding ligand binds, e.g., specifically with the second target molecule in the sample, the sample is contacted with a labeled-nucleic acid probe. The labeled- nucleic acid probe comprises a nucleotide sequence complementary to a at least a portion of anucleotide sequence of the nucleic acid tag conjugated to the second target-binding molecule. Generally, the complementary sequence is of sufficient length to allow the labeled-nucleic acid probe to bind, interact or hybridize with the nucleic acid tag conjugated to the second targetbinding molecule. The labeled-nucleic acid probe comprises a detectable label and the labeled- nucleic acid probe bound to the nucleic acid tag conjugated to the second target-binding molecule is detected via the detectable label. Detection of the labeled-nucleic acid probe indicates the presence and / or location of the second target molecule in the sample. Optionally, after detection of the labeled-nucleic acid probe indicating the presence and / or location of the second target molecule in the sample, the step of removing the nucleic acid tag and detecting a new different target molecule can be repeated one or more times, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times.

[0068] It is noted that presence and / or location of two or more different target molecules can be determined simultaneously by using a set of target binding molecules. The members of the set of target-binding ligands bind to different target molecules and are conjugated to a respective nucleic acid tag. The nucleic acid tag of each target-binding ligand in the set has a different sequence from other nucleic acid tags in the set and is conjugated to a different member of the target-binding ligands. Generally, in embodiments where presence and / or location of two or more different target molecules can be determined simultaneously, a first set of target molecule is imaged by contacting a sample, e.g., a preparation of cells or a tissue containing the target molecules with a first set of target-binding ligands. The members of the first set of target-binding ligands are capable of binding or interacting, e.g., specifically with a first set of target molecules in the sample, and wherein the members of the first set of target molecules are different. Members of the set of target-binding ligand molecules are conjugated to respective nucleic acid tag. The nucleic acid tag of each target-binding ligand has a different sequence from other nucleic acid tags that are conjugated to a different member of the set of target-binding ligands. After the first set of target-binding ligands bind, e.g., specifically with the first set of target molecules in the sample, the sample is contacted with a set of labeled- nucleic acid probes. Each member of the labeled-nucleic acid probes comprises a nucleotide sequence complementary to at least a portion of a nucleotide sequence of the nucleic acid tag conjugated to a member of the first set of target-binding ligands, i.e., different labeled-nucleic acid probes comprise different sequence so that they bind or hybridize to different nucleic acid tags. Each labeled-nucleic acid probes comprise a detectable label and the detectable labels of different labeled-nucleic acid probes are distinguishable from each other. In other words, the member of the set of labeled-nucleic acid probes are distinguishably-labeled nucleic acidprobes. The distinguishably-labeled nucleic acid probes bound to their complementary nucleic acid tags are detected via their respective detectable label. Detection of the distinguishably- labeled nucleic acid probes indicates the presence and / or location of the first set of target molecule in the sample. After detection of the distinguishably-labeled nucleic acid probes, the nucleic acid tags conjugated to the first set of target-binding ligands are removed. For example, the sample is contacted with a nuclease under conditions permitting digestion of the nucleic acid tags conjugated to the first set of target-binding ligands so that the labeled-nucleic acid probes no longer bind or hybridize with the nucleic acid tags conjugated to the first set of targetbinding ligands. After digestion, the sample is contacted with a second set of target-binding ligands. The members of the second set of target-binding ligands are capable of binding or interacting, e.g., specifically with a second set of target molecules in the sample. The members of the second set of target molecules are different from each other and from the members of the first set of target molecules. Members of the second set of target-binding ligands are conjugated to respective nucleic acid tag, where the nucleic acid tag of each target-binding ligand has a different sequence from other nucleic acid tags that are conjugated to a different member of the second set of target-binding ligands. After members of the second set of targetbinding ligands bind, e.g., specifically with the second set of target molecules in the sample, the sample is contacted with a second set of distinguishably-labeled nucleic acid probes. Each member of the labeled-nucleic acid probes comprises a nucleotide sequence complementary to at least a portion of a nucleotide sequence of the nucleic acid tag conjugated to a member of the second set of target-binding ligands, i.e., different labeled-nucleic acid probes comprise different sequence so that they bind or hybridize to different nucleic acid tags. Each labeled- nucleic acid probes comprise a detectable label and the detectable labels of different labeled- nucleic acid probes are distinguishable from each other. The distinguishably-labeled nucleic acid probes bound to their complementary nucleic acid tags are detected via their respective detectable label. Detection of the distinguishably-labeled nucleic acid probes indicates the presence and / or location of the second set of target molecule in the sample. Optionally, after detection of the labeled-nucleic acid probes indicating the presence and / or location of the second set of target molecules in the sample, the step of removing the nucleic acid tags and detecting a new different set of target molecules can be repeated one or more times, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times.

[0069] In an exemplary embodiment shown in FIG. 1A, cells or tissue samples are labeled with a first set of RNA probes conjugated with DNA barcode labels (target-binding ligands conjugated to a nucleic acid tag). The addition of imagers (labeled nucleic acid probes) andoptional rounds of imager exchanges allows for RNA imaging. The subsequent digestion with RNase releases the probes bound to RNA, making them easy to be washed off. The sample is then labeled with protein targets using DNA-barcoded antibodies (i.e., a second set of targetbinding ligands conjugated to a nucleic acid tag), enabling a second round of imaging. It is noted that the DNA barcode, i.e., the nucleic acid tag, on the antibodies (the second set of target-binding ligands) can match that of the RNA probes (the first set of target-binding ligands), resulting in 2x multiplexity.

[0070] In an exemplary embodiment shown in FIG. IB, cells or tissue samples are labeled with a first set of RNA probes conjugated with DNA barcode labels or antibodies conjugated with DNA barcode labels (target-binding ligands conjugated to a nucleic acid tag). The addition of imagers (labeled nucleic acid probes) and optional rounds of imager exchanges allows for nucleic acid (e.g., RNA) or protein imaging. The subsequent digestion with DNase digests all the DNA in the sample. This removes the DNA barcodes on the samples allowing for multiple rounds of labeling. Without wishing to be bound by a theory, compared with the embodiment shown in FIG. 1 A, this embodiment does not have the constrain of imaging RNA first then antibodies. Each imaging round can involve DNA-barcoded antibodies and / or DNA- barcoded RNA probes.

[0071] Some embodiments of the methods described herein are based, in part, on blocking the potential undesired sites in the sample to which labeled-nucleic acid probes can bind to reduce background in DNA-based imaging. See, for example FIGS. 3B and 3C. Generally, a first target molecule is imaged by contacting a sample, e.g., a preparation of cells or a tissue containing the target molecules with a with a set of target-binding ligand molecules. Members of the set of target-binding ligand molecules are conjugated to a respective nucleic acid tag. The nucleic acid tag of each target-binding ligand has a different sequence from other nucleic acid tags in the set of target-binding ligand molecules. Members of the set of target-binding ligands bind to different target molecules. After the target-binding ligands bind, e.g., specifically with their respective target molecules in the sample, the sample is contacted with a first set of blocking-nucleic acid probes and a first labeled-nucleic acid probe. It is noted that the sample can be contacted sequentially or simultaneously with the first set of blocking- nucleic acid probes and the first labeled-nucleic acid probe. Optionally, the concentration of the first labeled-nucleic acid probe is higher than the concentration of the blocking probe that binds to the same nucleic acid tag as the first labeled-nucleic acid probe. In some embodiments, the first set of blocking-nucleic acid probes and the first labeled-nucleic acid probe are comprised in a same composition, e.g., a first set of probes comprising: (i) the first set ofblocking-nucleic acid probes; and (ii) the first labeled-nucleic acid probe. Optionally, the concentration of the blocking probe that binds to the same nucleic acid tag as the first labeled- nucleic acid probe is lower than the concentration of the first labeled-nucleic acid probe in the first set of probes. Members of the first set of blocking-nucleic acid probes have a different nucleotide sequence from other members of the first set of blocking probes and said sequence is complementary to at least a portion of the nucleic acid tag of a different member of the set of target-binding ligand molecules. The complementary sequence of each blocking probe in the first set of blocking probes is of sufficient length to allow the blocking probe to bind, interact or hybridize with the nucleic acid tag conjugated to a target-binding ligand and inhibit or reduce binding, interaction or hybridization of the first labeled-nucleic acid to said nucleic acid tag. The first labeled-nucleic acid probe comprises a nucleotide sequence complementary to at least a portion of a nucleotide sequence of the nucleic acid tag conjugated to a first targetbinding ligand. The complementary sequence of the labeled-nucleic acid probe is of sufficient length to allow the labeled-nucleic acid probe to bind, interact or hybridize with the nucleic acid tag conjugated to the target-binding ligand. The first labeled-nucleic acid probe comprises a detectable label and the labeled-nucleic acid probe bound to the nucleic acid tag conjugated to the target-binding ligand is detected via the detectable label. Detection of the first labeled- nucleic acid probe indicates the presence and / or location of the first target molecule in the sample. After detection, the first set of blocking probes and the first labeled nucleic acid probe are removed from the nucleic acid tags and the sample, and the sample is contacted with a second set of blocking probes and a second labeled-nucleic acid probe. It is noted that the sample can be contacted sequentially or simultaneously with the second set of blocking-nucleic acid probes and the second labeled-nucleic acid probe. Optionally, the concentration of the second labeled-nucleic acid probe is higher than the concentration of the blocking probe that binds to the same nucleic acid tag as the second labeled-nucleic acid probe. In some embodiments, the second set of blocking-nucleic acid probes and the second labeled-nucleic acid probe are comprised in a same composition, e.g., a second set of probes comprising: (i) the second set of blocking-nucleic acid probes; and (ii) the second labeled-nucleic acid probe. Optionally, the concentration of the blocking probe that binds to the same nucleic acid tag as the second labeled-nucleic acid probe is lower than the concentration of the second labeled- nucleic acid probe in the second set of probes. Members of the second set of blocking-nucleic acid probes have a different nucleotide sequence from other members of the second set of blocking probes and said sequence is complementary to at least a portion of the nucleic acid tag of a different member of the set of target-binding ligand molecules. The complementarysequence of each blocking probe in the second set of blocking probes is of sufficient length to allow the blocking probe to bind, interact or hybridize with the nucleic acid tag conjugated to a target-binding ligand and inhibit or reduce binding, interaction or hybridization of the second labeled-nucleic acid probe to said nucleic acid tag. The second labeled-nucleic acid probe comprises a nucleotide sequence complementary to at least a portion of a nucleotide sequence of the nucleic acid tag conjugated to a second target-binding ligand. For clarity, the nucleotide sequence of the second labeled-nucleic acid probe is different from the nucleotide sequence of the second labeled-nucleic acid probe such that they bind, interact or hybridize with different nucleic acid tags. The second labeled-nucleic acid probe comprises a detectable label and the labeled-nucleic acid probe bound to the nucleic acid tag conjugated to the target-binding ligand is detected via the detectable label. Detection of the second labeled-nucleic acid probe indicates the presence and / or location of the second target molecule in the sample. It is noted that the detectable label of the second labeled-nucleic acid probe can be same or different from the detectable label of the first labeled-nucleic acid probe. Optionally, after detection of the second labeled-nucleic acid probe indicating the presence and / or location of the second target molecule in the sample, the step of removing the blocking probes and the labeled-nucleic acid probe and detecting a new different target molecule can be repeated one or more times, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times.

[0072] In some embodiments, a set of blocking probe does not comprise a blocking probe that binds, interacts or hybridizes with the same nucleic acid tag as a labeled-nucleic acid. In such cases, a first target molecule is imaged by contacting a sample, e.g., a preparation of cells or a tissue containing the target molecules with a with a set of target-binding ligand molecules. Members of the set of target-binding ligand molecules are conjugated to a respective nucleic acid tag. The nucleic acid tag of each target-binding ligand has a different sequence from other nucleic acid tags in the set of target-binding ligand molecules. Members of the set of targetbinding ligands bind to different target molecules. After the target-binding ligands bind, e.g., specifically with their respective target molecules in the sample, the sample is contacted with a first set of blocking-nucleic acid probes and a first labeled-nucleic acid probe. The first set of blocking-nucleic acid probes does not comprise a blocking probe that binds to the same nucleic acid tag as the first labeled-nucleic acid probe. It is noted that the sample can be contacted sequentially or simultaneously with the first set of blocking-nucleic acid probes and the first labeled-nucleic acid probe. Further, the concentration of the first labeled-nucleic acid probe can be same, higher or lower than the concentration of the blocking probes. In some embodiments, the first set of blocking-nucleic acid probes and the first labeled-nucleic acidprobe are comprised in a same composition, e.g., a first set of probes comprising: (i) the first set of blocking-nucleic acid probes; and (ii) the first labeled-nucleic acid probe. Members of the first set of blocking-nucleic acid probes have a different nucleotide sequence from other members of the first set of blocking probes and said sequence is complementary to at least a portion of the nucleic acid tag of a different member of the set of target-binding ligand molecules. The complementary sequence of each blocking probe in the first set of blocking probes is of sufficient length to allow the blocking probe to bind, interact or hybridize with the nucleic acid tag conjugated to a target-binding ligand and inhibit or reduce binding, interaction or hybridization of the first labeled-nucleic acid to said nucleic acid tag. The first labeled- nucleic acid probe comprises a nucleotide sequence complementary to at least a portion of a nucleotide sequence of the nucleic acid tag conjugated to a first target-binding ligand. The complementary sequence of the labeled-nucleic acid probe is of sufficient length to allow the labeled-nucleic acid probe to bind, interact or hybridize with the nucleic acid tag conjugated to the target-binding ligand. The first labeled-nucleic acid probe comprises a detectable label and the labeled-nucleic acid probe bound to the nucleic acid tag conjugated to the targetbinding ligand is detected via the detectable label. Detection of the first labeled-nucleic acid probe indicates the presence and / or location of the first target molecule in the sample. After detection, the first set of blocking probes and the first labeled nucleic acid probe are removed from the nucleic acid tags and the sample, and the sample is contacted with a second set of blocking probes and a second labeled-nucleic acid probe. It is noted that the sample can be contacted sequentially or simultaneously with the second set of blocking-nucleic acid probes and the second labeled-nucleic acid probe. The second set of blocking-nucleic acid probes does not comprise a blocking probe that binds to the same nucleic acid tag as the second labeled-nucleic acid probe. Further, the concentration of the second labeled-nucleic acid probe can be same, higher or lower than the concentration of the blocking probes. In some embodiments, the second set of blocking-nucleic acid probes and the second labeled-nucleic acid probe are comprised in a same composition, e.g., a second set of probes comprising: (i) the second set of blocking-nucleic acid probes; and (ii) the second labeled-nucleic acid probe. Members of the second set of blocking-nucleic acid probes have a different nucleotide sequence from other members of the second set of blocking probes and said sequence is complementary to at least a portion of the nucleic acid tag of a different member of the set of target-binding ligand molecules. The complementary sequence of each blocking probe in the second set of blocking probes is of sufficient length to allow the blocking probe to bind, interact or hybridize with the nucleic acid tag conjugated to a target-binding ligand and inhibit or reducebinding, interaction or hybridization of the second labeled-nucleic acid probe to said nucleic acid tag. The second labeled-nucleic acid probe comprises a nucleotide sequence complementary to at least a portion of a nucleotide sequence of the nucleic acid tag conjugated to a second target-binding ligand. For clarity, the nucleotide sequence of the second labeled- nucleic acid probe is different from the nucleotide sequence of the second labeled-nucleic acid probe such that they bind, interact or hybridize with different nucleic acid tags. The second labeled-nucleic acid probe comprises a detectable label and the labeled-nucleic acid probe bound to the nucleic acid tag conjugated to the target-binding ligand is detected via the detectable label. Detection of the second labeled-nucleic acid probe indicates the presence and / or location of the second target molecule in the sample. It is noted that the detectable label of the second labeled-nucleic acid probe can be same or different from the detectable label of the first labeled-nucleic acid probe. Optionally, after detection of the second labeled-nucleic acid probe indicating the presence and / or location of the second target molecule in the sample, the step of removing the blocking probes and the labeled-nucleic acid probe and detecting a new different target molecule can be repeated one or more times, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times.

[0073] As shown in FIG. 3A, the labeled-nucleic acid probes (target-binding ligands conjugated to a nucleic acid tag) are added without the blocking probes (blocking-nucleic acid probes). The labeled-nucleic acid probes not only bind to their complementary nucleic acid tags but may also bind to other nucleic acid tags if there are significant sequence complementarity leading to wrong labeling and data interpretation.

[0074] In an exemplary embodiment of the method shown in FIG. 3B, the labeled-nucleic acid probes are added with blocking probes to all nucleic acid tags that are present. The blocking probes have higher sequence complementarity to the nucleic acid tags that are not complementary to the labeled-nucleic acid probes. The blocking probes are only a fraction concentration of the labeled-nucleic acid probe. Without wishing to be bound by a theory, the labeled-nucleic acid probes can only bind to the designed nucleic acid tag as nonspecific bindings are displaced by the blocking probes. This is a very convenient protocol since one just need to make a master mix of all the blocking probes and add the appropriate labeled- nucleic acid probe to the mix as needed.

[0075] In an exemplary embodiment of the method shown in FIG. 3C, the labeled-nucleic acid probes are added with blocking probes to all other nucleic acid tags except for the nucleic acid tags to which the labeled-nucleic acid probes bind. The blocking probes have higher sequence complementarity to the nucleic acid tags that are not complementary to the labeled-nucleic acidprobes. The blocking probes can be added at a high concentration. Without wishing to be bound by a theory, the labeled-nucleic acid probes can only bind to the designed nucleic acid tag as nonspecific bindings are displaced by the blocking probes. This is useful in cases where the sequence complementarity is high between the labeled-nucleic acid probes and nonspecific DNA.

[0076] In some embodiments, the step of removing the blocking probes and labeled-nucleic acid from the nucleic acid tags and / or the sample comprises contacting the sample with an agent that dissociates hybridized nucleic acid strands, nucleic acid strands. For example, the sample can be contacted with a formamide solution (e.g., 50% formamide in IxPBS) which can dissociate the blocking probes and the labeled-nucleic acid probes from the nucleic acid tags. In some embodiments, the step of removing the blocking probes and labeled-nucleic acid from the nucleic acid tags and / or the sample comprises heating the sample, e.g., to a temperature sufficient, to dissociate the blocking probes and the labeled-nucleic acid probes from the nucleic acid tags.

[0077] In some embodiments, the step of removing the blocking probes and labeled-nucleic acid from the nucleic acid tags and / or the sample comprises contacting the sample with a nuclease, e.g., a DNase or an RNase, to cleave or digest the blocking probes and the labeled- nucleic acid probes.Target binding ligand

[0078] Embodiments of the various aspects described herein include target binding ligands. The terms “target-binding ligand”, “target-binding ligand molecule” and “target-binding molecule” are used interchangeably herein and refer to a molecule that binds to or interacts with a target molecule of interest. In other words, a target-binding ligand or molecule is a molecule that is binds, e.g., e.g., specifically binds, to a target molecule of interest. The targeting binding ligand can be a natural or synthetic molecule. A target-binding ligand can be a biomolecule, such as a polypeptide or a polynucleotide. In some embodiments, a targetbinding ligand is a polypeptide. Exemplary target-binding ligands include, but are not limited to, peptides, polypeptides, antibodies, antigen binding fragment of antibodies, antibody derivatives, antigens, receptors, a ligand for a receptor, oligonucleotides, and polynucleotides.

[0079] In some embodiments of any of the aspects described herein, the target-binding ligand binds to i.e., the target molecule is, a molecule selected from the non-limiting group of lipids, sugars, oligo- or poly- saccharides, amino acids, peptides or polypeptides, nucleosides,nucleotides, oligo- or poly- nucleotides, hormones, vitamins, small molecules, miRNAs, metabolites, and any combinations thereof.

[0080] By way of non-limiting examples, a target-binding ligand can be selected from antibodies, antigen binding fragments of antibodies, adnectins, ankyrins, other antibody mimetics and other protein scaffolds, nucleic acids (e.g., RNA and aptamers such as RNA or DNA aptamers), protein, peptide, binding partner, oligosaccharides, polysaccharides, lipopolysaccharides, cellular metabolites, cells, viruses, subcellular particles, haptens, pharmacologically active substances, alkaloids, steroids, vitamins, amino acids, avimers, peptidomimetics, hormone receptors, cytokine receptors, synthetic receptors, sugars or molecularly imprinted polymer. The target-binding ligand can be selective to a specific target or class of targets such as toxins and biomolecules. For example, the target can be ions, molecules, oligomers, polymers, proteins, peptides, nucleic acids, toxins, biological threat agents such as spore, viral, cellular and protein toxins, carbohydrates (e.g., mono-saccharides, disaccharides, oligosaccharides, polyols, and polysaccharides) and combinations of these (e.g., copolymers including these).

[0081] In some embodiments of any one of the aspects described herein, the target-binding ligand is an antibody or antigen binding fragment thereof. The term “antibody” refers to a protein that includes at least one immunoglobulin variable domain or immunoglobulin variable domain sequence that binds a target molecule. For example, an antibody can include an immunoglobulin heavy (H) chain variable region (abbreviated herein as VH), and an immunoglobulin light (L) chain variable region (abbreviated herein as VL). In another example, an antibody includes two heavy (H) chain variable regions and two light (L) chain variable regions. An antibody can include the structural features of IgA, IgG, IgE, IgD, IgM (as well as subtypes thereof). The term antibody as used herein includes any of a number of different constructs using one or more antigen-binding domains or fragments of an antibody to mediate binding to a target molecule. Thus, in addition to a complete IgA, IgG, IgE, IgD or IgM antibody, an antibody includes, but is 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. An affibody, which uses a non-antibody scaffold to support a diverse target-binding domain, can also be used as a target-binding ligand in the methods, compositions and kits described herein. As used herein, the terms “antibody” and “antibodies” include polyclonal antibodies, monoclonal antibodies, humanized or chimeric antibodies, single chain Fv antibody fragments, Fab fragments, and F(ab)2 fragments. The terms “antibody” and“antibodies” include intact antibody, or a binding fragment thereof that competes with the intact antibody for specific binding and includes chimeric, humanized, fully human, and bispecific antibodies.

[0082] As used herein, an “antigen-binding fragment” refers that portion of an antibody that is necessary and sufficient for binding to a given antigen. At a minimum, an antigen binding fragment of a conventional antibody will comprise six complementarity determining regions (CDRs) derived from the heavy and light chain polypeptides of an antibody arranged on a scaffold that permits them to selectively bind the antigen. A commonly used antigen-binding fragment includes the VH and VL domains of an antibody, which can be joined either via part of the constant domains of the heavy and light chains of an antibody, or, alternatively, by a linker, such as a peptide linker. Non-conventional antibodies, such as camelid and short antibodies have only heavy chain sequences, denoted, for example VHH. These can be used in a manner analogous to VH / VL-containing antigen-binding fragments. Non-limiting examples of antibody fragments encompassed by the term antigen-binding fragment include: (i) a Fab fragment, having VL, CL, VH and CHI domains; (ii) a Fab' fragment, which is a Fab fragment having one or more cysteine residues at the C-terminus of the CHI domain; (iii) an Fd fragment having VH and CHI domains; (iv) a Fd' fragment having VH and CHI domains and one or more cysteine residues at the C-terminus of the CHI domain; (v) an Fv fragment having the VL and VH domains of a single arm of an antibody; (vi) a dAb fragment (Ward et al., Nature 341, 544-546 (1989)) which consists of a VH domain; (vii) F(ab')2 fragments, a bivalent fragment including two Fab' fragments linked by a disulphide bridge at the hinge region; (viii) single chain antibody molecules (e.g., single chain Fv; scFv) (Bird et al., Science 242:423-426 (1988); and Huston et al., PNAS (USA) 85:5879-5883 (1988)); (ix) “diabodies” with two antigen binding sites, comprising a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain (see, e.g., EP 404,097; WO 93 / 11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); and “linear antibodies” comprising a pair of tandem Fd segments (VH-CH1-VH- CHI) which, together with complementary light chain polypeptides, form a pair of antigen binding regions (Zapata et al. Protein Eng. 8(10): 1057-1062 (1995); and U.S. Pat. No. 5,641,870). The molecules of Fv, scFv or diabody can be stabilized by incorporating disulfide bridges linking the VH and VL domains. Minibodies comprising a scFv fragment linked to a CH3 domain can also be obtained. Other examples of binding fragments are Fab’, which differs from Fab fragments by the addition of some residues at the carboxyl terminus of the CHI domain of the heavy chain, including one or more cysteines of the hinge region of the antibody, and Fab’-SH, which is aFab’ fragment in which the cysteine residue(s) of the constant domains carries a free thiol group. The binding fragments can be produced by recombinant DNA techniques. The binding fragments can also be produced by enzymatic or chemical cleavage of intact antibodies. Binding fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv, and single-chain antibodies.

[0083] In some embodiments, the target binding ligand can be a nucleic acid, e.g., the target binding ligand can be RNA. For example, when a target molecule is a nucleic acid, the target binding ligand can be an RNA.

[0084] In some embodiments of any one of the aspects described herein, the binding of the target-binding ligand to the target molecule is a specific binding. As used herein, the term “specific binding” refers to a physical interaction between two molecules, compounds, cells and / or particles wherein the first entity binds to the second, target entity with greater specificity and affinity than it binds to a third entity which is a nontarget. A reagent specific for a given target is one that exhibits specific binding for that target under the conditions of the assay being utilized. The specificity of a target-binding ligand can be determined based on affinity and / or avidity. The affinity, represented by the equilibrium constant for the dissociation (KD) of a target-binding ligand and its target, is a measure of the binding strength between the targetbinding ligand and its target. The lower the value of the KD, the stronger the binding strength between the target-binding ligand and its target. Alternatively, the affinity can also be expressed as the affinity constant (KA), which is 1 / KD). Accordingly, a target-binding ligand as described herein is said to be "specific for" or to “specifically bind” or “selectively bind” a first target compared to a second target when it binds to the first antigen with an affinity (as described above, and suitably expressed, for example as a KD value) that is at least 1000 times, 10000 times or more better than the affinity with target-binding ligand binds to another given molecule. Generally, a molecule that “specifically binds,” “selectively binds” or “is specific for” a given target will bind with a KD of 10-5 M (10000 nM) or less, e.g., 10-6 M, 10 7 M, 10 8 M, 10 9 M, 10-10 M, 10-11 M, 10-12 M, or less. Specific binding can be influenced by, for example, the affinity and avidity of the target-binding ligand and the concentration of the target-binding ligand. The person of ordinary skill in the art can determine appropriate conditions under which a target-binding ligand selectively binds the target using any suitable methods, such as titration of in a suitable binding assay. It should be understood in this context that where antibodies or antigen-binding fragments thereof are concerned, the specific binding is mediated by the CDRs of the antibody polypeptide, as opposed to any other portion of the antibody polypeptide. Antibody dissociation constants and affinities can be determined, forexample, by a surface plasmon resonance-based assay (such as the BIAcore assay; Forte Bio OctetTM analysis, enzyme-linked immunosorbent assay (ELISA); and competition assays (e.g., RIA’s), for example.

[0085] In some embodiments, the dissociation constant between the target-binding ligand and target molecule is at least about 200 nM, alternatively at least about 150 nM, alternatively at least about 100 nM, alternatively at least about 60 nM, alternatively at least about 50 nM, alternatively at least about 40 nM, alternatively at least about 30 nM, alternatively at least about 20 nM, alternatively at least about 10 nM, alternatively at least about 8 nM, alternatively at least about 6 nM, alternatively at least about 4 nM, alternatively at least about 2 nM, alternatively at least about 1 nM, or greater. In certain embodiments, the specific binding refers to binding where the target-binding ligand binds to its target molecule without substantially binding to any other species in the sample / test solution.

[0086] In some embodiments, the target binding ligand(s) used for detecting a first target molecule(s) can be different from the target binding ligand(s) used for detecting a second target molecule(s). For example, the target binding ligand(s) used for detecting a first target molecule(s) can be an RNA and the target binding ligand(s) used for detecting a second target molecule(s) can be a non-nucleic acid molecule, e.g., an antibody, an antigen binding fragment of an antibody, an antigen, a receptor, a ligand for a receptor, an enzyme, or other polypeptide or protein.

[0087] In some embodiments, the target binding ligand(s) used for detecting a first target molecule(s) can be same as the target binding ligand(s) used for detecting a second target molecule(s). For example, the target binding ligand(s) used for detecting a first target molecule(s) and the target binding ligand(s) used for detecting a second target molecule(s) can be independently a non-nucleic acid molecule. For example, the target binding ligand(s) used for detecting a first target molecule(s) and the target binding ligand(s) used for detecting a second target molecule(s) can be independently an antibody, an antigen binding fragment of an antibody, an antigen, a receptor, a ligand for a receptor, an enzyme, or other polypeptide or protein.

[0088] It is to be noted that the target-binding ligand(s) is added to the sample under conditions permitting specific binding of the target-binding ligand to its target molecule. Target-binding ligands as described herein can be contacted a sample according to methods known in the art. Where, for example, the target-binding ligand is an antibody or antigen-binding fragment thereof, methods widely applied in immunohistochemistry can be used to stain the sample for detection of the given target ligand. Thus, the methods for contacting the sample with thetarget-binding ligand can parallel those used with, e.g., fluorescently labeled antibodies or antibody fragments. Where performed in multiplex, the contacting or staining can comprise the addition of a set of target-binding ligands, each comprising a different nucleic acid tag. In instances where the target-binding ligand is not an antibody or antigen-binding fragment thereof, methods for contacting a sample with the target-binding ligand can be adapted from those known in the art for the given ligand. In particular, where the target molecule is a DNA or RNA molecule comprising a given sequence, conditions as used, e.g., for in situ hybridization can be used to permit binding of the target-binding ligand oligonucleotide(s) to the target sequence(s). In some embodiments, it can be beneficial to remove unbound targetbinding ligand prior to subsequent steps. This can be achieved, for example, by removal of target binding ligand-containing solution from the sample, followed by one or more rinsing steps in an appropriate solution lacking the target-binding ligand.

[0089] For detection, quantitation and / or imaging of different targets simultaneously at a detection step, a set of orthogonal target ligand-binding ligands, each specific for a different target of interest, and each conjugated to a different nucleic acid tag, can be used. The respective nucleic acid tags provide different, highly selective binding site(s) for a set of different, distinguishably-labeled probes that permit multiplex detection, quantitation and / or imaging of the members of a set of target molecules in a sample at each detection step.Nucleic acid tag

[0090] The target-binding ligands described herein are conjugated with a nucleic acid tag. The terms “nucleic acid tag”, “nucleic acid barcode” and “DNA barcode” are used interchangeably herein and refer to a nucleic acid sequence that can be used to unambiguously identify the target-binding ligand it is attached to. A nucleic acid tag described herein 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 in length. It is noted that a nucleic acid tag described herein can be DNA, RNA or a combination of DNA and RNA. In some embodiments, a nucleic acid tag described herein is DNA.

[0091] A nucleic acid tag can be linear or branched. Accordingly, in some embodiments, a nucleic acid tag described herein is a linear nucleic acid. In some other embodiments, a nucleic acid tag described herein is a branched nucleic acid.

[0092] In some embodiments, a nucleic acid tag described herein comprises two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more,ten or more, 15 or more, 20 or more, 25 or more, 30 or more, 35 or more, 40 or more, 45 or more, or 50 or more sites for binding with a labeled-nucleic acid probe. For example, the nucleic acid tag is a concatemer. As used herein the term “concatemer” refers to a nucleic acid molecule comprising two or more repeats of a given sequence in a head-to-tail, 5’ to 3’ orientation. Concatemers can include two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, 15 or more, 20 or more, 25 or more, 30 or more, 35 or more, 40 or more, 45 or more, or 50 or more repeats of the given sequence. The number of repeats for concatemers generated as described herein is determined by the number of cycles of strand separation and primer extension employed. The repeat units in a concatemer 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 in length. In some embodiments, the repeat units in a concatemer as described herein can be, for example, between 9-50 nucleotides in length, between 9-45 nucleotides in length, between 9-40 nucleotides in length, between 9-35 nucleotides in length, between 9-30 nucleotides in length, between 9-25 nucleotides in length, between 9-20 nucleotides in length, between 9-15 nucleotides in length, between 10-100 nucleotides in length, between 10-90 nucleotides in length, between 10-80 nucleotides in length, between 10- 70 nucleotides in length, between 10-60 nucleotides in length, between 10-50 nucleotides in length, between 10-40 nucleotides in length, between 10-30 nucleotides in length, between 10-20 nucleotides in length, between 11-100 nucleotides in length, between 11-90 nucleotides in length, between 11-80 nucleotides in length, between 11-70 nucleotides in length, between 11- 60 nucleotides in length, between 11-50 nucleotides in length, between 11-40 nucleotides in length, between 11-30 nucleotides in length, between 11-20 nucleotides in length, between 12- 100 nucleotides in length, between 12-90 nucleotides in length, between 12-80 nucleotides in length, between 12-70 nucleotides in length, between 12-60 nucleotides in length, between 12- 50 nucleotides in length, between 12-40 nucleotides in length, between 12-30 nucleotides in length, between 12-20 nucleotides in length, between 13-100 nucleotides in length, between 13-90 nucleotides in length, between 13-80 nucleotides in length, between 13-80 nucleotides in length, between 13-70 nucleotides in length, between 13-60 nucleotides in length, between13-50 nucleotides in length, between 13-40 nucleotides in length, between 13-30 nucleotides in length, between 13-20 nucleotides in length, between 14-100 nucleotides in length, between14-90 nucleotides in length, between 14-80 nucleotides in length, between 14-70 nucleotides in length, between 14-60 nucleotides in length, between 14-50 nucleotides in length, between14-40 nucleotides in length, between 14-30 nucleotides in length, between 14-20 nucleotides in length, between 15-100 nucleotides in length, between 15-90 nucleotides in length, between15-80 nucleotides in length, between 15-70 nucleotides in length, between 15-60 nucleotides in length, between 15-50 nucleotides in length, between 15-40 nucleotides in length, between 15-30 nucleotides in length, or between 15-20 nucleotides in length.

[0093] In some embodiments of any one of the aspects described herein, repeat units in a concatemer can be independently, for example, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 nucleotides in length.

[0094] In some embodiments where the nucleic acid tag is a concatemer, the labeled-nucleic acid probe comprises a sequence complementary to at least a part of the sequence repeated in the concatemer.

[0095] Exemplary methods for preparing target-binding ligands conjugated with a concatemer comprising repeats of a specific sequence, e.g., a primer sequence, are described for example, in PCT Application No. PCT / US2023 / 028205, filed July 20, 2023 and titled “METHOD FOR HIGHLY MULTIPLEXED, THERMAL CONTROLLABLE DNA EXTENSION AND ITS APPLICATIONS”, and PCT Application No. PCT / US2023 / 028207, filed July 20, 2023 and titled “ENGINEERING DYNAMIC DNA NANO-DEVICES TO AMPLIFY SIGNAL”, contents of both which are incorporated herein by reference in their entireties.

[0096] In some embodiments, the step of contacting the sample with the target-binding ligand(s) comprises contacting the sample with target-binding ligand(s) conjugated to an oligonucleotide primer and extending the oligonucleotide primer, e.g., using a single-stranded extender template. For example, the step of contacting the sample with a target-binding ligand comprises contacting the sample with a target-binding ligand conjugated to an oligonucleotide primer, adding to the sample a single-stranded extender template and a nucleic acid polymerase enzyme to extend the oligonucleotide primer using the single-stranded extender template.

[0097] In some embodiments, the step of contacting the sample with a target-binding ligand comprises: (i) contacting the sample with a target-binding ligand conjugated to an oligonucleotide primer; (ii) adding to the sample a single-stranded extender template and a nucleic acid polymerase enzyme; (iii) extending the oligonucleotide primer using the singlestranded extender template; (iv) heating the sample from (iii) to separate the single-stranded extender template from extended oligonucleotide primer produced in step (iii); and (v) repeating steps (ii) - (iv) at least once (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times), thereby generating a concatemer comprising repeats of the oligonucleotide primer sequence conjugated to the target-binding ligand.

[0098] It is noted that when the nucleic acid tag is concatemer, the overall length of the t nucleic acid tag is dependent on the number of repeats and their length. Thus, when a nucleic acid tag is not a concatemer, it can be, for example, between 9-100 nucleotides in length, between 9-95 nucleotides in length, between 9-90 nucleotides in length, between 9-85 nucleotides in length, between 9- 80 nucleotides in length, between 9-75 nucleotides in length, between 9-70 nucleotides in length, between 9-65 nucleotides in length, between 9-60 nucleotides in length, between 9-55 nucleotides in length, between 9-50 nucleotides in length, between 9-45 nucleotides in length, between 9-40 nucleotides in length, between 9-35 nucleotides in length, between 9-30 nucleotides in length, between 9-25 nucleotides in length, between 9-20 nucleotides in length, between 9-15 nucleotides in length, between 10-100 nucleotides in length, between 10-90 nucleotides in length, between 10-80 nucleotides in length, between 10- 70 nucleotides in length, between 10-60 nucleotides in length, between 10-50 nucleotides in length, between 10-40 nucleotides in length, between 10-30 nucleotides in length, between 10- 20 nucleotides in length, between 11-100 nucleotides in length, between 11-90 nucleotides in length, between 11-80 nucleotides in length, between 11-70 nucleotides in length, between 11- 60 nucleotides in length, between 11-50 nucleotides in length, between 11-40 nucleotides in length, between 11-30 nucleotides in length, between 11-20 nucleotides in length, between 12- 100 nucleotides in length, between 12-90 nucleotides in length, between 12-80 nucleotides in length, between 12-70 nucleotides in length, between 12-60 nucleotides in length, between 12- 50 nucleotides in length, between 12-40 nucleotides in length, between 12-30 nucleotides in length, between 12-20 nucleotides in length, between 13-100 nucleotides in length, between13-90 nucleotides in length, between 13-80 nucleotides in length, between 13-80 nucleotides in length, between 13-70 nucleotides in length, between 13-60 nucleotides in length, between13-50 nucleotides in length, between 13-40 nucleotides in length, between 13-30 nucleotides in length, between 13-20 nucleotides in length, between 14-100 nucleotides in length, between14-90 nucleotides in length, between 14-80 nucleotides in length, between 14-70 nucleotides in length, between 14-60 nucleotides in length, between 14-50 nucleotides in length, between14-40 nucleotides in length, between 14-30 nucleotides in length, between 14-20 nucleotides in length, between 15-100 nucleotides in length, between 15-90 nucleotides in length, between15-80 nucleotides in length, between 15-70 nucleotides in length, between 15-60 nucleotides in length, between 15-50 nucleotides in length, between 15-40 nucleotides in length, between15-30 nucleotides in length, or between 15-20 nucleotides in length.

[0099] As used herein, an oligonucleotide “primer” is an oligonucleotide that can be extended by a template-dependent nucleic acid polymerase when hybridized (e.g., via hydrogen- bondedbase pairing) to a template nucleic acid molecule. Generally, primers are conjugated with target-binding ligand in a manner that permits template-directed nucleic acid polymerase extension from the 3’ end of the primer. While various approaches for primer conjugation can be used, it is therefore important that the method used keep the 3 ’ nucleotide open for extension. Examples include conjugation via linking moiety attached at or near the 5’ end of the oligonucleotide prime

[0100] An oligonucleotide primer can be, for example, between 9-100 nucleotides in length, between 9-95 nucleotides in length, between 9-90 nucleotides in length, between 9-85 nucleotides in length, between 9- 80 nucleotides in length, between 9-75 nucleotides in length, between 9-70 nucleotides in length, between 9-65 nucleotides in length, between 9-60 nucleotides in length, between 9-55 nucleotides in length, between 9-50 nucleotides in length, between 9-45 nucleotides in length, between 9-40 nucleotides in length, between 9-35 nucleotides in length, between 9-30 nucleotides in length, between 9-25 nucleotides in length, between 9-20 nucleotides in length, between 9-15 nucleotides in length, between 10-100 nucleotides in length, between 10-90 nucleotides in length, between 10-80 nucleotides in length, between 10-70 nucleotides in length, between 10-60 nucleotides in length, between 10- 50 nucleotides in length, between 10-40 nucleotides in length, between 10-30 nucleotides in length, between 10-20 nucleotides in length, between 11-100 nucleotides in length, between11-90 nucleotides in length, between 11-80 nucleotides in length, between 11-70 nucleotides in length, between 11-60 nucleotides in length, between 11-50 nucleotides in length, between11-40 nucleotides in length, between 11-30 nucleotides in length, between 11-20 nucleotides in length, between 12-100 nucleotides in length, between 12-90 nucleotides in length, between12-80 nucleotides in length, between 12-70 nucleotides in length, between 12-60 nucleotides in length, between 12-50 nucleotides in length, between 12-40 nucleotides in length, between12-30 nucleotides in length, between 12-20 nucleotides in length, between 13-100 nucleotides in length, between 13-90 nucleotides in length, between 13-80 nucleotides in length, between13-80 nucleotides in length, between 13-70 nucleotides in length, between 13-60 nucleotides in length, between 13-50 nucleotides in length, between 13-40 nucleotides in length, between13-30 nucleotides in length, between 13-20 nucleotides in length, between 14-100 nucleotides in length, between 14-90 nucleotides in length, between 14-80 nucleotides in length, between14-70 nucleotides in length, between 14-60 nucleotides in length, between 14-50 nucleotides in length, between 14-40 nucleotides in length, between 14-30 nucleotides in length, between14-20 nucleotides in length, between 15-100 nucleotides in length, between 15-90 nucleotides in length, between 15-80 nucleotides in length, between 15-70 nucleotides in length, between15-60 nucleotides in length, between 15-50 nucleotides in length, between 15-40 nucleotides in length, between 15-30 nucleotides in length, or between 15-20 nucleotides in length.

[0101] In some embodiments of any one of the aspects described herein, the oligonucleotide primer can be, for example, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 nucleotides in length.

[0102] In some embodiments, a single-stranded extender template oligonucleotide as described herein has a 3’ modification or moiety (also referred to as a “stopper” moiety) that blocks or precludes template-dependent extension. Such a modification can also be referred to as a chain terminator, and includes, for example, a dideoxy nucleoside, 3’-CPR II CPG, 3 ’-phosphate CPG, 5’-OMe-dT-CE Phosphoramidite, 5’-amino-dT-CE Phosphoramidite, 2’3’-ddA-CE Phosphoramidite, 2’3’-ddC-CE Phosphoramidite, 2’3-ddG-CE Phosphoramidite, 2’3’-ddT-CE Phosphoramidite, 3’-dA-CPG, 3’-ddC-CPG, 3’-dC-CPG, 3’-dG-CPG, 3’-dT-CPG, 3’-Spacer- C3 CPG and the like.

[0103] a single-stranded extender template oligonucleotide can be, for example, between 9- 100 nucleotides in length, between 9-95 nucleotides in length, between 9-90 nucleotides in length, between 9-85 nucleotides in length, between 9- 80 nucleotides in length, between 9-75 nucleotides in length, between 9-70 nucleotides in length, between 9-65 nucleotides in length, between 9-60 nucleotides in length, between 9-55 nucleotides in length, between 9-50 nucleotides in length, between 9-45 nucleotides in length, between 9-40 nucleotides in length, between 9-35 nucleotides in length, between 9-30 nucleotides in length, between 9-25 nucleotides in length, between 9-20 nucleotides in length, between 9-15 nucleotides in length, between 10-100 nucleotides in length, between 10-90 nucleotides in length, between 10-80 nucleotides in length, between 10-70 nucleotides in length, between 10-60 nucleotides in length, between 10-50 nucleotides in length, between 10-40 nucleotides in length, between 10- 30 nucleotides in length, between 10-20 nucleotides in length, between 11-100 nucleotides in length, between 11-90 nucleotides in length, between 11-80 nucleotides in length, between 11- 70 nucleotides in length, between 11-60 nucleotides in length, between 11-50 nucleotides in length, between 11-40 nucleotides in length, between 11-30 nucleotides in length, between 11- 20 nucleotides in length, between 12-100 nucleotides in length, between 12-90 nucleotides in length, between 12-80 nucleotides in length, between 12-70 nucleotides in length, between 12- 60 nucleotides in length, between 12-50 nucleotides in length, between 12-40 nucleotides in length, between 12-30 nucleotides in length, between 12-20 nucleotides in length, between 13- 100 nucleotides in length, between 13-90 nucleotides in length, between 13-80 nucleotides in length, between 13-80 nucleotides in length, between 13-70 nucleotides in length, between 13-60 nucleotides in length, between 13-50 nucleotides in length, between 13-40 nucleotides in length, between 13-30 nucleotides in length, between 13-20 nucleotides in length, between 14- 100 nucleotides in length, between 14-90 nucleotides in length, between 14-80 nucleotides in length, between 14-70 nucleotides in length, between 14-60 nucleotides in length, between 14- 50 nucleotides in length, between 14-40 nucleotides in length, between 14-30 nucleotides in length, between 14-20 nucleotides in length, between 15-100 nucleotides in length, between 15-90 nucleotides in length, between 15-80 nucleotides in length, between 15-70 nucleotides in length, between 15-60 nucleotides in length, between 15-50 nucleotides in length, between 15-40 nucleotides in length, between 15-30 nucleotides in length, or between 15-20 nucleotides in length.

[0104] In some embodiments of any one of the aspects described herein, the single-stranded extender template oligonucleotide can be, for example, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 nucleotides in length.

[0105] It is to be understood that the single-stranded extender template and the nucleic acid polymerase enzyme are added to the sample under conditions permitting hybridization and extension of the oligonucleotide primer using the single-stranded extender template. As used herein, the term “conditions permitting hybridization and extension” of a given molecule, e.g., an oligonucleotide molecule, refers to conditions of temperature, salt, buffer and other reaction components sufficient for a template-directed polymerase-mediated primer extension reaction. Exact conditions for extension by a given polymerase vary with the enzyme chosen, but are known in the art and / or can be determined without undue experimentation. Conditions for hybridization, e.g., of a primer as described herein to a single-stranded extender template as described herein, will generally be those salt, buffer and other reaction component conditions appropriate for the chosen polymerase enzyme, with the annealing or hybridization temperature established by one of skill in the art primarily on the basis of the length of the concatemer repeat unit and degree of multiplexing in a given reaction. Thus, where performed in uniplex, with one given molecule, e.g., target-binding ligand being labeled with a single concatemeric sequence in the reaction, an annealing or hybridization temperature can be determined based on the Tm of the specific concatemer repeat sequence - generally the annealing temperature in a cycling reaction is about 5°C below the Tm for the hybridization of the repeat sequence to its complement under the salt and reaction component concentrations optimal for the enzyme of choice. Where performed in multiplex, the length of the repeat unit takes on added importance, as the Tm for various repeat sequences will vary. Primer / single-stranded extended templatedesign can take sequence variation into account to design primers and single-stranded extender templates that have Tm values that are relatively close to each other, generally on the order of within 5-7°C for all members of a set of primer / extender template sequences. Under these circumstances, a single annealing or hybridization temperature in a cycling reaction can permit efficient hybridization and extension of members of the set in the multiplex. In various embodiments of a method including a step of contacting a nucleic acid molecule with a singlestranded extender template under conditions permitting hybridization and extension of the nucleic acid molecule, the reaction can be incubated at an annealing temperature for a period of time (generally a matter of seconds to minutes) before raising the temperature to an optimum extension temperature for the polymerase enzyme. It should be understood that contacting a nucleic acid molecule with a single-stranded extender template “under conditions permitting hybridization and extension” of the nucleic acid molecule can include such an annealing / extending temperature shift. In some embodiments, annealing or hybridization occurs efficiently at a temperature at which the polymerase enzyme will be sufficiently active as not to require such a temperature shift; whether or not such a shift is needed in a given circumstance will be apparent to the one of ordinary skill in the art depending upon the enzyme used, and can also be determined empirically without undue experimentation.Labeled nucleic acid probe

[0106] The terms “labeled nucleic acid probe” and “labeled probe” are used interchangeably herein and refer to an oligonucleotide that includes a detectable label that is directly or indirectly detectable to provide a signal. A labeled-nucleic acid probe can be, for example, between 9-100 nucleotides in length, between 9-95 nucleotides in length, between 9-90 nucleotides in length, between 9-85 nucleotides in length, between 9- 80 nucleotides in length, between 9-75 nucleotides in length, between 9-70 nucleotides in length, between 9-65 nucleotides in length, between 9-60 nucleotides in length, between 9-55 nucleotides in length, between 9-50 nucleotides in length, between 9-45 nucleotides in length, between 9-40 nucleotides in length, between 9-35 nucleotides in length, between 9-30 nucleotides in length, between 9-25 nucleotides in length, between 9-20 nucleotides in length, between 9-15 nucleotides in length, between 10-100 nucleotides in length, between 10-90 nucleotides in length, between 10-80 nucleotides in length, between 10-70 nucleotides in length, between 10- 60 nucleotides in length, between 10-50 nucleotides in length, between 10-40 nucleotides in length, between 10-30 nucleotides in length, between 10-20 nucleotides in length, between 11- 100 nucleotides in length, between 11-90 nucleotides in length, between 11-80 nucleotides inlength, between 11-70 nucleotides in length, between 11-60 nucleotides in length, between 11- 50 nucleotides in length, between 11-40 nucleotides in length, between 11-30 nucleotides in length, between 11-20 nucleotides in length, between 12-100 nucleotides in length, between12-90 nucleotides in length, between 12-80 nucleotides in length, between 12-70 nucleotides in length, between 12-60 nucleotides in length, between 12-50 nucleotides in length, between12-40 nucleotides in length, between 12-30 nucleotides in length, between 12-20 nucleotides in length, between 13-100 nucleotides in length, between 13-90 nucleotides in length, between13-80 nucleotides in length, between 13-80 nucleotides in length, between 13-70 nucleotides in length, between 13-60 nucleotides in length, between 13-50 nucleotides in length, between13-40 nucleotides in length, between 13-30 nucleotides in length, between 13-20 nucleotides in length, between 14-100 nucleotides in length, between 14-90 nucleotides in length, between14-80 nucleotides in length, between 14-70 nucleotides in length, between 14-60 nucleotides in length, between 14-50 nucleotides in length, between 14-40 nucleotides in length, between14-30 nucleotides in length, between 14-20 nucleotides in length, between 15-100 nucleotides in length, between 15-90 nucleotides in length, between 15-80 nucleotides in length, between15-70 nucleotides in length, between 15-60 nucleotides in length, between 15-50 nucleotides in length, between 15-40 nucleotides in length, between 15-30 nucleotides in length, or between 15-20 nucleotides in length.

[0107] In some embodiments of any one of the aspects described herein, the labeled-nucleic acid probe can be, for example, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 nucleotides in length.

[0108] In some embodiments, a plurality of labeled-nucleic acid probes bind or hybridize to a nucleic acid tag.

[0109] It is noted a labeled-nucleic acid probe can comprises one, two, three, four or more detectable labels. Further, a detectable label can be at the 3 ’-end, 5 ’-end or at an internal position of the labeled-nucleic acid probe.

[0110] For multiplex approaches, probes labeled with distinguishable detectable labels can be used.Detectable label

[0111] As used herein, the term “detectable label” refers to a molecule or composition capable of producing a detectable signal indicative of the presence and / or location of a target. Detectable labels include any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. Exemplary detectablelabels include but are not limited to fluorophores (fluorescent molecules), chemiluminescent moieties, bioluminescent moieties, colloidal gold, colored glass or plastic (e.g., polystyrene, polypropylene, and latex) beads, radiolabels, nanoparticles, stable isotopes, radioisotopes (e.g., e.g.,32P,35S, etc), nucleotide chromophores, quantum dots, enzymes, echogenic substances, non-metallic isotopes, optical reporters, paramagnetic metal ions, and ferromagnetic metals. Means of detecting such labels are well known to those of skill in the art. Thus, for example, fluorescent markers can be detected using a photo-detector to detect emitted light, metals, e.g. a lanthanide metal can be detected in mass cytometry, radiolabels can be detected using photographic film or scintillation counters, fluorescent markers can be detected using a photodetector to detect emitted light. Enzymatic labels are typically detected by providing the enzyme with an enzyme substrate and detecting the reaction product produced by the action of the enzyme on the enzyme substrate, and calorimetric labels can be detected by visualizing the colored label. It is noted that methods of the preparation of labeled oligonucleotide probes bearing any of a number of different detectable moieties are known to those of skill in the art.

[0112] A wide variety of fluorescent reporter dyes are known in the art. Typically, the fluorophore is an aromatic or heteroaromatic compound and can be a pyrene, anthracene, naphthalene, acridine, stilbene, indole, benzindole, oxazole, thiazole, benzothiazole, cyanine, carbocyanine, salicylate, anthranilate, coumarin, fluorescein, rhodamine or other like compound.

[0088] Exemplary fluorophores include, but are not limited to, 1,5 IAEDANS; 1,8-ANS; 4- Methylumbelbferone; 5-carboxy-2,7-dichlorofluorescein; 5-Carboxyfluorescein (5-FAM); 5- Carboxynapthofluorescein (pH 10); 5-Carboxytetramethylrhodamine (5- TAMRA); 5-FAM (5- Carboxyfluorescein); 5-Hydroxy Tryptamine (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 Feulgen SITSA; Aequorin (Photoprotein); Alexa Fluor 350™; Alexa Fluor 430™; Alexa Fluor 488™; Alexa Fluor 532™; Alexa Fluor 546™; Alexa Fluor 568™; Alexa Fluor 594™; Alexa Fluor 633™; Alexa Fluor 647™; Alexa Fluor 660™; Alexa Fluor 680™; Alizarin Complexon; Alizarin Red; Allophycocyanin (APC); AMC, AMCA-S; AMCA (Aminomethylcoumarin); AMCA-X; Aminoactinomycin D; Aminocoumarin; Anilin Blue; Anthrocyl stearate; APC-Cy7; APTS; Astrazon Brilliant Red 4G; Astrazon Orange R; Astrazon Red 6B; Astrazon Yellow 7 GFF; Atabrine; ATTO-TAG™ CBQCA; ATTO-TAG™ FQ; Auramine; Aurophosphine G; Aurophosphine; BAO 9 (Bisaminophenyloxadiazole); BCECF(high pH); BCECF (low pH); Berberine Sulphate; Beta Factamase; BFP blue shifted GFP (Y66H); BG-647; Bimane; Bisbenzamide; Blancophor FFG; Blancophor SV; BOBO™ -1; BOBO™ -3; Bodipy 492 / 515; Bodipy 493 / 503; Bodipy 500 / 510; Bodipy 505 / 515; Bodipy 530 / 550; Bodipy 542 / 563; Bodipy 558 / 568; Bodipy 564 / 570; Bodipy 576 / 589; Bodipy 581 / 591; Bodipy 630 / 650-X; Bodipy 650 / 665- X; Bodipy 665 / 676; Bodipy FI; Bodipy FF ATP; Bodipy Fl -Ceramide; Bodipy R6G SE; Bodipy TMR; Bodipy TMR-X conjugate; Bodipy TMR-X, SE; Bodipy TR; Bodipy TR ATP; Bodipy TR- X SE; BO-PRO™ -1; BO-PRO™ -3; Brilliant Sulphoflavin FF; Calcein; Calcein Blue; Calcium Crimson™; Calcium Green; Calcium Green- 1 Ca2+Dye; Calcium Green-2 Ca2+; Calcium Green- 5N Ca2+; Calcium Green- C18 Ca2+; Calcium Orange; Cal cofluor White; Carboxy-X-rhodamine (5- ROX); Cascade Blue™; Cascade Yellow; Catecholamine; CFDA; CFP - Cyan Fluorescent Protein; Chlorophyll; Chromomycin A; Chromomycin A; CMFDA; Coelenterazine; Coelenterazine cp; Coelenterazine f; Coelenterazine fcp; Coelenterazine h; Coelenterazine hep; Coelenterazine ip; Coelenterazine O; Coumarin Phalloidin; CPM Methylcoumarin; CTC; Cy2™; Cy3.1 8; Cy3.5™; Cy3™; Cy5.1 8; Cy5.5™; Cy5™; Cy7™; Cyan GFP; cyclic AMP Fluorosensor (FiCRhR); d2; Dabcyl; Dansyl; Dansyl Amine; Dansyl Cadaverine; Dansyl Chloride; Dansyl DHPE; Dansyl fluoride; DAPI; Dapoxyl; Dapoxyl 2; Dapoxyl 3; DCFDA; DCFH (Dichlorodihydrofluorescein Diacetate); DDAO; DHR (Dihydorhodamine 123); Di-4- ANEPPS; Di-8-ANEPPS (non-ratio); DiA (4-D1-16-ASP); DIDS; Dihydorhodamine 123 (DHR); DiO (DiOC18(3)); DiR; DiR (DiIC18(7)); Dopamine; DsRed; DTAF; DY-630-NHS; DY-635-NHS; EBFP; ECFP; EGFP; EFF 97; Eosin; Erythrosin; Erythrosin ITC; Ethidium homodimer- 1 (EthD-1); Euchrysin; Europium (III) chloride; Europium; EYFP; Fast Blue; FDA; Feulgen (Pararosaniline); FITC; FF-645; Flazo Orange; Fluo-3; Fluo-4; Fluorescein Diacetate; Fluoro-Emerald; Fluoro-Gold (Hydroxystilbamidine); Fluor-Ruby; FluorX; FM 1- 43™; FM 4-46; Fura Red™ (high pH); Fura- 2, high calcium; Fura-2, low calcium; Genacryl Brilliant Red B; Genacryl Brilliant Yellow 10GF; Genacryl Pink 3G; Genacryl Yellow 5GF; GFP (S65T); GFP red shifted (rsGFP); GFP wild type, non-UV excitation (wtGFP); GFP wild type, UV excitation (wtGFP); GFPuv; Gloxalic Acid; Granular Blue; Haematoporphyrin; Hoechst 33258; Hoechst 33342; Hoechst 34580; HPTS; Hydroxycoumarin; Hydroxystilbamidine (FluoroGold); Hydroxytryptamine; Indodicarbocyanine (DiD); Indotricarbocyanine (DiR); Intrawhite Cf; JC-1; JO-JO-1; JO-PRO-1; LaserPro; Laurodan; LDS 751; Leucophor PAF; Leucophor SF; Leucophor WS; Lissamine Rhodamine; Lissamine Rhodamine B; LOLO-1; LO-PRO-1; Lucifer Yellow; Mag Green; Magdala Red (Phloxin B); Magnesium Green; Magnesium Orange; Malachite Green; Marina Blue; Maxiion BrilliantFlavin 10 GFF; Maxiion Brilliant Flavin 8 GFF; Merocyanin; Methoxy coumarin; Mitotracker Green FM; Mitotracker Orange; Mitotracker Red; Mitramycin; Monobromobimane; Monobromobimane (mBBr-GSH); Monochlorobimane; MPS (Methyl Green Pyronine Stilbene); NBD; NBD Amine; Nile Red; Nitrobenzoxadidole; Noradrenaline; Nuclear Fast Red; Nuclear Yellow; Nylosan Brilliant lavin E8G; Oregon Green™; Oregon Green 488-X; Oregon Green™ 488; Oregon Green™ 500; Oregon Green™ 514; Pacific Blue; Pararosanibne (Feulgen); PE-Cy5; PE-Cy7; PerCP; PerCP-Cy5.5; PE-TexasRed (Red 613); Phloxin B (Magdala 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; Proci on Yellow; Propidium lodid (PI); PyMPO; Pyrene; Pyronine; Pyronine B; Pyrozal Brilliant Flavin 7GF; QSY 7; Quinacrine Mustard; Resorufm; RH 414; Rhod- 2; Rhodamine; Rhodamine 110; Rhodamine 123; Rhodamine 5 GLD; Rhodamine 6G; Rhodamine B 540; Rhodamine B 200; Rhodamine B extra; Rhodamine BB; Rhodamine BG; Rhodamine Green; Rhodamine Phallicidine; Rhodamine Phalloidine; Rhodamine Red; Rhodamine WT; Rose Bengal; R- phycoerythrin (PE); red shifted GFP (rsGFP, S65T); S65A; S65C; S65L; S65T; Sapphire GFP; Serotonin; Sevron Brilliant Red 2B; Sevron Brilliant Red 4G; Sevron Brilliant Red B; Sevron Orange; Sevron Yellow L; sgBFP™; sgBFP™ (super glow BFP); sgGFP™; sgGFP™ (super glow GFP); SITS; SITS (Primuline); SITS (Stilbene Isothiosulphonic Acid); SPQ (6- methoxy- N-(3-sulfopropyl)-quinolinium); Stilbene; Sulphorhodamine B can C; Sulphorhodamine G Extra; Tetracycline; Tetramethylrhodamine; Texas Red™; Texas Red-X™ conjugate; Thiadicarbocyanine (DiSC3); Thiazine Red R; Thiazole Orange; Thioflavin 5; Thioflavin S; Thioflavin TCN; Thiolyte; Thiozole Orange; Tinopol CBS (Calcofluor White); TMR; TO- PRO-1; TO-PRO-3; TO-PRO-5; TOTO-1; TOTO-3; TnColor (PE-Cy5); TRITC (TetramethylRodaminelsoThioCyanate); True Blue; TruRed; Ultralite; Uranine B; Uvitex SFC; wt GFP; WW 781; XL665; X-Rhodamme; XRITC; Xylene Orange; Y66F; Y66H; Y66W; Yellow GFP; YFP; YO-PRO-1; YO-PRO-3; YOYO-1; and YOYO-3. Many suitable forms of these fluorescent compounds are available and can be used. Methods for fluorophore labeling of nucleic acids are known in the art. For multiplex approaches, probes labeled with fluorophores with distinguishable excitation and / or emission spectra can be used.

[0113] Other exemplary detectable labels include luminescent and bioluminescent markers ( e.g., biotin, luciferase (e.g.., bacterial, firefly, click beetle and the like), luciferin, and aequorin), radiolabels (e.g.., 3H, 1251, 35S, 14C, or 32P), enzymes (e.g.., galactosidases, glucorinidases, phosphatases (e.g.., alkaline phosphatase), peroxidases (e.g.., horseradish peroxidase), andcholinesterases), and calorimetric labels such as colloidal gold or colored glass or plastic (e.g.., polystyrene, polypropylene, and latex) beads. Patents teaching the use of such labels include U.S. Pat. Nos. 3,817,837, 3,850,752, 3,939,350, 3,996,345, 4,277,437, 4,275,149, and 4,366,241, each of which are incorporated herein by reference in their entireties.

[0114] In some embodiments, the detectable label is a fluorophore.Blocking probes

[0115] The terms “block nucleic acid probe” and “blocking probe” are used interchangeably herein and refer to an oligonucleotide that inhibit or reduce non-specific binding of a labeled- nucleic acid probe to nucleic acid tags and other nucleic acids in a sample that are not complementary to the labeled-nucleic acid probe. Generally, the blocking probe comprises a nucleotide sequence that is not complementary to the nucleic acid tag sequence to which a labeled-nucleic acid probe binds. Each blocking probe can be, for example, between 9-100 nucleotides in length, between 9-95 nucleotides in length, between 9-90 nucleotides in length, between 9-85 nucleotides in length, between 9- 80 nucleotides in length, between 9-75 nucleotides in length, between 9-70 nucleotides in length, between 9-65 nucleotides in length, between 9-60 nucleotides in length, between 9-55 nucleotides in length, between 9-50 nucleotides in length, between 9-45 nucleotides in length, between 9-40 nucleotides in length, between 9-35 nucleotides in length, between 9-30 nucleotides in length, between 9-25 nucleotides in length, between 9-20 nucleotides in length, between 9-15 nucleotides in length, between 10-100 nucleotides in length, between 10-90 nucleotides in length, between 10-80 nucleotides in length, between 10-70 nucleotides in length, between 10-60 nucleotides in length, between 10-50 nucleotides in length, between 10-40 nucleotides in length, between 10- 30 nucleotides in length, between 10-20 nucleotides in length, between 11-100 nucleotides in length, between 11-90 nucleotides in length, between 11-80 nucleotides in length, between 11- 70 nucleotides in length, between 11-60 nucleotides in length, between 11-50 nucleotides in length, between 11-40 nucleotides in length, between 11-30 nucleotides in length, between 11- 20 nucleotides in length, between 12-100 nucleotides in length, between 12-90 nucleotides in length, between 12-80 nucleotides in length, between 12-70 nucleotides in length, between 12- 60 nucleotides in length, between 12-50 nucleotides in length, between 12-40 nucleotides in length, between 12-30 nucleotides in length, between 12-20 nucleotides in length, between 13- 100 nucleotides in length, between 13-90 nucleotides in length, between 13-80 nucleotides in length, between 13-80 nucleotides in length, between 13-70 nucleotides in length, between 13- 60 nucleotides in length, between 13-50 nucleotides in length, between 13-40 nucleotides inlength, between 13-30 nucleotides in length, between 13-20 nucleotides in length, between 14- 100 nucleotides in length, between 14-90 nucleotides in length, between 14-80 nucleotides in length, between 14-70 nucleotides in length, between 14-60 nucleotides in length, between 14- 50 nucleotides in length, between 14-40 nucleotides in length, between 14-30 nucleotides in length, between 14-20 nucleotides in length, between 15-100 nucleotides in length, between 15-90 nucleotides in length, between 15-80 nucleotides in length, between 15-70 nucleotides in length, between 15-60 nucleotides in length, between 15-50 nucleotides in length, between 15-40 nucleotides in length, between 15-30 nucleotides in length, or between 15-20 nucleotides in length.

[0116] In some embodiments of any one of the aspects described herein, each blocking probe can be, for example, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 nucleotides in length.Binding of probes

[0117] Generally, the method described herein comprises a step of contacting nucleic acid tags with labeled-nucleic acid probes and / or blocking probes. It is to be understood that said contacting is under conditions permitting annealing or hybridizing together of two nucleic acids, e.g., a nucleic acid tag and labeled-nucleic acid probe, or a nucleic acid tag and blocking probe. As used herein, the term “hybridizing”, “hybridize”, “hybridization”, “annealing”, or “anneal” are used interchangeably in reference to the pairing of complementary nucleic acids using any process by which a strand of nucleic acid joins with a complementary strand through base pairing to form a hybridization complex. In other words, the term “hybridization” refers to the process in which two single-stranded polynucleotides bind non-covalently to form a stable double-stranded polynucleotide. The term “hybridization” may also refer to triplestranded hybridization. The resulting (usually) double-stranded polynucleotide is a “hybrid” or “duplex.”

[0001] “Hybridization conditions” will typically include salt concentrations of less than about 1 M, more usually less than about 500 mM and even more usually less than about 200 mM. Hybridization temperatures can be as low as 5°C, but are typically greater than 22° C more typically greater than about 30°C, and often in excess of about 37°C. Hybridizations are usually performed under stringent conditions, i.e., conditions under which a strand will hybridize to its intended target strands. Stringent conditions are sequence-dependent and are different in different circumstances. Longer fragments may require higher hybridization temperatures for specific hybridization. As other factors may affect the stringency ofhybridization, including base composition and length of the complementary strands, presence of organic solvents and extent of base mismatching, the combination of parameters is more important than the absolute measure of any one alone. Generally, stringent conditions are selected to be about 5°C lower than the Tm for the specific sequence at a defined ionic strength and pH. Exemplary stringent conditions include salt concentration of at least 0.01 M to no more than 1 M Na ion concentration (or other salts) at a pH 7.0 to 8.3 and a temperature of at least 25° C. For example, conditions of 5*SSPE (750 mM NaCl, 50 mM Na phosphate, 5 mM EDTA, pH 7.4) and a temperature of 25-30° C are suitable for allele-specific probe hybridizations. For stringent conditions, see for example, Sambrook, Fritsche and Maniatis, Molecular Cloning A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press (1989) and Anderson Nucleic Acid Hybridization, 1st Ed., BIOS Scientific Publishers Limited (1999). “Hybridizing specifically to” or “specifically hybridizing to” or like expressions refer to the binding, duplexing, or hybridizing of a molecule substantially to or only to a particular nucleotide sequence or sequences under stringent conditions when that sequence is present in a complex mixture.

[0002] In some embodiments of any of the aspects, nucleic acid strands described herein, e.g., nucleic acid tag and labeled-nucleic acid probe, or a nucleic acid tag and blocking probe, can be annealed together (e.g. by cooling from 80°C to 20°C over a period of time, e.g., 1 hour), or they can be combined together isothermally (e.g. at room temperature, 37°C, 46°C, etc.)Target molecule

[0118] The methods, compositions, kits and / or systems described herein can be used for detecting / imaging any desired molecule (i.e., target or target molecule) in a sample. For example, any target of interest can be detected / imaged using the methods, compositions, kits and / or systems described herein. Examples of targets include, but are not limited to, proteins, nucleic acids (e.g., DNA, RNA, microRNAs), lipids, saccharides (e.g., polysaccharides), small molecules, and antigens. In some embodiments, the target is a biomolecule. As used herein, a “biomolecule” is any molecule that is produced by a living organism, including large macromolecules such as proteins (e.g., antibodies), nucleic acids (e.g., DNA and RNA such as mRNA), polysaccharides, lipids and as well as small molecules such as primary metabolites, secondary metabolites, and natural products. In some embodiments, the target is a protein, e.g., an antibody. In some embodiments, the target is RNA, e.g., mRNA. In some embodiments, the target is DNA. In some embodiments, the target is a small molecule.

[0119] In some embodiments, the target is a protein such as, for example, proteins of a cellular environment (e.g., intracellular or membrane proteins). Examples of proteins include, without limitation, fibrous proteins such as cytoskeletal proteins (e.g., actin, arp2 / 3, coronin, dystrophin, FtsZ, keratin, myosin, nebulin, spectrin, tau, titin, tropomyosin, tubulin and collagen) and extracellular matrix proteins (e.g., collagen, elastin, f-spondin, pikachurin, and fibronectin); globular proteins such as plasma proteins (e.g., serum amyloid P component and serum albumin), coagulation factors (e.g., complement proteins, Cl -inhibitor and C3- convertase, Factor VIII, Factor XIII, fibrin, Protein C, Protein S, Protein Z, Protein Z-related protease inhibitor, thrombin, Von Willebrand Factor) and acute phase proteins such as C- reactive protein; hemoproteins; cell adhesion proteins (e.g., cadherin, ependymin, integrin, Ncam and selectin); transmembrane transport proteins (e.g., CFTR, glycophorin D and scramblase) such as ion channels (e.g., ligand-gated ion channels such nicotinic acetylcholine receptors and GABAa receptors, and voltage-gated ion channels such as potassium, calcium and sodium channels), synport / antiport proteins (e.g., glucose transporter); hormones and growth factors (e.g., epidermal growth factor (EGF), fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), peptide hormones such as insulin, insulin-like growth factor and oxytocin, and steroid hormones such as androgens, estrogens and progesterones); receptors such as transmembrane receptors (e.g., G-protein-coupled receptor, rhodopsin) and intracellular receptors (e.g., estrogen receptor); DNA-binding proteins (e.g., histones, protamines, CI protein); transcription regulators (e.g., c-myc, FOXP2, FOXP3, MyoD and P53); immune system proteins (e.g., immunoglobulins, major histocompatibility antigens and T cell receptors); nutrient storage / transport proteins (e.g., ferritin); chaperone proteins; and enzymes.

[0120] Additional examples of targets include, without limitation, DNA, RNA, cDNA, or the DNA product of RNA subjected to reverse transcription, A23187 (Calcimycin, Calcium Ionophore), Abamectine, Abietic acid, Acetic acid, Acetylcholine, Actin, Actinomycin D, Adenosine, Adenosine diphosphate (ADP), Adenosine monophosphate (AMP), Adenosine triphosphate (ATP), Adenylate cyclase, Adonitol, Adrenaline, epinephrine, Adrenocorticotropic hormone (ACTH), Aequorin, Aflatoxin, Agar, Alamethicin, Alanine, Albumins, Aldosterone, Aleurone, Alphaamanitin, Allantoin, Allethrin, a-Amanatin, Amino acid, Amylase, Anabolic steroid, Anethole, Angiotensinogen, Anisomycin, Antidiuretic hormone (ADH), Arabinose, Arginine, Ascomycin, Ascorbic acid (vitamin C), Asparagine, Aspartic acid, Asymmetric dimethylarginine, Atrial -natriuretic peptide (ANP), Auxin, Avidin, Azadirachtin A — C35H44O16, Bacteriocin, Beauvericin, Bicuculline, Bilirubin, Biopolymer,Biotin (Vitamin H), Brefeldin A, Brassinolide, Brucine, Cadaverine, Caffeine, Calciferol (Vitamin D), Calcitonin, Calmodulin, Calmodulin, Calreticulin, Camphor — (C10H16O), Cannabinol, Capsaicin, Carbohydrase, Carbohydrate, Carnitine, Carrageenan, Casein, Caspase, Cellulase, Cellulose — (C6H10O5), Cerulenin, Cetrimonium bromide (Cetrimide) — C19H42BrN, Chelerythrine, Chromomycin A3, Chaparonin, Chitin, -Chloralose, Chlorophyll, Cholecystokinin (CCK), Cholesterol, Choline, Chondroitin sulfate, Cinnamaldehyde, Citral, Citric acid, Citrinin, Citronellal, Citronellol, Citrulline, Cobalamin (vitamin B12), Coenzyme, Coenzyme Q, Colchicine, Collagen, Coniine, Corticosteroid, Corticosterone, Corticotropin releasing hormone (CRH), Cortisol, Creatine, Creatine kinase, Crystallin, a-Cyclodextrin, Cyclodextrin glycosyltransferase, Cyclopamine, Cyclopiazonic acid, Cysteine, Cystine, Cytidine, Cytochalasin, Cytochalasin E, Cytochrome, Cytochrome C, Cytochrome c oxidase, Cytochrome c peroxidase, Cytokine, Cytosine — C4H5N3O, Deoxy cholic acid, DON (DeoxyNivalenol), Deoxyribofuranose, Deoxyribose, Deoxyribose nucleic acid (DNA), Dextran, Dextrin, DNA, Dopamine, Enzyme, Ephedrine, Epinephrine — C9H13NO3, Erucic acid— CH3(CH2)7CH=CH(CH2)11C00H, Erythritol, Erythropoietin (EPO), Estradiol, Eugenol, Fatty acid, Fibrin, Fibronectin, Folic acid (Vitamin M), Follicle stimulating hormone (FSH), Formaldehyde, Formic acid, Formnoci, Fructose, Fumonisin Bl, Gamma globulin, Galactose, Gamma globulin, Gamma-aminobutyric acid, Gamma-butyrolactone, Gammahydroxybutyrate (GHB), Gastrin, Gelatin, Geraniol, Globulin, Glucagon, Glucosamine, Glucose — C6H12O6, Glucose oxidase, Gluten, Glutamic acid, Glutamine, Glutathione, Gluten, Glycerin (glycerol), Glycine, Glycogen, Glycolic acid, Glycoprotein (e.g., glycoprotein enzymes such as prostate-specific antigen (PSA)), Gonadotropin-releasing hormone (GnRH), Granzyme, Green fluorescent protein, Growth hormone, Growth hormone releasing hormone (GHRH), GTPase, Guanine, Guanosine, Guanosine triphosphate (+GTP), Haptoglobin, Hematoxylin, Heme, Hemerythrin, Hemocyanin, Hemoglobin, Hemoprotein, Heparan sulfate, High density lipoprotein, HDL, Histamine, Histidine, Histone, Histone methyltransferase, HLA antigen, Homocysteine, Hormone, human chorionic gonadotropin (hCG), Human growth hormone, Hyaluronate, Hyaluronidase, Hydrogen peroxide, 5- Hydroxymethylcytosine, Hydroxyproline, 5-Hydroxytryptamine, Indigo dye, Indole, Inosine, Inositol, Insulin, Insulin-like growth factor, Integral membrane protein, Integrase, Integrin, Intein, Interferon, Inulin, lonomycin, Ionone, Isoleucine, Iron-sulfur cluster, K252a, K252b, KT5720, KT5823, Keratin, Kinase, Lactase, Lactic acid, Lactose, Lanolin, Lauric acid, Leptin, Leptomycin B, Leucine, Lignin, Limonene, Linalool, Linoleic acid, Linolenic acid, Lipase, Lipid, Lipid anchored protein, Lipoamide, Lipoprotein, Low density lipoprotein, LDL,Luteinizing hormone (LH), Lycopene, Lysine, Lysozyme, Malic acid, Maltose, Melatonin, Membrane protein, Metalloprotein, Metallothionein, Methionine, Mimosine, Mithramycin A, Mitomycin C, Monomer, Mycophenolic acid, Myoglobin, Myosin, Natural phenols, Nucleic Acid, Ochratoxin A, Oestrogens, Oligopeptide, Oligomycin, Orcin, Orexin, Ornithine, Oxalic acid, Oxidase, Oxytocin, p53, PABA, Paclitaxel, Palmitic acid, Pantothenic acid (vitamin B5), parathyroid hormone (PTH), Paraprotein, Pardaxin, Parthenolide, Patulin, Paxilline, Penicillic acid, Penicillin, Penitrem A, Peptidase, Pepsin, Peptide, Perimycin, Peripheral membrane protein, Perosamine, Phenethylamine, Phenylalanine, Phosphagen, phosphatase, Phospholipid, Phenylalanine, Phytic acid, Plant hormones, Polypeptide, Polyphenols, Polysaccharides, Porphyrin, Prion, Progesterone, Prolactin (PRL), Proline, Propionic acid, Protamine, Protease, Protein, Proteinoid, Putrescine, Pyrethrin, Pyridoxine or pyridoxamine (Vitamin B6), Pyrrolysine, Pyruvic acid, Quinone, Radicicol, Raffinose, Renin, Retinene, Retinol (Vitamin A), Rhodopsin (visual purple), Riboflavin (vitamin B2), Ribofuranose, Ribose, Ribozyme, Ricin, RNA — Ribonucleic acid, RuBisCO, Safrole, Salicylaldehyde, Salicylic acid, Salvinorin A — C23H28O8, Saponin, Secretin, Selenocysteine, Selenomethionine, Selenoprotein, Serine, Serine kinase, Serotonin, Skatole, Signal recognition particle, Somatostatin, Sorbic acid, Squalene, Staurosporin, Stearic acid, Sterigmatocystin, Sterol, Strychnine, Sucrose (sugar), Sugars (in general), superoxide, tau protein, T2 Toxin, Tannic acid, Tannin, Tartaric acid, Taurine, Tetrodotoxin, Thaumatin, Topoisomerase, Tyrosine kinase, Taurine, Testosterone, Tetrahydrocannabinol (THC), Tetrodotoxin, Thapsigargin, Thaumatin, Thiamine (vitamin Bl) — C12H17C1N4OS.HC1, Threonine, Thrombopoietin, Thymidine, Thymine, Triacsin C, Thyroid-stimulating hormone (TSH), Thyrotropin-releasing hormone (TRH), Thyroxine (T4), Tocopherol (Vitamin E), Topoisomerase, Triiodothyronine (T3), Transmembrane receptor, Trichostatin A, Trophic hormone, Trypsin, Tryptophan, Tubulin, Tunicamycin, Tyrosine, Ubiquitin, Uracil, Urea, Urease, Uric acid — C5H4N4O3, Uridine, Valine, Valinomycin, Vanabins, Vasopressin, Verruculogen, Vitamins (in general), Vitamin A (retinol), Vitamin B, Vitamin Bl (thiamine), Vitamin B2 (riboflavin), Vitamin B3 (niacin or nicotinic acid), Vitamin B4 (adenine), Vitamin B5 (pantothenic acid), Vitamin B6 (pyridoxine or pyridoxamine), Vitamin B12 (cobalamin), Vitamin C (ascorbic acid), Vitamin D (calciferol), Vitamin E (tocopherol), Vitamin F, Vitamin H (biotin), Vitamin K (naphthoquinone), Vitamin M (folic acid), Wortmannin and Xylose-45.Sample

[0121] Described herein are methods, compositions, kits, and systems for detection of a target in a sample. In some embodiments, the sample can be a biological sample. The term “biological sample” as used herein can denote a sample taken or isolated from a biological organism. Exemplary biological samples include tissue samples, such as liver, spleen, kidney, lung, intestine, thymus, colon, tonsil, testis, skin, brain, heart, muscle, and pancreas tissue. Other exemplary biological samples include, but are not limited to, biopsies, bone marrow samples, organ samples, skin fragments and organisms. Materials obtained from clinical or forensic settings are also within the intended meaning of the term biological sample. In one embodiment, the sample is derived from a human, animal or plant. In one embodiment, the biological sample is a tissue sample, preferably an organ tissue sample. In one embodiment, samples are human. The sample can be obtained, for example, from autopsy, biopsy, muscle punch, or from surgery. It can be a solid tissue or solid tumor such as parenchyme, connective or fatty tissue, heart or skeletal muscle, smooth muscle, skin, brain, nerve, kidney, liver, spleen, breast, carcinoma (e.g., bowel, nasopharynx, breast, lung, stomach etc.), cartilage, lymphoma, meningioma, placenta, prostate, thymus, tonsil, umbilical cord or uterus. The tissue can be a tumor (benign or malignant), cancerous or precancerous tissue. The sample can be obtained from an animal or human subject affected by disease or other pathology or suspected of same (normal or diseased), or considered normal or healthy.

[0122] In some embodiments, the sample is a cell, e.g., a biological cell.

[0123] In some embodiments, the sample is a tissue sample.

[0124] Samples, including cell and / or tissue samples, can be fixed to avoid deterioration over subsequent staining and detection steps. The sample can be fixed as soon after collection as possible. There are many different types of fixatives known in the art. Exemplary fixatives include, but are not limited to, paraformaldehyde (PF A) at various concentrations (commonly between 1% and 5%, e.g., 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%), formaldehyde at various concentrations (e.g., 2%, 2.5%, 3%, 3.5%, 4%, 4.5% or 5% formaldehyde), 10% neutral buffered formalin, Bouin’s solution, methanol, acetone, glutaraldehyde, etc. A biological samples, e.g., cell and / or tissue samples, can be treated or processed so as to minimize nucleic acid degradation, where, for example, RNA is the target molecule. One of ordinary skill in the art will be able to determine the fixative best suited for the cell or tissue sample and technique to be performed. In some embodiments, the fixative that can be used is 4% paraformaldehyde.

[0125] In some embodiments, the sample, e.g., cell and / or tissue sample, can be embedded in a medium facilitating, for example, sectioning for histology and / or imaging. Paraffin-embedding of fixed samples is well known in the art. Briefly, however, after a sample, e.g., a tissue sample, has been fixed, it undergoes pre-embedding of paraffin to replace the water content of the sample with paraffin. Embedding in paraffin involves dehydration of tissues in increasing concentrations of alcohol, and then gradual replacement of alcohol by a paraffin solvent. Examples of a paraffin solvent include xylene. After pre-embedding, the sample is then embedded with melted paraffin using a mold, and hardened. Alternatives to paraffin wax include, but are not limited to epoxy, acrylic, agar, gelatin, and celloidin. One of ordinary skill in the art will be able to determine applicable embedding parameters.

[0126] After hardening, a paraffin-embedded tissue sample undergoes sectioning, wherein the sample is cut into thin slices, or sections, to be placed on a slide. These sections can generally be around 5pm thick, though they can be thinner or thicker depending upon tissue type, target molecule, and label / label detection used, among other parameters. One of ordinary skill in the art will be able to determine how thick of a section is needed. Once the sections are cut, they are transferred to a warm water bath and placed on a charged slide. Slides are dried, allowing for the removal of excess wax.

[0127] In some embodiments, after the samples are placed onto a slide, the sample can undergo staining. Stains provide contrast to sections of sample, making viewing structures of the sample easier. Exemplary stains include, but are not limited to alcian blue, aldehyde fuchsin, alkaline phosphatase, Bielshowsky stain, Congo red, crystal violet, eosin, Fontana- Masson, Giesmsa, Haematoxylin, Luna stain, Nissl, Period Acid Schiff (PAS), Red Oil 3, Reticulin stain, Sudan black, toluidine blue, and van Gieson. One of ordinary skill in the art will be able to determine what stain works best for the sample and technique to be performed. The term “staining” is also used in reference to the detection of particular target molecules, e.g., using target-binding ligands or molecules as described herein applied to cell or tissue samples. Additional details on histology can be found, for example, in Ross, M. H. et al. Histology: a text and atlas with correlated cell and molecular biology (7th ed.) Wolters Kluwer. (ISBN: 978-1451187427).Nuclease

[0128] Embodiments of the various aspects described herein include digestion or cleavage with a nuclease. The nuclease can be an endonuclease or exonuclease. The nuclease can be an RNA specific or DNA specific nuclease. The nucleases used can include naturally occurring nucleases as well variants thereof and modified nucleases.

[0129] In some embodiments of any one of the aspects described herein, the nuclease is an RNase. An RNase can be selected from eukaryotic RNases (e.g., mammalian RNases,bacterial RNases, or fungal RNases) or prokaryotic RNases. Exemplary RNases include, but are not limited to, RNase H, RNase I, RNase A, RNase B, RNase C, RNase HI, RNAse HII, RNase If, RNase Tl, RNase T2, RNase III, RNase L, RNase P, RNase PhyM, RNase U2, and RNase V. In some embodiments, RNase is RNase H or RNase I.

[0130] It is noted that a mixture of RNases can be used, or different types of RNases can be used in different steps of a method described herein. In some embodiments, a mix of RNase H and RNase I is used.

[0131] In some embodiments of any one of the aspects described herein, the nuclease is an DNase. The DNase can be selected from eukaryotic DNases (e.g., mammalian DNases, bacterial DNases, or fungal DNases) or prokaryotic DNases. Exemplary DNases include, but are not limited to DNase I, DNase II, DNase III, DNase IV, DNase V, DNase VI, and DNase VII. In some embodiments, the DNase is DNase I.

[0132] It is noted that kinetics of digestion or cleavage by a nuclease can be controlled by varying temperature, time, and buffer / salt conditions, for example. Nuclease, like most enzymes, are sensitive to many buffer conditions, including ionic strength, pH and types of metal ions present (e.g., sodium ions vs. magnesium ions). Thus, the temperature at which digestion or cleavage by the nuclease is performed can vary from, for example, 4°C to 65°C (e.g., 4°C, 25°C, 37°C, 42°C or 65°C). In some embodiments of the various aspects, the temperature at which the step of digestion or cleavage by the nuclease is performed is at about 4-25°C, 4-30°C, 4-35°C, 4-40°C, 4-45°C, 4-50°C, 4-55°C, 4-60°C, 10-25°C, 10-30°C, 10- 35°C, 10-40°C, 10-45°C, 10-50°C, 10-55°C, 10-60°C, 25-30°C, 25-35°C, 25-40°C, 25-45°C, 25-50°C, 25-55°C, 25-60°C, 25-65°C, 35-40°C, 35-45°C, 35-50°C, 35-55°C, 35-60°C, or 35- 65°C. In some embodiments of the various aspects, the step of digestion or cleavage by the nuclease is performed at about room temperature. In some other embodiments, the step of digestion or cleavage by the nuclease is performed at about 37°C.

[0003] The step of digestion or cleavage by the nuclease can be performed for about 30 minutes to about 24 hours. In some embodiments of any of the aspects, the step of digestion or cleavage by the nuclease step can be carried out for about 10 min, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 min, about 40 min, about 45 min, about 50 min, about 55 min, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 18 hours or about 24 hours.

[0004] Exemplary buffer formulations for digestion or cleavage by a nuclease include but are not limited to: Thermopol, NEB buffers 1,2, 3, 4, CutSmart, or customs buffers made with0.5 to 2X PBS or 5 to 200 mM Tris-HCl or 5-200 mM Potassium Acetate or 5-200 mM Magnesium Acetate, 5-200 mM Tris-Acetate or 5-200mM bis-tris-propance HCL. The buffers can be used with one or more of the following additives to modulate enzyme activity: 1-50 mM KC1, 1-20 mM MgSO4, 1-20 mM MgC12, 1-5 mM DTT, 0-500 pg / ml BSA, 1-500 NaCl, 0.01% to 0.5% Triton X-100 at pH values of 6-9.5.Extension of primer

[0133] In some embodiments of the various aspects described herein, the method comprises a step of extending the oligonucleotide primer, e.g., using a single-stranded extender template with a polymerase. Method of synthesizing nucleic acid strand using polymerases are well known in the art and available to one of ordinary skill in the art. It is noted that kinetics of extension with a polymerase (i.e., polymerization) can be controlled by varying temperature, time, buffer / salt conditions, and deoxyribonucleotide triphosphate (dNTP) concentrations, for example. Polymerases, like most enzymes, are sensitive to many buffer conditions, including ionic strength, pH and types of metal ions present (e.g., sodium ions vs. magnesium ions). Thus, the temperature at which polymerization step is performed can vary from, for example, 4°C to 65°C (e.g., 4°C, 25°C, 37°C, 42°C or 65°C). In some embodiments of the various aspects, the temperature at which the polymerization step is performed is about 4-25°C, 4- 30°C, 4-35°C, 4-40°C, 4-45°C, 4-50°C, 4-55°C, 4-60°C, 10-25C, 10-30°C, 10-35°C, 10-40°C, 10-45°C, 10-50°C, 10-55°C, 10-60°C, 25-30°C, 25-35°C, 25-40°C, 25-45°C, 25-50°C, 25- 55°C, 25-60°C, 25-65°C, 35-40°C, 35-45°C, 35-50°C, 35-55°C, 35-60°C, or 35-65°C. In some embodiments of the various aspects, the polymerization step is performed at about room temperature. In some other embodiments, the polymerization step is performed at about 37 °C.

[0134] An extension or polymerization step can be performed (incubated) for about 30 minutes to about 24 hours. In some embodiments of any of the aspects, the extension or polymerization step can be carried out for about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 18 hours or about 24 hours.

[0135] In some embodiments of the various aspects described herein, the extension or polymerization step comprises incubating under conditions that result in nucleic acidpolymerization, strand displacement and annealing, for a time sufficient to produce a synthesized strand, e.g., a concatemer.

[0136] The polymerase used to extend oligonucleotide primers and generate concatemers can be any of a number of template-dependent nucleic acid polymerases. In one embodiment, the polymerase is thermostable, such that it can withstand heating to a temperature and for a time sufficient to denature or dissociate double-stranded nucleic acids, retaining template-dependent polymerization activity when the reaction mixture is cooled to a temperature permitting annealing of extender template(s) to oligonucleotide primer(s) and primer extension. Different thermostable polymerases have different reaction buffer and extension temperature optima; these parameters are known to those of ordinary skill in the art and / or described in product literature for given polymerases. Non-limiting examples of thermostable polymerases useful 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), USBTM CycleSeqTM 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); 1-5 Hi-Fi DNA Polymerase (PDP-100, McLab, San Francisco, CA); 1-5 Hotstart DNA Polymerase (I5HD-100, McLab, San Francisco, CA); DNA polymerase, thermotoga neapolitana (DPTN-100, McLab, San Francisco, CA); Pfu DNA Polymerase (AD-200, McLab, San Franscisco, CA); Pfu DNA Polymerase (Cat. No. 600135, Agilent Technologies, Wood Dale, IL); PfuTurbo DNA Polymerase (Cat. No. 600252, Agilent Technologies, Wood Dale, IL) and the like. One of ordinary skill in the art can identify additional polymerases that would function in the methods, compositions and kits described herein and can adjust reaction conditions as may be needed for any given polymerase.

[0137] In some embodiments of any of the aspects, the polymerase is a DNA polymerase such as a DNA polymerase having DNA strand displacement activity (a strand displacing polymerase). “Strand displacement” describes the ability to displace downstream DNA encountered during synthesis. Examples of polymerases having DNA strand displacement activity that can be used as provided herein include, without limitation, phi29 DNA polymerase (e.g., NEB #M0269), Bst DNA polymerase, large fragment (e.g., NEB #M0275), or Bsu DNApolymerase, large fragment (e.g., NEB #M0330). Other polymerases having strand displacement activity can be used.

[0138] Exemplary buffers that can be used for the extension step include but are not limited to: Thermopol® buffer; NEB® buffers 1,2, 3, 4; CutSmart® buffer; Isothermal Amplification Buffer; and the like. Custom buffers can be made with 0.5 to 2X PBS; 5 to 200 mM Tris-HCl; 5-200 mM Potassium Acetate; 5-200 mM Magnesium Acetate; 5-200 mM Tris- Acetate; or 5- 200 mM Bis-Tris-Propane-HCl can be used with the addition one or all of these additives to modulate the enzyme activity (e.g., 1-50 mM KC1, 1-20 mM MgSO4, 1-20 mM MgC12, 1-5 mM DTT, 0-500 ug / ml BSA, 1-500 NaCl, 0.01% to 0.5% Triton X-100 at pH values of 6-9.5). The buffer can also include dNTPs (e.g., dATP, dCTP, dGTP and dTTP). When only 2-3 types of dNTPs are used, the omitted nucleotides serve as stoppers for the polymerase action, optionally with functional modifications.Kits

[0139] In another aspect, provided herein is a kit for detecting, quantifying, and / or imaging target molecules. Generally, the kit comprises at least one, e.g., 1, 2, 3, 4, 5 or more of the components, e.g., target-binding ligand conjugated to a nucleic acid tag, target-binding ligand conjugated to an oligonucleotide primer, labeled-nucleic acid probe, nuclease, single-stranded extender template, and polymerase, described herein.

[0140] In some embodiments, the kit comprises: (i) a target-binding ligand conjugated with a nucleic acid tag; (ii) a labeled-nucleic acid probe, wherein the labeled-nucleic acid probe comprises a detectable label and has a nucleotide sequence complementary to at least a portion of the nucleic acid tag; and (iii) a nuclease.

[0141] In some embodiments, the kit comprises: (i) a target-binding ligand conjugated with a nucleic acid tag; (ii) a labeled-nucleic acid probe, wherein the labeled-nucleic acid probe comprises a detectable label and has a nucleotide sequence complementary to at least a portion of the nucleic acid tag; and (iii) a set of blocking probes, optionally, the set of blocking probes does not comprise a blocking probe having a sequence complementary to the nucleic acid tag conjugated to the target-binding ligand.

[0142] In some embodiments, the kit comprises: (i) a target-binding ligand conjugated with a nucleic acid tag; (ii) a labeled-nucleic acid probe, wherein the labeled-nucleic acid probe comprises a detectable label and has a nucleotide sequence complementary to at least a portion of the nucleic acid tag; (iii) a nuclease; and (iv) a set of blocking probes, optionally, the set ofblocking probes does not comprise a blocking probe having a sequence complementary to the nucleic acid tag conjugated to the target-binding ligand.

[0143] In some embodiments, the kit comprises: (i) a target-binding ligand conjugated with an oligonucleotide primer; (ii) a single-stranded extender template; (iii) a polymerase; (iv) a labeled-nucleic acid probe, wherein the labeled-nucleic acid probe comprises a detectable label and has a nucleotide sequence complementary to at least a portion of the oligonucleotide primer or the extender template; and (v) a nuclease, optionally, the kit further comprises one or more reagents for nucleic acid polymerization by a polymerase, such as deoxyribonucleotide triphosphates (dNTPs), salt and / or buffers.

[0144] In some embodiments, the kit comprises: (i) a target-binding ligand conjugated with an oligonucleotide primer; (ii) a single-stranded extender template; (iii) a polymerase; (iv) a labeled-nucleic acid probe, wherein the labeled-nucleic acid probe comprises a detectable label and has a nucleotide sequence complementary to at least a portion of the oligonucleotide primer or the extender template; and (v) a set of blocking probes, optionally, the set of blocking probes does not comprise a blocking probe having a sequence complementary to the nucleic acid tag conjugated to the target-binding ligand, optionally, the kit further comprises one or more reagents for nucleic acid polymerization by a polymerase, such as deoxyribonucleotide triphosphates (dNTPs), salt and / or buffers.

[0145] The kit described herein lend itself well to multiplex labeling, detection and / or imaging. Accordingly, in some embodiments, the kit comprises: (i) a first target-binding ligand conjugated with a first nucleic acid tag; (ii) a first labeled-nucleic acid probe, wherein the first labeled-nucleic acid probe comprises a detectable label and has a nucleotide sequence complementary to at least a portion of the first nucleic acid tag; (iii) a second target-binding ligand conjugated with a second nucleic acid tag, wherein the second target-binding ligand binds a target different from the first target-binding ligand; (iv) a second labeled-nucleic acid probe, wherein the second labeled-nucleic acid probe comprises a detectable label and has a nucleotide sequence complementary to at least a portion of the second nucleic acid tag; and (v) a nuclease. The first detectable label and the second detectable label can be distinguishable from each other. In some cases, the first detectable label and the second detectable label are same, i.e., not distinguishable from each other.

[0146] In some embodiments, the kit comprises: (i) a first target-binding ligand conjugated with a first nucleic acid tag; (ii) a first labeled-nucleic acid probe, wherein the first labeled- nucleic acid probe comprises a detectable label and has a nucleotide sequence complementary to at least a portion of the first nucleic acid tag; (iii) a second target-binding ligand conjugatedwith a second nucleic acid tag, wherein the second target-binding ligand binds a target different from the first target-binding ligand; (iv) a second labeled-nucleic acid probe, wherein the second labeled-nucleic acid probe comprises a detectable label and has a nucleotide sequence complementary to at least a portion of the second nucleic acid tag; and (v) a set of blocking probes, optionally, the set of blocking probes does not comprise a blocking probe having a sequence complementary to the first nucleic acid tag and / or the second nucleic acid tag, and optionally, the kit further comprises a nuclease. The first detectable label and the second detectable label can be distinguishable from each other. In some cases, the first detectable label and the second detectable label are same, i.e., not distinguishable from each other.

[0147] In some embodiments, the kit comprises: (i) a first target-binding ligand conjugated with a first oligonucleotide primer; (ii) a first single-stranded extender template for template directed extension of the first oligonucleotide primer; (iii) a second target-binding ligand conjugated with a second oligonucleotide primer, wherein the second target-binding ligand binds a target different from the first target-binding ligand; (iv) a second single-stranded extender template for template directed extension of the second oligonucleotide primer; (v) a polymerase; (vi) a first labeled-nucleic acid probe comprising a first detectable label and having a nucleotide sequence complementary to at least a portion of the first oligonucleotide primer or the first extender template; (vii) a second labeled-nucleic acid probe comprising a second detectable label and having a nucleotide sequence complementary to at least a portion of the second oligonucleotide primer or the second extender template; and (viii) a nuclease. The first detectable label and the second detectable label can be distinguishable from each other. In some cases, the first detectable label and the second detectable label are same, i.e., not distinguishable from each other.

[0148] In some embodiments, the kit comprises: (i) a target-binding ligand conjugated with an oligonucleotide primer; ii) a first single-stranded extender template for template directed extension of the first oligonucleotide primer; (iii) a second target-binding ligand conjugated with a second oligonucleotide primer, wherein the second target-binding ligand binds a target different from the first target-binding ligand; (iv) a second single-stranded extender template for template directed extension of the second oligonucleotide primer; (v) a polymerase; (vi) a first labeled-nucleic acid probe comprising a first detectable label and having a nucleotide sequence complementary to at least a portion of the first oligonucleotide primer or the first extender template; (vii) a second labeled-nucleic acid probe comprising a second detectable label and having a nucleotide sequence complementary to at least a portion of the second oligonucleotide primer or the second extender template; and (viii) a set of blocking probes,optionally, the set of blocking probes does not comprise a blocking probe having a sequence complementary to the first nucleic acid tag and / or the second nucleic acid tag. The first detectable label and the second detectable label can be distinguishable from each other. In some cases, the first detectable label and the second detectable label are same, i.e., not distinguishable from each other.

[0149] In some embodiments, the kit further comprises one or more reagents for digestion or cleavage by a nuclease such salts and / or buffers.

[0150] In some embodiments, the kit further comprises means for detecting the detectable label, e.g., a signal produced by the detectable label.

[0151] In some embodiments, the kit further comprises instructions for use.System

[0152] In another aspect, provided herein is a system for detecting, quantifying, and / or imaging target molecules. Generally, the system comprises at least one, e.g., 1, 2, 3, 4, 5 or more of the components, e.g., target-binding ligand conjugated to a nucleic acid tag, target-binding ligand conjugated to an oligonucleotide primer, labeled-nucleic acid probe, nuclease, single-stranded extender template, and polymerase, described herein.

[0153] In some embodiments, the system comprises: (i) a target-binding ligand conjugated with a nucleic acid tag; (ii) a labeled-nucleic acid probe, wherein the labeled-nucleic acid probe comprises a detectable label and has a nucleotide sequence complementary to at least a portion of the nucleic acid tag; and (iii) a nuclease.

[0154] In some embodiments, the system comprises: (i) a target-binding ligand conjugated with a nucleic acid tag; (ii) a labeled-nucleic acid probe, wherein the labeled-nucleic acid probe comprises a detectable label and has a nucleotide sequence complementary to at least a portion of the nucleic acid tag; and (iii) a set of blocking probes, optionally, the set of blocking probes does not comprise a blocking probe having a sequence complementary to the nucleic acid tag conjugated to the target-binding ligand.

[0155] In some embodiments, the system comprises: (i) a target-binding ligand conjugated with a nucleic acid tag; (ii) a labeled-nucleic acid probe, wherein the labeled-nucleic acid probe comprises a detectable label and has a nucleotide sequence complementary to at least a portion of the nucleic acid tag; (iii) a nuclease; and (iv) a set of blocking probes, optionally, the set of blocking probes does not comprise a blocking probe having a sequence complementary to the nucleic acid tag conjugated to the target-binding ligand.

[0156] In some embodiments, the system comprises: (i) a target-binding ligand conjugated with an oligonucleotide primer; (ii) a single-stranded extender template; (iii) a polymerase; (iv) a labeled-nucleic acid probe, wherein the labeled-nucleic acid probe comprises a detectable label and has a nucleotide sequence complementary to at least a portion of the oligonucleotide primer or the extender template; and (v) a nuclease, optionally, the system further comprises one or more reagents for nucleic acid polymerization by a polymerase, such as deoxyribonucleotide triphosphates (dNTPs), salt and / or buffers.

[0157] In some embodiments, the system comprises: (i) a target-binding ligand conjugated with an oligonucleotide primer; (ii) a single-stranded extender template; (iii) a polymerase; (iv) a labeled-nucleic acid probe, wherein the labeled-nucleic acid probe comprises a detectable label and has a nucleotide sequence complementary to at least a portion of the oligonucleotide primer or the extender template; and (v) a set of blocking probes, optionally, the set of blocking probes does not comprise a blocking probe having a sequence complementary to the nucleic acid tag conjugated to the target-binding ligand, optionally, the system further comprises one or more reagents for nucleic acid polymerization by a polymerase, such as deoxyribonucleotide triphosphates (dNTPs), salt and / or buffers.

[0158] The system described herein lend itself well to multiplex labeling, detection and / or imaging. Accordingly, in some embodiments, the system comprises: (i) a first target-binding ligand conjugated with a first nucleic acid tag; (ii) a first labeled-nucleic acid probe, wherein the first labeled-nucleic acid probe comprises a detectable label and has a nucleotide sequence complementary to at least a portion of the first nucleic acid tag; (iii) a second target-binding ligand conjugated with a second nucleic acid tag, wherein the second target-binding ligand binds a target different from the first target-binding ligand; (iv) a second labeled-nucleic acid probe, wherein the second labeled-nucleic acid probe comprises a detectable label and has a nucleotide sequence complementary to at least a portion of the second nucleic acid tag; and (v) a nuclease. The first detectable label and the second detectable label can be distinguishable from each other. In some cases, the first detectable label and the second detectable label are same, i.e., not distinguishable from each other.

[0159] In some embodiments, the system comprises: (i) a first target-binding ligand conjugated with a first nucleic acid tag; (ii) a first labeled-nucleic acid probe, wherein the first labeled- nucleic acid probe comprises a detectable label and has a nucleotide sequence complementary to at least a portion of the first nucleic acid tag; (iii) a second target-binding ligand conjugated with a second nucleic acid tag, wherein the second target-binding ligand binds a target different from the first target-binding ligand; (iv) a second labeled-nucleic acid probe, wherein thesecond labeled-nucleic acid probe comprises a detectable label and has a nucleotide sequence complementary to at least a portion of the second nucleic acid tag; and (v) a set of blocking probes, optionally, the set of blocking probes does not comprise a blocking probe having a sequence complementary to the first nucleic acid tag and / or the second nucleic acid tag, and optionally, the system further comprises a nuclease. The first detectable label and the second detectable label can be distinguishable from each other. In some cases, the first detectable label and the second detectable label are same, i.e., not distinguishable from each other.

[0160] In some embodiments, the system comprises: (i) a first target-binding ligand conjugated with a first oligonucleotide primer; (ii) a first single-stranded extender template for template directed extension of the first oligonucleotide primer; (iii) a second target-binding ligand conjugated with a second oligonucleotide primer, wherein the second target-binding ligand binds a target different from the first target-binding ligand; (iv) a second single-stranded extender template for template directed extension of the second oligonucleotide primer; (v) a polymerase; (vi) a first labeled-nucleic acid probe comprising a first detectable label and having a nucleotide sequence complementary to at least a portion of the first oligonucleotide primer or the first extender template; (vii) a second labeled-nucleic acid probe comprising a second detectable label and having a nucleotide sequence complementary to at least a portion of the second oligonucleotide primer or the second extender template; and (viii) a nuclease. The first detectable label and the second detectable label can be distinguishable from each other. In some cases, the first detectable label and the second detectable label are same, i.e., not distinguishable from each other.

[0161] In some embodiments, the system comprises: (i) a target-binding ligand conjugated with an oligonucleotide primer; ii) a first single-stranded extender template for template directed extension of the first oligonucleotide primer; (iii) a second target-binding ligand conjugated with a second oligonucleotide primer, wherein the second target-binding ligand binds a target different from the first target-binding ligand; (iv) a second single-stranded extender template for template directed extension of the second oligonucleotide primer; (v) a polymerase; (vi) a first labeled-nucleic acid probe comprising a first detectable label and having a nucleotide sequence complementary to at least a portion of the first oligonucleotide primer or the first extender template; (vii) a second labeled-nucleic acid probe comprising a second detectable label and having a nucleotide sequence complementary to at least a portion of the second oligonucleotide primer or the second extender template; and (viii) a set of blocking probes, optionally, the set of blocking probes does not comprise a blocking probe having a sequence complementary to the first nucleic acid tag and / or the second nucleic acid tag. Thefirst detectable label and the second detectable label can be distinguishable from each other. In some cases, the first detectable label and the second detectable label are same, i.e., not distinguishable from each other.

[0162] In some embodiments, the system further comprises one or more reagents for digestion or cleavage by a nuclease such salts and / or buffers.

[0163] In some embodiments, the system further comprises means for detecting the detectable label, e.g., a signal produced by the detectable label.

[0164] In some embodiments, the system further comprises means for detecting the detectable label.Reaction mixture

[0165] In another aspect, provided herein is a rection mixture. Generally, the reaction mixture comprises at least one, e.g., 1, 2, 3, 4, 5 or more of the components, e.g., target-binding ligand conjugated to a nucleic acid tag, target-binding ligand conjugated to an oligonucleotide primer, labeled-nucleic acid probe, nuclease, single-stranded extender template, and polymerase, described herein.

[0166] In some embodiments, the reaction mixture comprises: (i) a target-binding ligand conjugated with a nucleic acid tag; (ii) a labeled-nucleic acid probe, wherein the labeled- nucleic acid probe comprises a detectable label and has a nucleotide sequence complementary to at least a portion of the nucleic acid tag; and (iii) a nuclease.

[0167] In some embodiments, the reaction mixture comprises: (i) a target-binding ligand conjugated with a nucleic acid tag; (ii) a labeled-nucleic acid probe, wherein the labeled- nucleic acid probe comprises a detectable label and has a nucleotide sequence complementary to at least a portion of the nucleic acid tag; and (iii) a set of blocking probes, optionally, the set of blocking probes does not comprise a blocking probe having a sequence complementary to the nucleic acid tag conjugated to the target-binding ligand.

[0168] In some embodiments, the reaction mixture comprises: (i) a target-binding ligand conjugated with a nucleic acid tag; (ii) a labeled-nucleic acid probe, wherein the labeled- nucleic acid probe comprises a detectable label and has a nucleotide sequence complementary to at least a portion of the nucleic acid tag; (iii) a nuclease; and (iv) a set of blocking probes, optionally, the set of blocking probes does not comprise a blocking probe having a sequence complementary to the nucleic acid tag conjugated to the target-binding ligand.

[0169] In some embodiments, the reaction mixture comprises: (i) a target-binding ligand conjugated with an oligonucleotide primer; (ii) a single-stranded extender template; (iii) apolymerase; (iv) a labeled-nucleic acid probe, wherein the labeled-nucleic acid probe comprises a detectable label and has a nucleotide sequence complementary to at least a portion of the oligonucleotide primer or the extender template; and (v) a nuclease, optionally, the reaction mixture further comprises one or more reagents for nucleic acid polymerization by a polymerase, such as deoxyribonucleotide triphosphates (dNTPs), salt and / or buffers.

[0170] In some embodiments, the reaction mixture comprises: (i) a target-binding ligand conjugated with an oligonucleotide primer; (ii) a single-stranded extender template; (iii) a polymerase; (iv) a labeled-nucleic acid probe, wherein the labeled-nucleic acid probe comprises a detectable label and has a nucleotide sequence complementary to at least a portion of the oligonucleotide primer or the extender template; and (v) a set of blocking probes, optionally, the set of blocking probes does not comprise a blocking probe having a sequence complementary to the nucleic acid tag conjugated to the target-binding ligand, optionally, the reaction mixture further comprises one or more reagents for nucleic acid polymerization by a polymerase, such as deoxyribonucleotide triphosphates (dNTPs), salt and / or buffers.

[0171] In some embodiments, the reaction mixture comprises: (i) a first target-binding ligand conjugated with a first nucleic acid tag; (ii) a first labeled-nucleic acid probe, wherein the first labeled-nucleic acid probe comprises a detectable label and has a nucleotide sequence complementary to at least a portion of the first nucleic acid tag; (iii) a second target-binding ligand conjugated with a second nucleic acid tag, wherein the second target-binding ligand binds a target different from the first target-binding ligand; (iv) a second labeled-nucleic acid probe, wherein the second labeled-nucleic acid probe comprises a detectable label and has a nucleotide sequence complementary to at least a portion of the second nucleic acid tag; and (v) a nuclease. The first detectable label and the second detectable label can be distinguishable from each other. In some cases, the first detectable label and the second detectable label are same, i.e., not distinguishable from each other.

[0172] In some embodiments, the reaction mixture comprises: (i) a first target-binding ligand conjugated with a first nucleic acid tag; (ii) a first labeled-nucleic acid probe, wherein the first labeled-nucleic acid probe comprises a detectable label and has a nucleotide sequence complementary to at least a portion of the first nucleic acid tag; (iii) a second target-binding ligand conjugated with a second nucleic acid tag, wherein the second target-binding ligand binds a target different from the first target-binding ligand; (iv) a second labeled-nucleic acid probe, wherein the second labeled-nucleic acid probe comprises a detectable label and has a nucleotide sequence complementary to at least a portion of the second nucleic acid tag; and (v) a set of blocking probes, optionally, the set of blocking probes does not comprise a blockingprobe having a sequence complementary to the first nucleic acid tag and / or the second nucleic acid tag, and optionally, the reaction mixture further comprises a nuclease. The first detectable label and the second detectable label can be distinguishable from each other. In some cases, the first detectable label and the second detectable label are same, i.e., not distinguishable from each other.

[0173] In some embodiments, the reaction mixture comprises: (i) a first target-binding ligand conjugated with a first oligonucleotide primer; (ii) a first single-stranded extender template for template directed extension of the first oligonucleotide primer; (iii) a second target-binding ligand conjugated with a second oligonucleotide primer, wherein the second target-binding ligand binds a target different from the first target-binding ligand; (iv) a second single-stranded extender template for template directed extension of the second oligonucleotide primer; (v) a polymerase; (vi) a first labeled-nucleic acid probe comprising a first detectable label and having a nucleotide sequence complementary to at least a portion of the first oligonucleotide primer or the first extender template; (vii) a second labeled-nucleic acid probe comprising a second detectable label and having a nucleotide sequence complementary to at least a portion of the second oligonucleotide primer or the second extender template; and (viii) a nuclease. The first detectable label and the second detectable label can be distinguishable from each other. In some cases, the first detectable label and the second detectable label are same, i.e., not distinguishable from each other.

[0174] In some embodiments, the reaction mixture comprises: (i) a target-binding ligand conjugated with an oligonucleotide primer; ii) a first single-stranded extender template for template directed extension of the first oligonucleotide primer; (iii) a second target-binding ligand conjugated with a second oligonucleotide primer, wherein the second target-binding ligand binds a target different from the first target-binding ligand; (iv) a second single-stranded extender template for template directed extension of the second oligonucleotide primer; (v) a polymerase; (vi) a first labeled-nucleic acid probe comprising a first detectable label and having a nucleotide sequence complementary to at least a portion of the first oligonucleotide primer or the first extender template; (vii) a second labeled-nucleic acid probe comprising a second detectable label and having a nucleotide sequence complementary to at least a portion of the second oligonucleotide primer or the second extender template; and (viii) a set of blocking probes, optionally, the set of blocking probes does not comprise a blocking probe having a sequence complementary to the first nucleic acid tag and / or the second nucleic acid tag. The first detectable label and the second detectable label can be distinguishable from each other. Insome cases, the first detectable label and the second detectable label are same, i.e., not distinguishable from each other.

[0175] In some embodiments, the reaction mixture further comprises one or more reagents for digestion or cleavage by a nuclease such salts and / or buffers.Composition

[0176] In another aspect, provided herein is a rection mixture. Generally, the composition comprises at least one, e.g., 1, 2, 3, 4, 5 or more of the components, e.g., target-binding ligand conjugated to a nucleic acid tag, target-binding ligand conjugated to an oligonucleotide primer, labeled-nucleic acid probe, nuclease, single-stranded extender template, and polymerase, described herein.

[0177] In some embodiments, the composition comprises: (i) a target-binding ligand conjugated with a nucleic acid tag; (ii) a labeled-nucleic acid probe, wherein the labeled- nucleic acid probe comprises a detectable label and has a nucleotide sequence complementary to at least a portion of the nucleic acid tag; and (iii) a nuclease.

[0178] In some embodiments, the composition comprises: (i) a target-binding ligand conjugated with a nucleic acid tag; (ii) a labeled-nucleic acid probe, wherein the labeled- nucleic acid probe comprises a detectable label and has a nucleotide sequence complementary to at least a portion of the nucleic acid tag; and (iii) a set of blocking probes, optionally, the set of blocking probes does not comprise a blocking probe having a sequence complementary to the nucleic acid tag conjugated to the target-binding ligand.

[0179] In some embodiments, the composition comprises: (i) a target-binding ligand conjugated with a nucleic acid tag; (ii) a labeled-nucleic acid probe, wherein the labeled- nucleic acid probe comprises a detectable label and has a nucleotide sequence complementary to at least a portion of the nucleic acid tag; (iii) a nuclease; and (iv) a set of blocking probes, optionally, the set of blocking probes does not comprise a blocking probe having a sequence complementary to the nucleic acid tag conjugated to the target-binding ligand.

[0180] In some embodiments, the composition comprises: (i) a target-binding ligand conjugated with an oligonucleotide primer; (ii) a single-stranded extender template; (iii) a polymerase; (iv) a labeled-nucleic acid probe, wherein the labeled-nucleic acid probe comprises a detectable label and has a nucleotide sequence complementary to at least a portion of the oligonucleotide primer or the extender template; and (v) a nuclease, optionally, the composition further comprises one or more reagents for nucleic acid polymerization by a polymerase, such as deoxyribonucleotide triphosphates (dNTPs), salt and / or buffers.

[0181] In some embodiments, the composition comprises: (i) a target-binding ligand conjugated with an oligonucleotide primer; (ii) a single-stranded extender template; (iii) a polymerase; (iv) a labeled-nucleic acid probe, wherein the labeled-nucleic acid probe comprises a detectable label and has a nucleotide sequence complementary to at least a portion of the oligonucleotide primer or the extender template; and (v) a set of blocking probes, optionally, the set of blocking probes does not comprise a blocking probe having a sequence complementary to the nucleic acid tag conjugated to the target-binding ligand, optionally, the composition further comprises one or more reagents for nucleic acid polymerization by a polymerase, such as deoxyribonucleotide triphosphates (dNTPs), salt and / or buffers.

[0182] In some embodiments, the composition comprises: (i) a first target-binding ligand conjugated with a first nucleic acid tag; (ii) a first labeled-nucleic acid probe, wherein the first labeled-nucleic acid probe comprises a detectable label and has a nucleotide sequence complementary to at least a portion of the first nucleic acid tag; (iii) a second target-binding ligand conjugated with a second nucleic acid tag, wherein the second target-binding ligand binds a target different from the first target-binding ligand; (iv) a second labeled-nucleic acid probe, wherein the second labeled-nucleic acid probe comprises a detectable label and has a nucleotide sequence complementary to at least a portion of the second nucleic acid tag; and (v) a nuclease. The first detectable label and the second detectable label can be distinguishable from each other. In some cases, the first detectable label and the second detectable label are same, i.e., not distinguishable from each other.

[0183] In some embodiments, the composition comprises: (i) a first target-binding ligand conjugated with a first nucleic acid tag; (ii) a first labeled-nucleic acid probe, wherein the first labeled-nucleic acid probe comprises a detectable label and has a nucleotide sequence complementary to at least a portion of the first nucleic acid tag; (iii) a second target-binding ligand conjugated with a second nucleic acid tag, wherein the second target-binding ligand binds a target different from the first target-binding ligand; (iv) a second labeled-nucleic acid probe, wherein the second labeled-nucleic acid probe comprises a detectable label and has a nucleotide sequence complementary to at least a portion of the second nucleic acid tag; and (v) a set of blocking probes, optionally, the set of blocking probes does not comprise a blocking probe having a sequence complementary to the first nucleic acid tag and / or the second nucleic acid tag, and optionally, the composition further comprises a nuclease. The first detectable label and the second detectable label can be distinguishable from each other. In some cases, the first detectable label and the second detectable label are same, i.e., not distinguishable from each other.

[0184] In some embodiments, the composition comprises: (i) a first target-binding ligand conjugated with a first oligonucleotide primer; (ii) a first single-stranded extender template for template directed extension of the first oligonucleotide primer; (iii) a second target-binding ligand conjugated with a second oligonucleotide primer, wherein the second target-binding ligand binds a target different from the first target-binding ligand; (iv) a second single-stranded extender template for template directed extension of the second oligonucleotide primer; (v) a polymerase; (vi) a first labeled-nucleic acid probe comprising a first detectable label and having a nucleotide sequence complementary to at least a portion of the first oligonucleotide primer or the first extender template; (vii) a second labeled-nucleic acid probe comprising a second detectable label and having a nucleotide sequence complementary to at least a portion of the second oligonucleotide primer or the second extender template; and (viii) a nuclease. The first detectable label and the second detectable label can be distinguishable from each other. In some cases, the first detectable label and the second detectable label are same, i.e., not distinguishable from each other.

[0185] In some embodiments, the composition comprises: (i) a target-binding ligand conjugated with an oligonucleotide primer; ii) a first single-stranded extender template for template directed extension of the first oligonucleotide primer; (iii) a second target-binding ligand conjugated with a second oligonucleotide primer, wherein the second target-binding ligand binds a target different from the first target-binding ligand; (iv) a second single-stranded extender template for template directed extension of the second oligonucleotide primer; (v) a polymerase; (vi) a first labeled-nucleic acid probe comprising a first detectable label and having a nucleotide sequence complementary to at least a portion of the first oligonucleotide primer or the first extender template; (vii) a second labeled-nucleic acid probe comprising a second detectable label and having a nucleotide sequence complementary to at least a portion of the second oligonucleotide primer or the second extender template; and (viii) a set of blocking probes, optionally, the set of blocking probes does not comprise a blocking probe having a sequence complementary to the first nucleic acid tag and / or the second nucleic acid tag. The first detectable label and the second detectable label can be distinguishable from each other. In some cases, the first detectable label and the second detectable label are same, i.e., not distinguishable from each other.

[0186] In some embodiments, the composition further comprises one or more reagents for digestion or cleavage by a nuclease such salts and / or buffers.Nucleic acid modifications

[0187] Nucleic acid molecules, e.g., nucleic acid tags, labeled-nucleic acid probes, blocking probes, oligonucleotide primers, and single-stranded extender templates, described herein can comprise one or more nucleic acid modifications. Exemplary nucleic acid modifications include, but are not limited to, nucleobase modifications, sugar modifications, inter-sugar linkage modifications, conjugates e.g., ligands), and any combinations thereof.

[0188] Exemplary modified nucleobases include, but are not limited to, inosine, xanthine, hypoxanthine, nubularine, isoguanisine, tubercidine, and substituted or modified analogs of adenine, guanine, cytosine and uracil, such as 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 5-halouracil, 5-(2-aminopropyl)uracil, 5-amino allyl uracil, 8-halo, amino, thiol, thioalkyl, hydroxyl and other 8-substituted adenines and guanines, 5 -trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine, 5- substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine, dihydrouracil, 3-deaza-5- azacytosine, 2-aminopurine, 5-alkyluracil, 7-alkylguanine, 5-alkyl cytosine, 7-deazaadenine,N6, N6-dimethyladenine, 2,6-diaminopurine, 5-amino-allyl-uracil, N3 -methyluracil, substituted 1,2,4-triazoles, 2-pyridinone, 5-nitroindole, 3 -nitropyrrole, 5-methoxyuracil, uracil-5-oxyacetic acid, 5 -methoxy carbonylmethyluracil, 5-methyl-2-thiouracil, 5- methoxy carbonylmethyl -2-thiouracil, 5-methylaminomethyl-2-thiouracil, 3-(3-amino- 3carboxypropyl)uracil, 3 -methylcytosine, 5-methylcytosine, N4-acetyl cytosine, 2- thiocytosine, N6-methyladenine, N6-isopentyladenine, 2-methylthio-N6-isopentenyladenine, N-methylguanines, or O-alkylated bases. Further purines and pyrimidines include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in the Concise Encyclopedia of PolymerScience and Engineering, pages 858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990, and those disclosed by Englisch et aL, Angewandte Chemie, International Edition, 1991, 30, 613.

[0189] In some embodiments, modified nucleobase can be selected from the group consisting of inosine, xanthine, hypoxanthine, nubularine, isoguanisine, tubercidine, 2-(halo)adenine, 2-(alkyl)adenine, 2-(propyl)adenine, 2-(amino)adenine, 2-(aminoalkyll)adenine,2-(aminopropyl)adenine, 2-(methylthio)-N6-(isopentenyl)adenine, 6-(alkyl)adenine,6-(methyl)adenine, 7-(deaza)adenine, 8-(alkenyl)adenine, 8-(alkyl)adenine.8-(alkynyl)adenine, 8-(amino)adenine, 8-(halo)adenine, 8-(hydroxyl)adenine.8-(thioalkyl)adenine, 8-(thiol)adenine, N6-(isopentyl)adenine, N6-(methyl)adenine.N6, N6-(dimethyl)adenine, 2-(alkyl)guanine,2-(propyl)guanine, 6-(alkyl)guanine,6-(methyl)guanine, 7-(alkyl)guanine, 7-(methyl)guanine, 7-(deaza)guanine, 8-(alkyl)guanine, 8-(alkenyl)guanine, 8-(alkynyl)guanine, 8-(amino)guanine, 8-(halo)guanine, 8- (hydroxyl)guanine, 8-(thioalkyl)guanine, 8-(thiol)guanine, N-(methyl)guanine, 2- (thio)cytosine, 3-(deaza)-5-(aza)cytosine, 3-(alkyl)cytosine, 3-(methyl)cytosine, 5- (alkyl)cytosine, 5-(alkynyl)cytosine, 5-(halo)cytosine, 5-(methyl)cytosine, 5-(propynyl)cytosine, 5-(propynyl)cytosine, 5-(trifluoromethyl)cytosine, 6-(azo)cytosine, N4-(acetyl)cytosine, 3-(3-amino-3-carboxypropyl)uracil, 5-ethynyl-2'-deoxyuridine, 2- (thio)uracil,5-(methyl)-2-(thio)uracil, 5-(methylaminomethyl)-2-(thio)uracil, 4-(thio)uracil,5-(methyl)-4-(thio)uracil, 5-(methylaminomethyl)-4-(thio)uracil,5-(methyl)-2,4-(dithio)uracil, 5-(methylaminomethyl)-2,4-(dithio)uracil, 5-(2- aminopropyl)uracil, 5-(alkyl)uracil, 5-(alkynyl)uracil, 5-(allylamino)uracil, 5-(aminoallyl)uracil, 5-(aminoalkyl)uracil, 5-(guanidiniumalkyl)uracil, 5-(l,3-diazole-l- alkyl)uracil, 5-(cyanoalkyl)uracil, 5-(dialkylaminoalkyl)uracil, 5-(dimethylaminoalkyl)uracil, 5-(halo)uracil, 5-(methoxy)uracil, uracil-5-oxyacetic acid, 5-(methoxycarbonylmethyl)-2- (thio)uracil, 5-(methoxycarbonyl-methyl)uracil, 5-(propynyl)uracil, 5-(propynyl)uracil, 5-(trifluoromethyl)uracil, 6-(azo)uracil, dihydrouracil, N3-(methyl)uracil, 5-uracil (i.e., pseudouracil), 2-(thio)pseudouracil,4-(thio)pseudouracil,2,4-(dithio)psuedouracil,5-(alkyl)pseudouracil, 5-(methyl)pseudouracil, 5-(alkyl)-2-(thio)pseudouracil, 5-(methyl)-2- (thio)pseudouracil, 5-(alkyl)-4-(thio)pseudouracil, 5-(methyl)-4-(thio)pseudouracil, 5-(alkyl)- 2,4-(dithio)pseudouracil, 5-(methyl)-2,4-(dithio)pseudouracil, 1 -substituted pseudouracil, 1 -substituted 2(thio)-pseudouracil, 1 -substituted 4-(thio)pseudouracil, 1 -substituted 2,4- (dithio)pseudouracil, 1 -(aminocarbonylethylenyl)-pseudouracil, 1 -(aminocarbonylethylenyl)- 2(thio)-pseudouracil, l-(aminocarbonylethylenyl)-4-(thio)pseudouracil,1-(aminocarbonylethylenyl)-2,4-(dithio)pseudouracil,1 -(aminoalkylaminocarbonylethylenyl)-pseudouracil, 1 -(aminoalkylamino- carbonylethylenyl)-2(thio)-pseudouracil, l-(aminoalkylaminocarbonylethylenyl)-4-(thio)pseudouracil, l-(aminoalkylaminocarbonylethylenyl)-2,4-(dithio)pseudouracil, 1,3- (diaza)-2-(oxo)-phenoxazin- 1 -yl, 1 -(aza)-2-(thio)-3 -(aza)-phenoxazin- 1 -yl, 1 ,3 -(diaza)-2- (oxo)-phenthiazin-l-yl, l-(aza)-2-(thio)-3-(aza)-phenthiazin-l-yl, 7-substituted l,3-(diaza)-2- (oxo)-phenoxazin-l-yl, 7-substituted l-(aza)-2-(thio)-3-(aza)-phenoxazin-l-yl, 7-substituted 1 ,3-(diaza)-2-(oxo)-phenthiazin-l -yl, 7-substituted 1 -(aza)-2-(thio)-3 -(aza)-phenthiazin- 1 -yl, 7-(aminoalkylhydroxy)-l,3-(diaza)-2-(oxo)-phenoxazin-l-yl, 7-(aminoalkylhydroxy)-l-(aza)-2-(thio)-3 -(aza)-phenoxazin- 1 -yl, 7-(aminoalkylhydroxy)- 1 ,3 -(diaza)-2-(oxo)-phenthiazin- 1 - yl, 7-(aminoalkylhydroxy)-l-(aza)-2-(thio)-3-(aza)-phenthiazin-l-yl, 7-(guanidiniumalkylhydroxy)-l,3-(diaza)-2-(oxo)-phenoxazin-l-yl, 7-(guanidiniumalkylhydroxy)-l-(aza)-2-(thio)-3-(aza)-phenoxazin-l-yl, 7-(guanidiniumalkyl- hydroxy)- 1 ,3 -(diaza)-2-(oxo)-phenthiazin- 1 -yl, 7-(guanidiniumalkylhydroxy)- 1 -(aza)-2-(thio)-3-(aza)-phenthiazin-l-yl, l,3,5-(triaza)-2,6-(dioxa)-naphthalene, inosine, xanthine, hypoxanthine, nubularine, tubercidine, isoguanisine, inosinyl, 2-aza-inosinyl, 7-deaza- inosinyl, nitroimidazolyl, nitropyrazolyl, nitrobenzimidazolyl, nitroindazolyl, aminoindolyl, pyrrolopyrimidinyl, 3-(methyl)isocarbostyrilyl, 5-(methyl)isocarbostyrilyl, 3-(methyl)-7- (propynyl)isocarbostyrilyl, 7-(aza)indolyl, 6-(methyl)-7-(aza)indolyl, imidizopyridinyl, 9- (methyl)-imidizopyridinyl, pyrrolopyrizinyl, isocarbostyrilyl, 7-(propynyl)isocarbostyrilyl, propynyl-7-(aza)indolyl, 2,4,5-(trimethyl)phenyl, 4-(methyl)indolyl, 4,6-(dimethyl)indolyl, phenyl, napthalenyl, anthracenyl, phenanthracenyl, pyrenyl, stilbenyl, tetracenyl, pentacenyl, difluorotolyl, 4-(fluoro)-6-(methyl)benzimidazole, 4-(methyl)benzimidazole, 6-(azo)thymine, 2-pyridinone, 5-nitroindole, 3 -nitropyrrole, 6-(aza)pyrimidine, 2-(amino)purine, 2,6- (diamino)purine, 5-substituted pyrimidines, N2-substituted purines, N6-substituted purines, Cosubstituted purines, substituted 1,2,4-triazoles, and any O-alkylated or N-alkylated derivatives thereof.

[0190] Exemplary sugar modifications include, but are not limited to, 2’ -Fluoro, 3 ’-Fluoro, 2’- OMe, 3’-0Me, 2’ -deoxy modifications, and acyclic nucleotides, e.g., peptide nucleic acids (PNA), unlocked nucleic acids (UNA) or glycol nucleic acid (GNA).

[0191] In some embodiments, a nucleic acid modification can include replacement or modification of an inter-sugar linkage. Exemplary inter-sugar linkage modifications include, but are not limited to, phosphotriesters, methylphosphonates, phosphoramidite, phosphorothioates, methylenemethylimino, thiodiester, thionocarbamate, siloxane, N,N'- dimethylhydrazine ( — CH2-N(CH3)-N(CH3)-), amide-3 (3'-CH2-C(=O)-N(H)-5') and amide- 4 (3'-CH2-N(H)-C(=O)-5'), hydroxylamino, siloxane (dialkylsiloxane), carboxamide, carbonate, carboxymethyl, carbamate, carboxylate ester, thioether, ethylene oxide linker, sulfide, sulfonate, sulfonamide, sulfonate ester, thioformacetal (3'-S-CH2-O-5'), formacetal (3 '-O-CH2-O-5'), oxime, methyleneimino, methykenecarbonylamino, methylenemethylimino (MMI, 3'-CH2-N(CH3)-O-5'), methylenehydrazo, methylenedimethylhydrazo, methyleneoxymethylimino, ethers (C3’-O-C5’), thioethers (C3’-S-C5’), thioacetamido (C3’- N(H)-C(=O)-CH2-S-C5’, C3’-O-P(O)-O-SS-C5’, C3’-CH2-NH-NH-C5’, 3'-NHP(O)(OCH3)- O-5' and 3'-NHP(O)(OCH3)-O-5’

[0192] In some embodiments, nucleic acid modifications can include peptide nucleic acids (PNA), bridged nucleic acids (BNA), morpholinos, locked nucleic acids (LNA), glycol nucleicacids (GNA), threose nucleic acids (TNA), or any other xeno nucleic acids (XNA) described in the art.

[0193] A nucleic acid modification can be located anywhere in the nucleic acid, e.g., nucleic acid tag, labeled-nucleic acid probe, blocking probe, oligonucleotide primer, or single-stranded extender template, comprising said modification. For example, the nucleic acid modification can be at the 5 ’-end, the 3 ’-end or at an internal position of the nucleic acid, e.g., nucleic acid tag, labeled-nucleic acid probe, blocking probe, oligonucleotide primer, or single-stranded extender template.

[0194] In some embodiments of any one of the aspects described herein, the nucleic acid tag comprises a nucleic acid modification capable of enhancing nuclease cleavage.

[0195] In some embodiments of any one of the aspects described herein, the nucleic acid tag comprises a nucleic acid modification capable of enhancing binding or hybridizing with a labeled-nucleic acid probe.

[0196] In some embodiments of any one of the aspects described herein, the nucleic acid tag comprises a nucleic acid modification capable of enhancing binding or hybridizing with a blocking probe.

[0197] In some embodiments of any one of the aspects described herein, the nucleic acid tag comprises a nucleic acid modification capable of inhibiting / reducing binding or hybridizing with a labeled-nucleic acid probe that is not fully complementary to the nucleic acid tag.Exemplary embodiments

[0198] The technology may be as described in any one of the following numbered Embodiments:

[0199] Embodiment 1 : A method of detecting a set of target molecule in situ in a preparation of cells or tissue, the method comprising: (a) contacting the preparation of cells or tissue with a first target-binding ligand conjugated to a first nucleic acid tag, under conditions permitting specific binding of the first target-binding ligand to a first target molecule; (b) contacting the first nucleic acid tag with a first labeled nucleic acid probe, wherein the first labeled nucleic acid probe comprises a nucleotide sequence complementary to at least a portion of the first nucleic acid tag; (c) detecting the labeled nucleic acid probe, wherein detection of the labeled nucleic acid probe indicates the presence and location of the target molecule in the preparation of cells or tissue; (d) adding to the preparation of cells or tissue a nuclease under conditions permitting digestion of the first nucleic acid tag and / or the labeled nucleic acid probe; (e) contacting the preparation of cells or tissue from (d) with a second target-binding ligandconjugated to a second nucleic acid tag, under conditions permitting specific binding of the second target-binding ligand to a second target molecule; (f) contacting the second nucleic acid tag with a second labeled nucleic acid probe, , wherein the second labeled nucleic acid probe comprises a nucleotide sequence complementary to at least a portion of the second nucleic acid tag; (g) detecting the second labeled nucleic acid probe, wherein detection of the second labeled nucleic acid probe indicates the presence and location of the second target molecule in the preparation of cells or tissue; and (h) optionally, repeating steps (d)-(g) one or more times.

[0200] Embodiment 2: A method of detecting a set of target molecules in situ in a preparation of cells or tissue, the method comprising: (a) contacting the preparation of cells or tissue with a first set of target-binding ligand molecules under conditions permitting specific binding of the target-binding ligand molecules to respective first target molecules present in the preparation of cells or tissue, wherein members of the set of target-binding ligand molecules are conjugated to respective first nucleic acid tag, wherein the first nucleic acid tag of each target-binding ligand has a different sequence from other first nucleic acid tags in the set, and is conjugated to a different member of a first set of target-binding ligand molecules; (b) contacting the first nucleic acid tags with a first set of distinguishably-labeled nucleic acid probes, wherein each labeled nucleic acid probe in the set comprises a nucleotide sequence complementary to at least a portion of a respective first nucleic acid tag; (c) detecting the first labeled nucleic acid probes, wherein detection of the distinguishably-labeled nucleic acid probes indicate the presence and location of the first target molecules in the preparation of cells or tissue; (d) adding to the preparation of cells or tissue a nuclease under conditions permitting digestion of the first nucleic acid tags and / or the labeled nucleic acid probes; (e) contacting the preparation of cells or tissue with a second set of target-binding ligand molecules under conditions permitting specific binding of the target-binding ligand molecules to respective second target molecules present in the preparation of cells or tissue, wherein members of the set of target-binding ligand molecules are conjugated to respective second nucleic acid tag, wherein the second nucleic acid tag of each target-binding ligand has a different sequence from other second nucleic acid tags in the set, and is conjugated to a different member of a second set of target-binding ligand molecules; (f) contacting the first nucleic acid tags with a second set of distinguishably-labeled nucleic acid probes, wherein each labeled nucleic acid probe in the set comprises a nucleotide sequence complementary to at least a portion of a respective second nucleic acid tag; (g) detecting the second labeled nucleic acid probes, wherein detection of the distinguishably-labeled nucleic acid probes indicate the presence and location of thesecond target molecules in the preparation of cells or tissue; and (h) optionally, repeating steps (d)-(g).

[0201] Embodiment 3: The method of Embodiment 2, wherein the step of contacting the first nucleic acid tags with the first set of distinguishably-labeled nucleic acid probes comprises contacting the first nucleic acid tags with a set of probe, wherein the first set of probes comprises a first set of blocking-nucleic acid probes and the first set of distinguishably-labeled nucleic acid probes, wherein each blocking-nucleic acid probe has a different sequence from other blocking-nucleic acid probes and said sequence is complementary to at least a portion of the nucleic acid tag of a different member of the set of target-binding ligand molecules, and wherein a concentration of the blocking-nucleic acid probes in the first set of blocking-nucleic acid probes is lower than a concentration of the labeled nucleic acid probes in the first set of blocking-nucleic acid probes.

[0202] Embodiment 4: The method of Embodiment 2, wherein the step of contacting the first nucleic acid tags with the first set of distinguishably-labeled nucleic acid probes comprises contacting the first nucleic acid tags with a set of probe, wherein the first set of probes comprises a first set of blocking-nucleic acid probes and the first set of distinguishably-labeled nucleic acid probes, wherein each blocking-nucleic acid probe has a different sequence from other blocking-nucleic acid probes and said sequence is complementary to at least a portion of the nucleic acid tag of a different member of the set of target-binding ligand molecules, and wherein the set of blocking-nucleic acid probes does not comprise a blocking-nucleic acid probe having a sequence complementary to the nucleic acid tag having a sequence complementary to the distinguishably-labeled nucleic acid probes.

[0203] Embodiment 5:The method of any one of Embodiments 2-4, wherein the step of contacting the second nucleic acid tags with the second set of distinguishably-labeled nucleic acid probes comprises contacting the second nucleic acid tags with a set of probe, wherein the second set of probes comprises a second set of blocking-nucleic acid probes and the second set of distinguishably-labeled nucleic acid probes, wherein each blocking-nucleic acid probe has a different sequence from other blocking-nucleic acid probes and said sequence is complementary to at least a portion of the nucleic acid tag of a different member of the set of target-binding ligand molecules, and wherein a concentration of the blocking-nucleic acid probes in the second set of blocking-nucleic acid probes is lower than a concentration of the labeled nucleic acid probes in the second set of blocking-nucleic acid probes.

[0204] Embodiment 6: The method of any one of Embodiments 2-4, wherein the step of contacting the second nucleic acid tags with the second set of distinguishably-labeled nucleicacid probes comprises contacting the second nucleic acid tags with a set of probe, wherein the second set of probes comprises a second set of blocking-nucleic acid probes and the second set of distinguishably-labeled nucleic acid probes, wherein each blocking-nucleic acid probe has a different sequence from other blocking-nucleic acid probes and said sequence is complementary to at least a portion of the nucleic acid tag of a different member of the set of target-binding ligand molecules, and wherein the set of blocking-nucleic acid probes does not comprise a blocking-nucleic acid probe having a sequence complementary to the nucleic acid tag having a sequence complementary to the distinguishably-labeled nucleic acid probes.

[0205] Embodiment 7: The method of any one of Embodiments 1-6, wherein the first and second target molecules are different.

[0206] Embodiment 8: The method of any one of Embodiments 1-6, wherein the first and second target molecules are same.

[0207] Embodiment 9: The method of any one of Embodiments 1-8, wherein the first or second target-binding ligand comprises a protein.

[0208] Embodiment 10: The method of any one of Embodiments 1-9, wherein the first or second target-binding ligand comprises an antibody or antigen-binding fragment thereof.

[0209] Embodiment 11 : The method of any one of Embodiments 1-8, wherein the first or second target-binding ligand comprises RNA.

[0210] Embodiment 12: The method of any one of Embodiments 1-11, wherein the first or second nucleic acid tag is DNA.

[0211] Embodiment 13: The method of any one of Embodiments 1-12, wherein the nuclease is a DNase.

[0212] Embodiment 14: The method of Embodiment 13, wherein the first nucleic acid tag is DNA conjugated with a protein.

[0213] Embodiment 15: The method of Embodiment 13, wherein the first nucleic acid tag is DNA conjugated with the antibody or antigen-binding fragment thereof.

[0214] Embodiment 16: The method of Embodiment 13, wherein the first nucleic acid tag is DNA conjugated with an RNA.

[0215] Embodiment 17: The method of any one of Embodiments 1-12, wherein the first target-binding ligand is an RNA.

[0216] Embodiment 18: The method of Embodiment 17, wherein the nuclease is an RNase.

[0217] Embodiment 19: The method of Embodiment 18, wherein the first target-binding ligand is an RNA.

[0218] Embodiment 20: The method of Embodiment 19, wherein the second target-binding ligand comprises protein.

[0219] Embodiment 21 : The method of Embodiment 20, wherein the second nucleic acid tag is DNA conjugated with protein.

[0220] Embodiment 22: The method of Embodiment 20, wherein the second nucleic acid tag is DNA conjugated with an antibody or antigen-binding fragment thereof.

[0221] Embodiment 23: The method of Embodiment 20, wherein the second nucleic acid tag is DNA conjugated with an RNA.

[0222] Embodiment 24: The method of any one of Embodiments 1-23, wherein the first nucleic acid tag is a concatemer comprising repeats of a first oligonucleotide primer sequence.

[0223] Embodiment 25 : The method of Embodiment 24, wherein the step of contacting the preparation of cells or tissue with the first target-binding ligand comprises: (i) contacting the preparation of cells or tissue with a first target-binding ligand conjugated to a first oligonucleotide primer, under conditions permitting specific binding of the first target-binding ligand to the target molecule; (ii) adding to the preparation of cells or tissue a reaction mixture comprising a first single-stranded extender template and a nucleic acid polymerase enzyme under conditions permitting hybridization and extension of the first oligonucleotide primer using the first single-stranded extender template; (iii) heating the reaction mixture of step (ii) to separate the first single-stranded extender template from extended first oligonucleotide primer produced in step (ii); and (iv) repeating steps (ii) and (iii) at least once, thereby generating a concatemer comprising repeats of the first oligonucleotide primer sequence conjugated to the first target-binding ligand.

[0224] Embodiment 26: The method of any one of Embodiments 1-25, wherein the second nucleic acid tag is a concatemer comprising repeats of a second oligonucleotide primer sequence.

[0225] Embodiment 27 : The method of Embodiment 26, wherein the step of contacting the preparation of cells or tissue with the second target-binding ligand comprises: (i) contacting the preparation of cells or tissue with a second target-binding ligand conjugated to a second oligonucleotide primer, under conditions permitting specific binding of the second targetbinding ligand to the target molecule; (ii) adding to the preparation of cells or tissue a reaction mixture comprising a second single-stranded extender template and a nucleic acid polymerase enzyme under conditions permitting hybridization and extension of the second oligonucleotide primer using the second single-stranded extender template; (iii) heating the reaction mixture of step (ii) to separate the second single-stranded extender template from extended secondoligonucleotide primer produced in step (ii); and (iv) repeating steps (ii) and (iii) at least once, thereby generating a concatemer comprising repeats of the second oligonucleotide primer sequence conjugated to the second target-binding ligand.

[0226] Embodiment 28: The method of any one of Embodiments 1-27, wherein the first nucleic acid tag is a branched nucleic acid, optionally, the first nucleic acid tag is a branched DNA.

[0227] Embodiment 29: The method of any one of Embodiments 1-28, wherein a plurality of labeled nucleic acid probe molecules hybridizes to first or second nucleic acid tag.

[0228] Embodiment 30: The method of any one of Embodiments 1-28, wherein a single labeled nucleic acid probe molecules hybridizes to first or second nucleic acid tag.

[0229] Embodiment 31 : A method of detecting a set of target molecules in situ in a preparation of cells or tissue, the method comprising: (a) contacting the preparation of cells or tissue with a set of target-binding ligand molecules under conditions permitting specific binding of the target-binding ligand molecules to target molecules present in the preparation of cells or tissue, wherein members of the set of target-binding ligand molecules are conjugated to respective nucleic acid tag, wherein the nucleic acid tag of each target-binding ligand has a different sequence from other first nucleic acid tags in the set; (b) contacting the nucleic acid tags with a first set of probes, wherein the first set of probes comprises a set of blocking-nucleic acid probes and a first labeled-nucleic acid probe, wherein each blocking-nucleic acid probe has a different sequence from other blocking probes and said sequence is complementary to at least a portion of the nucleic acid tag of a different member of the set of target-binding ligand molecules, wherein the first labeled-nucleic acid probe comprises a sequence complementary to at least a portion of the nucleic acid tag conjugated to a first target-binding ligand in the set of target-binding ligand molecules and the sequence of the first labeled-nucleic acid probe is identical to the sequence of a first blocking-nucleic acid probe in the set of blocking-nucleic acid probes, wherein a concentration of the blocking-nucleic acid probes is lower than a concentration of the first labeled nucleic acid probe in the first set of blocking-nucleic acid probes; (c) detecting the first labeled nucleic acid probe, wherein detection of the first labeled nucleic acid probe indicates the presence and location of a first target molecule in the preparation of cells or tissue; (d) contacting the nucleic acid tags with a second set of probes, wherein the second set of probes comprises a set of blocking-nucleic acid probes and a second labeled-nucleic acid probe, wherein each blocking-nucleic acid probe has a different sequence from other blocking probes and said sequence is complementary to at least a portion of the nucleic acid tag of a different member of the set of target-binding ligand molecules, whereinthe second labeled-nucleic acid probe comprises a sequence complementary to at least a portion of the nucleic acid tag conjugated to a second target-binding ligand in the set of target-binding ligand molecules and the sequence of the second labeled-nucleic acid probe is identical to the sequence of a second blocking-nucleic acid probe in the set of blocking-nucleic acid probes, wherein a concentration of the blocking-nucleic acid probes is lower than a concentration of the second labeled nucleic acid probe in the second set of blocking-nucleic acid probes; (e) detecting the second labeled nucleic acid probe, wherein detection of the second labeled nucleic acid probe indicates the presence and location of a second target molecule in the preparation of cells or tissue; and (f) optionally, repeating steps (d)-(e) one or more times.

[0230] Embodiment 32: A method of detecting a set of target molecules in situ in a preparation of cells or tissue, the method comprising: (a) contacting the preparation of cells or tissue with a set of target-binding ligand molecules under conditions permitting specific binding of the target-binding ligand molecules to target molecules present in the preparation of cells or tissue, wherein members of the set of target-binding ligand molecules are conjugated to respective nucleic acid tag, wherein the nucleic acid tag of each target-binding ligand has a different sequence from other first nucleic acid tags in the set; (b) contacting the nucleic acid tags with a first set of probes, wherein the first set of probes comprises a set of blocking-nucleic acid probes and a first labeled-nucleic acid probe, wherein each blocking-nucleic acid probe has a different sequence from other blocking-nucleic acid probes and said sequence is complementary to at least a portion of the nucleic acid tag of a different member of the set of target-binding ligand molecules, wherein the first labeled-nucleic acid probe comprises a sequence complementary to at least a portion of the nucleic acid tag conjugated to a first targetbinding ligand in the set of target-binding ligand molecules, and wherein the set of blocking- nucleic acid probes does not comprise a blocking-nucleic acid probe having a sequence complementary to the nucleic acid tag conjugated to the first target-binding ligand; (c) detecting the first labeled nucleic acid probe, wherein detection of the first labeled nucleic acid probe indicates the presence and location of a first target molecule in the preparation of cells or tissue; contacting the nucleic acid tags with a second set of probes, wherein the second set of probes comprises a set of blocking-nucleic acid probes and a second labeled-nucleic acid probe, wherein each blocking-nucleic acid probe has a different sequence from other blocking- nucleic acid probes and said sequence is complementary to at least a portion of the nucleic acid tag of a different member of the set of target-binding ligand molecules, wherein the second labeled-nucleic acid probe comprises a sequence complementary to at least a portion of the nucleic acid tag conjugated to a second target-binding ligand in the set of target-binding ligandmolecules, and wherein the set of blocking-nucleic acid probes does not comprise a blocking- nucleic acid probe having a sequence complementary to the nucleic acid tag conjugated to the second target-binding ligand; (e) detecting the second labeled nucleic acid probe, wherein detection of the second labeled nucleic acid probe indicates the presence and location of a second target molecule in the preparation of cells or tissue; and (f) optionally, repeating steps (d)-(e) one or more times.

[0231] Embodiment 33: The method of Embodiment 31 or 32, wherein a concentration of the blocking probes is higher than a concentration of the labeled-nucleic acid probes.

[0232] Embodiment 34: The method of Embodiment 31 or 32, wherein a concentration of the blocking probes is lower than a concentration of the labeled-nucleic acid probes.

[0233] Embodiment 35: The method of any one of Embodiments 31-34, wherein at least one, e.g., each nucleic acid tag is a concatemer comprising repeats of a respective first oligonucleotide primer sequence.

[0234] Embodiment 36: The method of Embodiment 35, wherein the step of contacting the preparation of cells or tissue with a set of target-binding ligand molecules comprises: (i) contacting the preparation of cells or tissue with a set of target-binding ligand molecules under conditions permitting specific binding of the target-binding ligand molecules to target molecules present in the preparation of cells or tissue, wherein members of the set of targetbinding ligand molecules are conjugated to respective first oligonucleotide primer members of a set of orthogonal first oligonucleotide primer and first single-stranded extender template oligonucleotide pairs, wherein the first oligonucleotide primer of each pair has a different sequence from other first oligonucleotide primers in the set, and is conjugated to a different member of the set of target-binding ligand molecules; (ii) contacting the preparation of cells or tissue with the set of target-binding ligand molecules under conditions permitting specific binding of the target-binding ligand molecules to target molecules present in the preparation of cells or tissue; (iii) adding to the preparation of cells or tissue a reaction mixture comprising first single-stranded extender templates of the set of orthogonal first oligonucleotide primer and first single-stranded extender template oligonucleotide pairs, and a nucleic acid polymerase, under conditions permitting hybridization and extension of the first oligonucleotide primers using the respective first single-stranded extender templates; (iv) heating the reaction mixture of step (iii) to separate the first single-stranded extender templates from extended first oligonucleotide primers produced in step (iii); and (v) repeating steps (iii) and (iv) at least once, thereby generating, on respective members of the set of target-bindingligand molecules, a concatemer comprising repeats of the respective first oligonucleotide primer sequence, conjugated to the target-binding ligand molecule.

[0235] Embodiment 37: The method of any one of Embodiments 31-34, wherein at least one, e.g., each nucleic acid tag is a branched nucleic acid, optionally, the branched nucleic acid is DNA.

[0236] Embodiment 38: The method of any one of Embodiments 31-37, wherein at least one, e.g., each target-binding ligand comprises a protein.

[0237] Embodiment 39: The method of any one of Embodiments 31-37, wherein at least one, e.g., each target-binding ligand comprises an antibody or antigen-binding fragment thereof.

[0238] Embodiment 40: The method of any one of Embodiments 31-37, wherein at least one, e.g., each target-binding ligand comprises RNA.

[0239] Embodiment 41 : The method of any one of Embodiments 31 -40, wherein a plurality of labeled nucleic acid probe molecules hybridizes to a plurality of concatemer repeats conjugated to the target-binding ligands.

[0240] Embodiment 42: The method of any one of Embodiments 1-41, wherein the preparation of cells or tissue is fixed.

[0241] Embodiment 43: The method of Embodiment 42, wherein the preparation of tissue is paraffin embedded.

[0242] Embodiment 44: A kit comprising: (i) a target-binding ligand conjugated with a nucleic acid tag; (ii) a labeled-nucleic acid probe, wherein the labeled-nucleic acid probe comprises a detectable label and has a nucleotide sequence complementary to at least a portion of the nucleic acid tag; and (iii) a nuclease.

[0243] Embodiment 45: A kit comprising: (i) a first target-binding ligand conjugated with a first nucleic acid tag; (ii) a first labeled-nucleic acid probe, wherein the first labeled-nucleic acid probe comprises a detectable label and has a nucleotide sequence complementary to at least a portion of the first nucleic acid tag; (iii) a second target-binding ligand conjugated with a second nucleic acid tag, wherein the second target-binding ligand binds a target different from the first target-binding ligand; (iv) a second labeled-nucleic acid probe, wherein the second labeled-nucleic acid probe comprises a detectable label and has a nucleotide sequence complementary to at least a portion of the second nucleic acid tag; and (v) a nuclease.

[0244] Embodiment 46: The kit of Embodiment 45, wherein the detectable label of the first labeled-nucleic acid probe and the second labeled-nucleic acid probe are distinguishable from each other.

[0245] Embodiment 47 : The kit of Embodiment 45, wherein the detectable label of the first labeled-nucleic acid probe and the second labeled-nucleic acid probe are the same.

[0246] Embodiment 48: A kit comprising: (i) a target-binding ligand conjugated with a nucleic acid tag; (ii) a labeled-nucleic acid probe, wherein the labeled-nucleic acid probe comprises a detectable label and has a nucleotide sequence complementary to at least a portion of the nucleic acid tag; and (iii) a set of blocking probes, optionally, the set of blocking probes does not comprise a blocking probe having a sequence complementary to the nucleic acid tag conjugated to the target-binding ligand, and optionally, the kit further comprises a nuclease.

[0247] Embodiment 49: A kit comprising: (i) a first target-binding ligand conjugated with a first nucleic acid tag; (ii) a first labeled-nucleic acid probe, wherein the first labeled-nucleic acid probe comprises a detectable label and has a nucleotide sequence complementary to at least a portion of the first nucleic acid tag; (iii) a second target-binding ligand conjugated with a second nucleic acid tag, wherein the second target-binding ligand binds a target different from the first target-binding ligand; (iv) a second labeled-nucleic acid probe, wherein the second labeled-nucleic acid probe comprises a detectable label and has a nucleotide sequence complementary to at least a portion of the second nucleic acid tag; and (v) a set of blocking probes, optionally, the set of blocking probes does not comprise a blocking probe having a sequence complementary to the first nucleic acid tag and / or the second nucleic acid tag, and optionally, the kit further comprises a nuclease.

[0248] Embodiment 50: The kit of Embodiment 49, wherein the detectable label of the first labeled-nucleic acid probe and the second labeled-nucleic acid probe are distinguishable from each other.

[0249] Embodiment 51 : The kit of Embodiment 49, wherein the detectable label of the first labeled-nucleic acid probe and the second labeled-nucleic acid probe are the same.

[0250] Embodiment 52: A kit comprising: (i) a target-binding ligand conjugated with an oligonucleotide primer; (ii) a single-stranded extender template; (iii) a polymerase; (iv) a labeled-nucleic acid probe, wherein the labeled-nucleic acid probe comprises a detectable label and has a nucleotide sequence complementary to at least a portion of the oligonucleotide primer or the extender template; and (v) a nuclease.

[0251] Embodiment 53 : A kit comprising: (i) a first target-binding ligand conjugated with a first oligonucleotide primer; (ii) a first single-stranded extender template for template directed extension of the first oligonucleotide primer; (iii) a second target-binding ligand conjugated with a second oligonucleotide primer, wherein the second target-binding ligand binds a target different from the first target-binding ligand; (iv) a second single-strandedextender template for template directed extension of the second oligonucleotide primer; (v) a polymerase; (vi) a first labeled-nucleic acid probe comprising a first detectable label and having a nucleotide sequence complementary to at least a portion of the first oligonucleotide primer or the first extender template; (vii) a second labeled-nucleic acid probe comprising a second detectable label and having a nucleotide sequence complementary to at least a portion of the second oligonucleotide primer or the second extender template; and (viii) a nuclease.

[0252] Embodiment 54: The kit of Embodiment 53, wherein the detectable label of the first labeled-nucleic acid probe and the second labeled-nucleic acid probe are distinguishable from each other.

[0253] Embodiment 55 : The kit of Embodiment 53, wherein the detectable label of the first labeled-nucleic acid probe and the second labeled-nucleic acid probe are the same.

[0254] Embodiment 56: A kit comprising: (i) a first target-binding ligand conjugated with a first oligonucleotide primer; (ii) a first single-stranded extender template ; (iii) a polymerase; (iv) a labeled-nucleic acid probe, wherein the labeled-nucleic acid probe comprises a detectable label and has a nucleotide sequence complementary to at least a portion of the oligonucleotide primer or the extender template; and (v) a set of blocking probes, optionally, the set of blocking probes does not comprise a blocking probe having a sequence complementary to the nucleic acid tag conjugated to the target-binding ligand.

[0255] Embodiment 57: A kit comprising: (i) a target-binding ligand conjugated with an oligonucleotide primer; ii) a first single-stranded extender template for template directed extension of the first oligonucleotide primer; (iii) a second target-binding ligand conjugated with a second oligonucleotide primer, wherein the second target-binding ligand binds a target different from the first target-binding ligand; (iv) a second single-stranded extender template for template directed extension of the second oligonucleotide primer; (v) a polymerase; (vi) a first labeled-nucleic acid probe comprising a first detectable label and having a nucleotide sequence complementary to at least a portion of the first oligonucleotide primer or the first extender template; (vii) a second labeled-nucleic acid probe comprising a second detectable label and having a nucleotide sequence complementary to at least a portion of the second oligonucleotide primer or the second extender template; and (viii) a set of blocking probes, optionally, the set of blocking probes does not comprise a blocking probe having a sequence complementary to the first nucleic acid tag and / or the second nucleic acid tag.

[0256] Embodiment 58: The kit of Embodiment 57, wherein the detectable label of the first labeled-nucleic acid probe and the second labeled-nucleic acid probe are distinguishable from each other.

[0257] Embodiment 59: The kit of Embodiment 57, wherein the detectable label of the first labeled-nucleic acid probe and the second labeled-nucleic acid probe are the same.

[0258] Embodiment 60: The kit of any one of Embodiments 52-59, wherein the kit further comprises one or more components for template dependent extension of the oligonucleotide primer with the polymerase.

[0259] Embodiment 61 : The kit of any one of Embodiments 52-60, wherein the kit further comprises dNTPs.

[0260] Embodiment 62: The kit of any of one of Embodiments 52-61, wherein the kit further comprises a buffer for polymerization with the polymerase.

[0261] Embodiment 63 : The kit of any one of Embodiments 44-62, wherein the kit further comprises a buffer for digestion or cleavage with the nuclease.

[0262] Embodiment 64: The kit of any one of Embodiments 44-63, wherein the kit further comprises means for detecting or imagining a signal produced by the detectable label.

[0263] Embodiment 65 : A system for detecting targets in a sample, comprising: (i) a targetbinding ligand conjugated with a nucleic acid tag; (ii) a labeled-nucleic acid probe, wherein the labeled-nucleic acid probe comprises a detectable label and has a nucleotide sequence complementary to at least a portion of the nucleic acid tag; and (iii) a nuclease.

[0264] Embodiment 66: A system comprising: (i) a first target-binding ligand conjugated with a first nucleic acid tag; (ii) a first labeled-nucleic acid probe, wherein the first labeled- nucleic acid probe comprises a detectable label and has a nucleotide sequence complementary to at least a portion of the first nucleic acid tag; (iii) a second target-binding ligand conjugated with a second nucleic acid tag, wherein the second target-binding ligand binds a target different from the first target-binding ligand; (iv) a second labeled-nucleic acid probe, wherein the second labeled-nucleic acid probe comprises a detectable label and has a nucleotide sequence complementary to at least a portion of the second nucleic acid tag; and (v) a nuclease.

[0265] Embodiment 67: The system of Embodiment 66, wherein the detectable label of the first labeled-nucleic acid probe and the second labeled-nucleic acid probe are distinguishable from each other.

[0266] Embodiment 68: The system of Embodiment 66, wherein the detectable label of the first labeled-nucleic acid probe and the second labeled-nucleic acid probe are the same.

[0267] Embodiment 69: A system comprising: (i) a target-binding ligand conjugated with a nucleic acid tag; (ii) a labeled-nucleic acid probe, wherein the labeled-nucleic acid probe comprises a detectable label and has a nucleotide sequence complementary to at least a portion of the nucleic acid tag; and (iii) a set of blocking probes, optionally, the set of blocking probesdoes not comprise a blocking probe having a sequence complementary to the nucleic acid tag conjugated to the target-binding ligand, and optionally, the system further comprises a nuclease.

[0268] Embodiment 70: A system comprising: (i) a first target-binding ligand conjugated with a first nucleic acid tag; (ii) a first labeled-nucleic acid probe, wherein the first labeled- nucleic acid probe comprises a detectable label and has a nucleotide sequence complementary to at least a portion of the first nucleic acid tag; (iii) a second target-binding ligand conjugated with a second nucleic acid tag, wherein the second target-binding ligand binds a target different from the first target-binding ligand; (iv) a second labeled-nucleic acid probe, wherein the second labeled-nucleic acid probe comprises a detectable label and has a nucleotide sequence complementary to at least a portion of the second nucleic acid tag; and (v) a set of blocking probes, optionally, the set of blocking probes does not comprise a blocking probe having a sequence complementary to the first nucleic acid tag and / or the second nucleic acid tag, and optionally, the system further comprises a nuclease.

[0269] Embodiment 71 : The system of Embodiment 70, wherein the detectable label of the first labeled-nucleic acid probe and the second labeled-nucleic acid probe are distinguishable from each other.

[0270] Embodiment 72: The system of Embodiment 70, wherein the detectable label of the first labeled-nucleic acid probe and the second labeled-nucleic acid probe are the same.

[0271] Embodiment 73: A system comprising: (i) a target-binding ligand conjugated with an oligonucleotide primer; (ii) a single-stranded extender template; (iii) a polymerase; (iv) a labeled-nucleic acid probe, wherein the labeled-nucleic acid probe comprises a detectable label and has a nucleotide sequence complementary to at least a portion of the oligonucleotide primer or the extender template; and (v) a nuclease.

[0272] Embodiment 74: A system comprising: (i) a first target-binding ligand conjugated with a first oligonucleotide primer; (ii) a first single-stranded extender template for template directed extension of the first oligonucleotide primer; (iii) a second target-binding ligand conjugated with a second oligonucleotide primer, wherein the second target-binding ligand binds a target different from the first target-binding ligand; (iv) a second single-stranded extender template for template directed extension of the second oligonucleotide primer; (v) a polymerase; (vi) a first labeled-nucleic acid probe comprising a first detectable label and having a nucleotide sequence complementary to at least a portion of the first oligonucleotide primer or the first extender template; (vii) a second labeled-nucleic acid probe comprising a seconddetectable label and having a nucleotide sequence complementary to at least a portion of the second oligonucleotide primer or the second extender template; and (viii) a nuclease.

[0273] Embodiment 75: The system of Embodiment 74, wherein the detectable label of the first labeled-nucleic acid probe and the second labeled-nucleic acid probe are distinguishable from each other.

[0274] Embodiment 76: The system of Embodiment 74, wherein the detectable label of the first labeled-nucleic acid probe and the second labeled-nucleic acid probe are the same.

[0275] Embodiment 77: A system comprising: (i) a first target-binding ligand conjugated with a first oligonucleotide primer; (ii) a first single-stranded extender template ; (iii) a polymerase; (iv) a labeled-nucleic acid probe, wherein the labeled-nucleic acid probe comprises a detectable label and has a nucleotide sequence complementary to at least a portion of the oligonucleotide primer or the extender template; and (v) a set of blocking probes, optionally, the set of blocking probes does not comprise a blocking probe having a sequence complementary to the nucleic acid tag conjugated to the target-binding ligand.

[0276] Embodiment 78: A system comprising: (i) a target-binding ligand conjugated with an oligonucleotide primer; ii) a first single-stranded extender template for template directed extension of the first oligonucleotide primer; (iii) a second target-binding ligand conjugated with a second oligonucleotide primer, wherein the second target-binding ligand binds a target different from the first target-binding ligand; (iv) a second single-stranded extender template for template directed extension of the second oligonucleotide primer; (v) a polymerase; (vi) a first labeled-nucleic acid probe comprising a first detectable label and having a nucleotide sequence complementary to at least a portion of the first oligonucleotide primer or the first extender template; (vii) a second labeled-nucleic acid probe comprising a second detectable label and having a nucleotide sequence complementary to at least a portion of the second oligonucleotide primer or the second extender template; and (viii) a set of blocking probes, optionally, the set of blocking probes does not comprise a blocking probe having a sequence complementary to the first nucleic acid tag and / or the second nucleic acid tag.

[0277] Embodiment 79: The system of Embodiment 78, wherein the detectable label of the first labeled-nucleic acid probe and the second labeled-nucleic acid probe are distinguishable from each other.

[0278] Embodiment 80: The system of Embodiment 78, wherein the detectable label of the first labeled-nucleic acid probe and the second labeled-nucleic acid probe are the same.

[0279] Embodiment 81 : The system of any one of Embodiments 73-80, wherein the system further comprises one or more components for template dependent extension of the oligonucleotide primer with the polymerase.

[0280] Embodiment 82: The system of any one of Embodiments 73-81, wherein the system further comprises dNTPs.

[0281] Embodiment 83: The system of any of one of Embodiments 73-82, wherein the system further comprises a buffer for polymerization with the polymerase.

[0282] Embodiment 84: The system of any one of Embodiments 65-83, wherein the system further comprises a buffer for digestion or cleavage with the nuclease.

[0283] Embodiment 85 : The system of any one of Embodiments 65-84, wherein the system further comprises means for detecting or imagining a signal produced by the detectable label.

[0284] Embodiment 86: A reaction mixture comprising: (i) a target-binding ligand conjugated with a nucleic acid tag; (ii) a labeled-nucleic acid probe, wherein the labeled- nucleic acid probe comprises a detectable label and has a nucleotide sequence complementary to at least a portion of the nucleic acid tag; and (iii) a nuclease.

[0285] Embodiment 87: A reaction mixture comprising: (i) a first target-binding ligand conjugated with a first nucleic acid tag; (ii) a first labeled-nucleic acid probe, wherein the first labeled-nucleic acid probe comprises a detectable label and has a nucleotide sequence complementary to at least a portion of the first nucleic acid tag; (iii) a second target-binding ligand conjugated with a second nucleic acid tag, wherein the second target-binding ligand binds a target different from the first target-binding ligand; (iv) a second labeled-nucleic acid probe, wherein the second labeled-nucleic acid probe comprises a detectable label and has a nucleotide sequence complementary to at least a portion of the second nucleic acid tag; and (v) a nuclease, and wherein the detectable label of the first labeled-nucleic acid probe and the second labeled-nucleic acid probe are distinguishable from each other.

[0286] Embodiment 88: A reaction mixture comprising: (i) a target-binding ligand conjugated with a nucleic acid tag; (ii) a labeled-nucleic acid probe, wherein the labeled- nucleic acid probe comprises a detectable label and has a nucleotide sequence complementary to at least a portion of the nucleic acid tag; and (iii) a set of blocking probes, optionally, the set of blocking probes does not comprise a blocking probe having a sequence complementary to the nucleic acid tag conjugated to the target-binding ligand, and optionally, the reaction mixture further comprises a nuclease.

[0287] Embodiment 89: A reaction mixture comprising: (i) a first target-binding ligand conjugated with a first nucleic acid tag; (ii) a first labeled-nucleic acid probe, wherein the firstlabeled-nucleic acid probe comprises a detectable label and has a nucleotide sequence complementary to at least a portion of the first nucleic acid tag; (iii) a second target-binding ligand conjugated with a second nucleic acid tag, wherein the second target-binding ligand binds a target different from the first target-binding ligand; (iv) a second labeled-nucleic acid probe, wherein the second labeled-nucleic acid probe comprises a detectable label and has a nucleotide sequence complementary to at least a portion of the second nucleic acid tag; and (v) a set of blocking probes, optionally, the set of blocking probes does not comprise a blocking probe having a sequence complementary to the first nucleic acid tag and / or the second nucleic acid tag, wherein the detectable label of the first labeled-nucleic acid probe and the second labeled-nucleic acid probe are distinguishable from each other, and optionally, the reaction mixture further comprises a nuclease.

[0288] Embodiment 90: A reaction mixture comprising: (i) a target-binding ligand conjugated with an oligonucleotide primer; (ii) a single-stranded extender template; (iii) a polymerase; (iv) a labeled-nucleic acid probe, wherein the labeled-nucleic acid probe comprises a detectable label and has a nucleotide sequence complementary to at least a portion of the oligonucleotide primer or the extender template; and (v) a nuclease.

[0289] Embodiment 91 : A reaction mixture comprising: (i) a first target-binding ligand conjugated with a first oligonucleotide primer; (ii) a first single-stranded extender template for template directed extension of the first oligonucleotide primer; (iii) a second target-binding ligand conjugated with a second oligonucleotide primer, wherein the second target-binding ligand binds a target different from the first target-binding ligand; (iv) a second single-stranded extender template for template directed extension of the second oligonucleotide primer; (v) a polymerase; (vi) a first labeled-nucleic acid probe comprising a first detectable label and having a nucleotide sequence complementary to at least a portion of the first oligonucleotide primer or the first extender template; (vii) a second labeled-nucleic acid probe comprising a second detectable label and having a nucleotide sequence complementary to at least a portion of the second oligonucleotide primer or the second extender template; and (viii) a nuclease, and wherein the detectable label of the first labeled-nucleic acid probe and the second labeled- nucleic acid probe are distinguishable from each other.

[0290] Embodiment 92: A reaction mixture comprising: (i) a first target-binding ligand conjugated with a first oligonucleotide primer; (ii) a first single-stranded extender template ; (iii) a polymerase; (iv) a labeled-nucleic acid probe, wherein the labeled-nucleic acid probe comprises a detectable label and has a nucleotide sequence complementary to at least a portion of the oligonucleotide primer or the extender template; and (v) a set of blocking probes,optionally, the set of blocking probes does not comprise a blocking probe having a sequence complementary to the nucleic acid tag conjugated to the target-binding ligand.

[0291] Embodiment 93: A reaction mixture comprising: (i) a target-binding ligand conjugated with an oligonucleotide primer; ii) a first single-stranded extender template for template directed extension of the first oligonucleotide primer; (iii) a second target-binding ligand conjugated with a second oligonucleotide primer, wherein the second target-binding ligand binds a target different from the first target-binding ligand; (iv) a second single-stranded extender template for template directed extension of the second oligonucleotide primer; (v) a polymerase; (vi) a first labeled-nucleic acid probe comprising a first detectable label and having a nucleotide sequence complementary to at least a portion of the first oligonucleotide primer or the first extender template; (vii) a second labeled-nucleic acid probe comprising a second detectable label and having a nucleotide sequence complementary to at least a portion of the second oligonucleotide primer or the second extender template; and (viii) a set of blocking probes, wherein the detectable label of the first labeled-nucleic acid probe and the second labeled-nucleic acid probe are distinguishable from each other, and optionally, the set of blocking probes does not comprise a blocking probe having a sequence complementary to the first nucleic acid tag and / or the second nucleic acid tag.

[0292] Embodiment 94: The reaction mixture of any one of Embodiments 90-93, wherein the reaction mixture further comprises one or more components for template dependent extension of the oligonucleotide primer with the polymerase.

[0293] Embodiment 95: The reaction mixture of any one of Embodiments 90-94, wherein the reaction mixture further comprises dNTPs.

[0294] Embodiment 96: The reaction mixture of any of one of Embodiments 90-95, wherein the reaction mixture further comprises a buffer for polymerization with the polymerase.

[0295] Embodiment 97: The reaction mixture of any one of Embodiments 86-96, wherein the reaction mixture further comprises a buffer for digestion or cleavage with the nuclease.

[0296] Embodiment 98: The reaction mixture of any one of Embodiments 86-97, wherein the target-binding ligand is bound to the target.

[0297] Embodiment 99: The reaction mixture of any one of Embodiments 86-98, wherein the labeled-nucleic acid probe and the nucleic acid tag are bound or hybridized with each other.

[0298] Embodiment 100: A composition comprising: (i) a target-binding ligand conjugated with a nucleic acid tag; (ii) a labeled-nucleic acid probe, wherein the labeled-nucleic acid probecomprises a detectable label and has a nucleotide sequence complementary to at least a portion of the nucleic acid tag; and (iii) a nuclease.

[0299] Embodiment 101 : A composition comprising: (i) a first target-binding ligand conjugated with a first nucleic acid tag; (ii) a first labeled-nucleic acid probe, wherein the first labeled-nucleic acid probe comprises a detectable label and has a nucleotide sequence complementary to at least a portion of the first nucleic acid tag; (iii) a second target-binding ligand conjugated with a second nucleic acid tag, wherein the second target-binding ligand binds a target different from the first target-binding ligand; (iv) a second labeled-nucleic acid probe, wherein the second labeled-nucleic acid probe comprises a detectable label and has a nucleotide sequence complementary to at least a portion of the second nucleic acid tag; and (v) a nuclease, and wherein the detectable label of the first labeled-nucleic acid probe and the second labeled-nucleic acid probe are distinguishable from each other.

[0300] Embodiment 102: A composition comprising: (i) a target-binding ligand conjugated with a nucleic acid tag; (ii) a labeled-nucleic acid probe, wherein the labeled-nucleic acid probe comprises a detectable label and has a nucleotide sequence complementary to at least a portion of the nucleic acid tag; and (iii) a set of blocking probes, optionally, the set of blocking probes does not comprise a blocking probe having a sequence complementary to the nucleic acid tag conjugated to the target-binding ligand, and optionally, the composition further comprises a nuclease.

[0301] Embodiment 103: A composition comprising: (i) a first target-binding ligand conjugated with a first nucleic acid tag; (ii) a first labeled-nucleic acid probe, wherein the first labeled-nucleic acid probe comprises a detectable label and has a nucleotide sequence complementary to at least a portion of the first nucleic acid tag; (iii) a second target-binding ligand conjugated with a second nucleic acid tag, wherein the second target-binding ligand binds a target different from the first target-binding ligand; (iv) a second labeled-nucleic acid probe, wherein the second labeled-nucleic acid probe comprises a detectable label and has a nucleotide sequence complementary to at least a portion of the second nucleic acid tag; and (v) a set of blocking probes, optionally, the set of blocking probes does not comprise a blocking probe having a sequence complementary to the first nucleic acid tag and / or the second nucleic acid tag, wherein the detectable label of the first labeled-nucleic acid probe and the second labeled-nucleic acid probe are distinguishable from each other, and optionally, the composition further comprises a nuclease.

[0302] Embodiment 104: A composition comprising: (i) a target-binding ligand conjugated with an oligonucleotide primer; (ii) a single-stranded extender template; (iii) a polymerase; (iv)a labeled-nucleic acid probe, wherein the labeled-nucleic acid probe comprises a detectable label and has a nucleotide sequence complementary to at least a portion of the oligonucleotide primer or the extender template; and (v) a nuclease.

[0303] Embodiment 105: A composition comprising: (i) a first target-binding ligand conjugated with a first oligonucleotide primer; (ii) a first single-stranded extender template for template directed extension of the first oligonucleotide primer; (iii) a second target-binding ligand conjugated with a second oligonucleotide primer, wherein the second target-binding ligand binds a target different from the first target-binding ligand; (iv) a second single-stranded extender template for template directed extension of the second oligonucleotide primer; (v) a polymerase; (vi) a first labeled-nucleic acid probe comprising a first detectable label and having a nucleotide sequence complementary to at least a portion of the first oligonucleotide primer or the first extender template; (vii) a second labeled-nucleic acid probe comprising a second detectable label and having a nucleotide sequence complementary to at least a portion of the second oligonucleotide primer or the second extender template; and (viii) a nuclease, and wherein the detectable label of the first labeled-nucleic acid probe and the second labeled- nucleic acid probe are distinguishable from each other.

[0304] Embodiment 106: A composition comprising: (i) a first target-binding ligand conjugated with a first oligonucleotide primer; (ii) a first single-stranded extender template ; (iii) a polymerase; (iv) a labeled-nucleic acid probe, wherein the labeled-nucleic acid probe comprises a detectable label and has a nucleotide sequence complementary to at least a portion of the oligonucleotide primer or the extender template; and (v) a set of blocking probes, optionally, the set of blocking probes does not comprise a blocking probe having a sequence complementary to the nucleic acid tag conjugated to the target-binding ligand.

[0305] Embodiment 107: A composition comprising: (i) a target-binding ligand conjugated with an oligonucleotide primer; ii) a first single-stranded extender template for template directed extension of the first oligonucleotide primer; (iii) a second target-binding ligand conjugated with a second oligonucleotide primer, wherein the second target-binding ligand binds a target different from the first target-binding ligand; (iv) a second single-stranded extender template for template directed extension of the second oligonucleotide primer; (v) a polymerase; (vi) a first labeled-nucleic acid probe comprising a first detectable label and having a nucleotide sequence complementary to at least a portion of the first oligonucleotide primer or the first extender template; (vii) a second labeled-nucleic acid probe comprising a second detectable label and having a nucleotide sequence complementary to at least a portion of the second oligonucleotide primer or the second extender template; and (viii) a set of blockingprobes, wherein the detectable label of the first labeled-nucleic acid probe and the second labeled-nucleic acid probe are distinguishable from each other, and optionally, the set of blocking probes does not comprise a blocking probe having a sequence complementary to the first nucleic acid tag and / or the second nucleic acid tag.

[0306] Embodiment 108: The composition of any one of Embodiments 104-107, wherein the composition further comprises one or more components for template dependent extension of the oligonucleotide primer with the polymerase.

[0307] Embodiment 109: The composition of any one of Embodiments 104-108, wherein the composition further comprises dNTPs.

[0308] Embodiment 110: The composition of any of one of Embodiments 104-109, wherein the composition further comprises a buffer for polymerization with the polymerase.

[0309] Embodiment 111 : The composition of any one of Embodiments 100-110, wherein the composition further comprises a buffer for digestion or cleavage with the nuclease.

[0310] Embodiment 112: The composition of any one of Embodiments 100-111, wherein the target-binding ligand is bound to the target.

[0311] Embodiment 113: The composition of any one of Embodiments 100-112, wherein the labeled-nucleic acid probe and the nucleic acid tag are bound or hybridized with each other.

[0312] Specific elements of any of the disclosed embodiments can be combined or substituted for elements in other embodiments. Furthermore, while advantages associated with certain embodiments of the disclosure have been described in the context of these embodiments, other embodiments may also exhibit such advantages, and not all embodiments need necessarily exhibit such advantages to fall within the scope of the disclosure.Some selected definitions

[0313] For convenience, certain terms employed herein, in the specification, examples and appended claims are collected herein. Unless stated otherwise, or implicit from context, the following terms and phrases include the meanings provided below. Unless explicitly stated otherwise, or apparent from context, the terms and phrases below do not exclude the meaning that the term or phrase has acquired in the art to which it pertains. The definitions are provided to aid in describing particular embodiments, and are not intended to limit the claimed invention, because the scope of the invention is limited only by the claims. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0314] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as those commonly understood to one of ordinary skill in the art to which this invention pertains. Although any known methods, devices, and materials may be used in the practice or testing of the invention, the methods, devices, and materials in this regard are described herein.

[0315] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0316] Certain ranges are presented herein with numerical values being preceded by the term “about.” The term “about” is used herein to provide literal support for the exact number that it precedes, as well as a number that is near to or approximately the number that the term precedes. In determining whether a number is near to or approximately a specifically recited number, the near or approximating unrecited number may be a number which, in the context in which it is presented, provides the substantial equivalent of the specifically recited number.

[0317] As used herein the term “comprising” or “comprises” is used in reference to compositions, methods, and respective component(s) thereof, that are essential to the invention, yet open to the inclusion of unspecified elements, whether essential or not. In other words, except where the context requires otherwise, the term “comprise” and variations of the term, such as “comprising”, “comprises” and “comprised”, are not intended to exclude further additives, components, integers or steps.

[0318] The singular terms “a,” “an,” and “the” include plural referents unless context clearly indicates otherwise. Similarly, the word “or” is intended to include “and” unless the context clearly indicates otherwise. It is further noted that the claims can be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation.

[0319] As used herein, the term “binding” or “bound” generally refers to a reversible binding of one molecule to molecule via, e.g., van der Waals force, hydrophobic force, hydrogenbonding, and / or electrostatic force. The binding interaction between two molecules can be described by a dissociation constant (Ka) or association constant (K).

[0320] As used herein, the term “conjugated” refers to the linkage of, for example, an oligonucleotide to a target-binding ligand in a manner that is stable through steps of thermal cycling to generate concatemers or ordered nucleic acid extension products as described herein. Conjugates can include covalent linkages. In various embodiments, conjugates can include a linker molecule between the target-binding ligand and the conjugated oligonucleotide.

[0321] As used herein, “contacting” refers to any suitable means for delivering, or exposing, an agent, or nucleic acid provided herein to at least one component as provided herein (e.g., sample, a target binding molecule, etc.). In some embodiments, contacting comprises physical human activity, e.g., an injection; an act of dispensing, mixing, and / or decanting; and / or manipulation of a delivery device or machine.

[0322] All patents and other publications identified are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications that might be used in connection with 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 such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicants and does not constitute any admission as to the correctness of the dates or contents of these documents.

Claims

CLAIMSWhat is claimed is:

1. A method of detecting a set of target molecules in situ in a preparation of cells or tissue, the method comprising: a. contacting the preparation of cells or tissue with a first target-binding ligand conjugated to a first nucleic acid tag, under conditions permitting specific binding of the first target-binding ligand to a first target molecule; b. contacting the first nucleic acid tag with a first labeled nucleic acid probe, wherein the first labeled nucleic acid probe comprises a nucleotide sequence complementary to at least a portion of the first nucleic acid tag; c. detecting the labeled nucleic acid probe, wherein detection of the labeled nucleic acid probe indicates the presence and location of the target molecule in the preparation of cells or tissue; d. adding to the preparation of cells or tissue a nuclease under conditions permitting cleavage or digestion of the first nucleic acid tag and / or the labeled nucleic acid probe; e. contacting the preparation of cells or tissue from (d) with a second target-binding ligand conjugated to a second nucleic acid tag, under conditions permitting specific binding of the second target-binding ligand to a second target molecule; f. contacting the second nucleic acid tag with a second labeled nucleic acid probe, , wherein the second labeled nucleic acid probe comprises a nucleotide sequence complementary to at least a portion of the second nucleic acid tag; g. detecting the second labeled nucleic acid probe, wherein detection of the second labeled nucleic acid probe indicates the presence and location of the second target molecule in the preparation of cells or tissue; and h. optionally, repeating steps (d)-(g) one or more times.

2. A method of detecting a set of target molecules in situ in a preparation of cells or tissue, the method comprising: a. contacting the preparation of cells or tissue with a first set of target-binding ligand molecules under conditions permitting specific binding of the target-binding ligand molecules to respective first target molecules present in the preparation of cells or tissue, wherein members of the set of target-binding ligand molecules are conjugated to respective first nucleic acid tag, wherein the first nucleic acid tag of each target-binding ligand has a different sequencefrom other first nucleic acid tags in the set, and is conjugated to a different member of a first set of target-binding ligand molecules; b. contacting the first nucleic acid tags with a first set of distinguishably-labeled nucleic acid probes, wherein each labeled nucleic acid probe in the set comprises a nucleotide sequence complementary to at least a portion of a respective first nucleic acid tag; c. detecting the first labeled nucleic acid probes, wherein detection of the distinguishably- labeled nucleic acid probes indicate the presence and location of the first target molecules in the preparation of cells or tissue; d. adding to the preparation of cells or tissue a nuclease under conditions permitting digestion of the first nucleic acid tags and / or the labeled nucleic acid probes; e. contacting the preparation of cells or tissue with a second set of target-binding ligand molecules under conditions permitting specific binding of the target-binding ligand molecules to respective second target molecules present in the preparation of cells or tissue, wherein members of the set of target-binding ligand molecules are conjugated to respective second nucleic acid tag, wherein the second nucleic acid tag of each target-binding ligand has a different sequence from other second nucleic acid tags in the set, and is conjugated to a different member of a second set of target-binding ligand molecules; f. contacting the first nucleic acid tags with a second set of distinguishably-labeled nucleic acid probes, wherein each labeled nucleic acid probe in the set comprises a nucleotide sequence complementary to at least a portion of a respective second nucleic acid tag; g. detecting the second labeled nucleic acid probes, wherein detection of the distinguishably-labeled nucleic acid probes indicate the presence and location of the second target molecules in the preparation of cells or tissue; and h. optionally, repeating steps (d)-(g).

3. The method of claim 2, wherein the step of contacting the first nucleic acid tags with the first set of distinguishably-labeled nucleic acid probes comprises contacting the first nucleic acid tags with a set of probe, wherein the first set of probes comprises a first set of blocking- nucleic acid probes and the first set of distinguishably-labeled nucleic acid probes, wherein each blocking-nucleic acid probe has a different sequence from other blocking-nucleic acid probes and said sequence is complementary to at least a portion of the nucleic acid tag of a different member of the set of target-binding ligand molecules, and wherein a concentration of the blocking-nucleic acid probes in the first set of blocking-nucleic acid probes is lower than a concentration of the labeled nucleic acid probes in the first set of blocking-nucleic acid probes.

4. The method of claim 2, wherein the step of contacting the first nucleic acid tags with the first set of distinguishably-labeled nucleic acid probes comprises contacting the first nucleic acid tags with a set of probe, wherein the first set of probes comprises a first set of blocking- nucleic acid probes and the first set of distinguishably-labeled nucleic acid probes, wherein each blocking-nucleic acid probe has a different sequence from other blocking-nucleic acid probes and said sequence is complementary to at least a portion of the nucleic acid tag of a different member of the set of target-binding ligand molecules, and wherein the set of blocking-nucleic acid probes does not comprise a blocking-nucleic acid probe having a sequence complementary to the nucleic acid tag having a sequence complementary to the distinguishably-labeled nucleic acid probes.

5. The method of any one of claims 2-4, wherein the step of contacting the second nucleic acid tags with the second set of distinguishably-labeled nucleic acid probes comprises contacting the second nucleic acid tags with a set of probe, wherein the second set of probes comprises a second set of blocking-nucleic acid probes and the second set of distinguishably- labeled nucleic acid probes, wherein each blocking-nucleic acid probe has a different sequence from other blocking-nucleic acid probes and said sequence is complementary to at least a portion of the nucleic acid tag of a different member of the set of target-binding ligand molecules, and wherein a concentration of the blocking-nucleic acid probes in the second set of blocking- nucleic acid probes is lower than a concentration of the labeled nucleic acid probes in the second set of blocking-nucleic acid probes.

6. The method of any one of claims 2-4, wherein the step of contacting the second nucleic acid tags with the second set of distinguishably-labeled nucleic acid probes comprises contacting the second nucleic acid tags with a set of probe, wherein the second set of probes comprises a second set of blocking-nucleic acid probes and the second set of distinguishably- labeled nucleic acid probes, wherein each blocking-nucleic acid probe has a different sequence from other blocking-nucleic acid probes and said sequence is complementary to at least a portion of the nucleic acid tag of a different member of the set of target-binding ligand molecules, and wherein the set of blocking-nucleic acid probes does not comprise a blocking-nucleic acid probe having a sequence complementary to the nucleic acid tag having a sequence complementary to the distinguishably-labeled nucleic acid probes.

7. The method of any one of claims 1-6, wherein the first and second target molecules are different.

8. The method of any one of claims 1-6, wherein the first and second target molecules are same.

9. The method of any one of claims 1-8, wherein the first or second target-binding ligand comprises a protein.

10. The method of any one of claims 1-9, wherein the first or second target-binding ligand comprises an antibody or antigen-binding fragment thereof.

11. The method of any one of claims 1-8, wherein the first or second target-binding ligand comprises RNA.

12. The method of any one of claims 1-11, wherein the first or second nucleic acid tag is DNA.

13. The method of any one of claims 1-12, wherein the nuclease is a DNase.

14. The method of claim 13, wherein the first nucleic acid tag is DNA conjugated with a protein.

15. The method of claim 13, wherein the first nucleic acid tag is DNA conjugated with the antibody or antigen-binding fragment thereof.

16. The method of claim 13, wherein the first nucleic acid tag is DNA conjugated with an RNA.

17. The method of any one of claims 1-12, wherein the first target-binding ligand is an RNA.

18. The method of claim 17, wherein the nuclease is an RNase.

19. The method of claim 18, wherein the first target-binding ligand is an RNA.

20. The method of claim 19, wherein the second target-binding ligand comprises protein.

21. The method of claim 20, wherein the second nucleic acid tag is DNA conjugated with protein.

22. The method of claim 20, wherein the second nucleic acid tag is DNA conjugated with an antibody or antigen-binding fragment thereof.

23. The method of claim 20, wherein the second nucleic acid tag is DNA conjugated with an RNA.

24. The method of any one of claims 1-23, wherein the first nucleic acid tag is a concatemer comprising repeats of a first oligonucleotide primer sequence.

25. The method of claim 24, wherein the step of contacting the preparation of cells or tissue with the first target-binding ligand comprises:i. contacting the preparation of cells or tissue with a first target-binding ligand conjugated to a first oligonucleotide primer, under conditions permitting specific binding of the first target-binding ligand to the target molecule; ii. adding to the preparation of cells or tissue a reaction mixture comprising a first singlestranded extender template and a nucleic acid polymerase enzyme under conditions permitting hybridization and extension of the first oligonucleotide primer using the first single-stranded extender template; iii. heating the reaction mixture of step (ii) to separate the first single-stranded extender template from extended first oligonucleotide primer produced in step (ii); and iv. repeating steps (ii) and (iii) at least once, thereby generating a concatemer comprising repeats of the first oligonucleotide primer sequence conjugated to the first target-binding ligand.

26. The method of any one of claims 1-23, wherein the second nucleic acid tag is a concatemer comprising repeats of a second oligonucleotide primer sequence.

27. The method of claim 26, wherein the step of contacting the preparation of cells or tissue with the second target-binding ligand comprises: i. contacting the preparation of cells or tissue with a second target-binding ligand conjugated to a second oligonucleotide primer, under conditions permitting specific binding of the second target-binding ligand to the target molecule; ii. adding to the preparation of cells or tissue a reaction mixture comprising a second singlestranded extender template and a nucleic acid polymerase enzyme under conditions permitting hybridization and extension of the second oligonucleotide primer using the second single-stranded extender template; iii. heating the reaction mixture of step (ii) to separate the second single-stranded extender template from extended second oligonucleotide primer produced in step (ii); and iv. repeating steps (ii) and (iii) at least once, thereby generating a concatemer comprising repeats of the second oligonucleotide primer sequence conjugated to the second targetbinding ligand.

28. The method of any one of claims 1-27, wherein the first nucleic acid tag is a branched nucleic acid, optionally, the first nucleic acid tag is a branched DNA.

29. The method of any one of claims 1-28, wherein a plurality of labeled nucleic acid probe molecules hybridizes to first or second nucleic acid tag.

30. The method of any one of claims 1-28, wherein a single labeled nucleic acid probe molecules hybridizes to first or second nucleic acid tag.

31. A method of detecting a set of target molecules in situ in a preparation of cells or tissue, the method comprising: a. contacting the preparation of cells or tissue with a set of target-binding ligand molecules under conditions permitting specific binding of the target-binding ligand molecules to target molecules present in the preparation of cells or tissue, wherein members of the set of targetbinding ligand molecules are conjugated to respective nucleic acid tag, wherein the nucleic acid tag of each target-binding ligand has a different sequence from other first nucleic acid tags in the set; b. contacting the nucleic acid tags with a first set of probes, wherein the first set of probes comprises a set of blocking-nucleic acid probes and a first labeled-nucleic acid probe, wherein each blocking-nucleic acid probe has a different sequence from other blocking probes and said sequence is complementary to at least a portion of the nucleic acid tag of a different member of the set of target-binding ligand molecules, wherein the first labeled-nucleic acid probe comprises a sequence complementary to at least a portion of the nucleic acid tag conjugated to a first target-binding ligand in the set of targetbinding ligand molecules and the sequence of the first labeled-nucleic acid probe is identical to the sequence of a first blocking-nucleic acid probe in the set of blocking-nucleic acid probes, wherein a concentration of the blocking-nucleic acid probes is lower than a concentration of the first labeled nucleic acid probe in the first set of blocking-nucleic acid probes; c. detecting the first labeled nucleic acid probe, wherein detection of the first labeled nucleic acid probe indicates the presence and location of a first target molecule in the preparation of cells or tissue; d. contacting the nucleic acid tags with a second set of probes, wherein the second set of probes comprises a set of blocking-nucleic acid probes and a second labeled-nucleic acid probe, wherein each blocking-nucleic acid probe has a different sequence from other blocking probes and said sequence is complementary to at least a portion of the nucleic acid tag of a different member of the set of target-binding ligand molecules, wherein the second labeled-nucleic acid probe comprises a sequence complementary to at least a portion of the nucleic acid tag conjugated to a second target-binding ligand in the set of targetbinding ligand molecules and the sequence of the second labeled-nucleic acid probe is identical to the sequence of a second blocking-nucleic acid probe in the set of blocking-nucleic acid probes, andoptionally, wherein a concentration of the blocking-nucleic acid probes is lower than a concentration of the second labeled nucleic acid probe in the second set of blocking-nucleic acid probes; e. detecting the second labeled nucleic acid probe, wherein detection of the second labeled nucleic acid probe indicates the presence and location of a second target molecule in the preparation of cells or tissue; and f. optionally, repeating steps (d)-(e) one or more times.

32. A method of detecting a set of target molecules in situ in a preparation of cells or tissue, the method comprising: a. contacting the preparation of cells or tissue with a set of target-binding ligand molecules under conditions permitting specific binding of the target-binding ligand molecules to target molecules present in the preparation of cells or tissue, wherein members of the set of targetbinding ligand molecules are conjugated to respective nucleic acid tag, wherein the nucleic acid tag of each target-binding ligand has a different sequence from other first nucleic acid tags in the set; b. contacting the nucleic acid tags with a first set of probes, wherein the first set of probes comprises a set of blocking-nucleic acid probes and a first labeled-nucleic acid probe, wherein each blocking-nucleic acid probe has a different sequence from other blocking-nucleic acid probes and said sequence is complementary to at least a portion of the nucleic acid tag of a different member of the set of target-binding ligand molecules, wherein the first labeled-nucleic acid probe comprises a sequence complementary to at least a portion of the nucleic acid tag conjugated to a first target-binding ligand in the set of targetbinding ligand molecules, and wherein the set of blocking-nucleic acid probes does not comprise a blocking-nucleic acid probe having a sequence complementary to the nucleic acid tag conjugated to the first targetbinding ligand; c. detecting the first labeled nucleic acid probe, wherein detection of the first labeled nucleic acid probe indicates the presence and location of a first target molecule in the preparation of cells or tissue; d. contacting the nucleic acid tags with a second set of probes, wherein the second set of probes comprises a set of blocking-nucleic acid probes and a second labeled-nucleic acid probe, wherein each blocking-nucleic acid probe has a different sequence from other blocking-nucleic acid probes and said sequence is complementary to at least a portion of the nucleic acid tag of a different member of the set of target-binding ligand molecules,wherein the second labeled-nucleic acid probe comprises a sequence complementary to at least a portion of the nucleic acid tag conjugated to a second target-binding ligand in the set of targetbinding ligand molecules, and wherein the set of blocking-nucleic acid probes does not comprise a blocking-nucleic acid probe having a sequence complementary to the nucleic acid tag conjugated to the second targetbinding ligand; e. detecting the second labeled nucleic acid probe, wherein detection of the second labeled nucleic acid probe indicates the presence and location of a second target molecule in the preparation of cells or tissue; and f. optionally, repeating steps (d)-(e) one or more times.

33. The method of claim 31 or 32, wherein a concentration of the blocking probes is higher than a concentration of the labeled-nucleic acid probes.

34. The method of claim 31 or 32, wherein a concentration of the blocking probes is lower than a concentration of the labeled-nucleic acid probes.

35. The method of any one of claims 31-34, wherein at least one, e.g., each nucleic acid tag is a concatemer comprising repeats of a respective first oligonucleotide primer sequence.

36. The method of claim 35, wherein the step of contacting the preparation of cells or tissue with a set of target-binding ligand molecules comprises: i. contacting the preparation of cells or tissue with a set of target-binding ligand molecules under conditions permitting specific binding of the target-binding ligand molecules to target molecules present in the preparation of cells or tissue, wherein members of the set of targetbinding ligand molecules are conjugated to respective first oligonucleotide primer members of a set of orthogonal first oligonucleotide primer and first single-stranded extender template oligonucleotide pairs, wherein the first oligonucleotide primer of each pair has a different sequence from other first oligonucleotide primers in the set, and is conjugated to a different member of the set of target-binding ligand molecules; ii. contacting the preparation of cells or tissue with the set of target-binding ligand molecules under conditions permitting specific binding of the target-binding ligand molecules to target molecules present in the preparation of cells or tissue; iii. adding to the preparation of cells or tissue a reaction mixture comprising first singlestranded extender templates of the set of orthogonal first oligonucleotide primer and first single-stranded extender template oligonucleotide pairs, and a nucleic acid polymerase, under conditions permitting hybridization and extension of the first oligonucleotide primers using the respective first single-stranded extender templates;iv. heating the reaction mixture of step (iii) to separate the first single-stranded extender templates from extended first oligonucleotide primers produced in step (iii); and v. repeating steps (iii) and (iv) at least once, thereby generating, on respective members of the set of target-binding ligand molecules, a concatemer comprising repeats of the respective first oligonucleotide primer sequence, conjugated to the target-binding ligand molecule.

37. The method of any one of claims 31-34, wherein at least one, e.g., each nucleic acid tag is a branched nucleic acid, optionally, the branched nucleic acid is DNA.

38. The method of any one of claims 31-37, wherein at least one, e.g., each target-binding ligand comprises a protein.

39. The method of any one of claims 31-37, wherein at least one, e.g., each target-binding ligand comprises an antibody or antigen-binding fragment thereof.

40. The method of any one of claims 31-37, wherein at least one, e.g., each target-binding ligand comprises RNA.

41. The method of any one of claims 31-40, wherein a plurality of labeled nucleic acid probe molecules hybridizes to a plurality of concatemer repeats conjugated to the targetbinding ligands.

42. The method of any one of claims 1-41, wherein the preparation of cells or tissue is fixed.

43. The method of claim 42, wherein the preparation of tissue is paraffin embedded.

44. A kit comprising: (i) a target-binding ligand conjugated with a nucleic acid tag; (ii) a labeled-nucleic acid probe, wherein the labeled-nucleic acid probe comprises a detectable label and has a nucleotide sequence complementary to at least a portion of the nucleic acid tag; and (iii) a nuclease.

45. A kit comprising: (i) a first target-binding ligand conjugated with a first nucleic acid tag; (ii) a first labeled-nucleic acid probe, wherein the first labeled-nucleic acid probe comprises a detectable label and has a nucleotide sequence complementary to at least a portion of the first nucleic acid tag; (iii) a second target-binding ligand conjugated with a second nucleic acid tag, wherein the second target-binding ligand binds a target different from the first target-binding ligand; (iv) a second labeled-nucleic acid probe, wherein the second labeled- nucleic acid probe comprises a detectable label and has a nucleotide sequence complementary to at least a portion of the second nucleic acid tag; and (v) a nuclease.

46. The kit of claim 45, wherein the detectable label of the first labeled-nucleic acid probe and the second labeled-nucleic acid probe are distinguishable from each other.

47. The kit of claim 45, wherein the detectable label of the first labeled-nucleic acid probe and the second labeled-nucleic acid probe are the same.

48. A kit comprising: (i) a target-binding ligand conjugated with a nucleic acid tag; (ii) a labeled-nucleic acid probe, wherein the labeled-nucleic acid probe comprises a detectable label and has a nucleotide sequence complementary to at least a portion of the nucleic acid tag; and (iii) a set of blocking probes, optionally, the set of blocking probes does not comprise a blocking probe having a sequence complementary to the nucleic acid tag conjugated to the target-binding ligand, and optionally, the kit further comprises a nuclease.

49. A kit comprising: (i) a first target-binding ligand conjugated with a first nucleic acid tag; (ii) a first labeled-nucleic acid probe, wherein the first labeled-nucleic acid probe comprises a detectable label and has a nucleotide sequence complementary to at least a portion of the first nucleic acid tag; (iii) a second target-binding ligand conjugated with a second nucleic acid tag, wherein the second target-binding ligand binds a target different from the first target-binding ligand; (iv) a second labeled-nucleic acid probe, wherein the second labeled- nucleic acid probe comprises a detectable label and has a nucleotide sequence complementary to at least a portion of the second nucleic acid tag; and (v) a set of blocking probes, optionally, the set of blocking probes does not comprise a blocking probe having a sequence complementary to the first nucleic acid tag and / or the second nucleic acid tag, and optionally, the kit further comprises a nuclease.

50. The kit of claim 49, wherein the detectable label of the first labeled-nucleic acid probe and the second labeled-nucleic acid probe are distinguishable from each other.

51. The kit of claim 49, wherein the detectable label of the first labeled-nucleic acid probe and the second labeled-nucleic acid probe are the same.

52. A kit comprising: (i) a target-binding ligand conjugated with an oligonucleotide primer; (ii) a single-stranded extender template; (iii) a polymerase; (iv) a labeled-nucleic acid probe, wherein the labeled-nucleic acid probe comprises a detectable label and has a nucleotide sequence complementary to at least a portion of the oligonucleotide primer or the extender template; and (v) a nuclease.

53. A kit comprising: (i) a first target-binding ligand conjugated with a first oligonucleotide primer; (ii) a first single-stranded extender template for template directed extension of the first oligonucleotide primer; (iii) a second target-binding ligand conjugated with a second oligonucleotide primer, wherein the second target-binding ligand binds a target different from the first target-binding ligand; (iv) a second single-stranded extender template for template directed extension of the second oligonucleotide primer; (v) a polymerase; (vi) a first labeled-nucleic acid probe comprising a first detectable label and having a nucleotide sequence complementary to at least a portion of the first oligonucleotide primer or the first extender template; (vii) a second labeled-nucleic acid probe comprising a second detectable label and having a nucleotide sequence complementary to at least a portion of the second oligonucleotide primer or the second extender template; and (viii) a nuclease.

54. The kit of claim 53, wherein the detectable label of the first labeled-nucleic acid probe and the second labeled-nucleic acid probe are distinguishable from each other.

55. The kit of claim 53, wherein the detectable label of the first labeled-nucleic acid probe and the second labeled-nucleic acid probe are the same.

56. A kit comprising: (i) a first target-binding ligand conjugated with a first oligonucleotide primer; (ii) a first single-stranded extender template ; (iii) a polymerase; (iv) a labeled-nucleic acid probe, wherein the labeled-nucleic acid probe comprises a detectable label and has a nucleotide sequence complementary to at least a portion of the oligonucleotide primer or the extender template; and (v) a set of blocking probes, optionally, the set of blocking probes does not comprise a blocking probe having a sequence complementary to the nucleic acid tag conjugated to the target-binding ligand.

57. A kit comprising: (i) a target-binding ligand conjugated with an oligonucleotide primer; ii) a first single-stranded extender template for template directed extension of the first oligonucleotide primer; (iii) a second target-binding ligand conjugated with a second oligonucleotide primer, wherein the second target-binding ligand binds a target different from the first target-binding ligand; (iv) a second single-stranded extender template for template directed extension of the second oligonucleotide primer; (v) a polymerase; (vi) a first labeled- nucleic acid probe comprising a first detectable label and having a nucleotide sequence complementary to at least a portion of the first oligonucleotide primer or the first extender template; (vii) a second labeled-nucleic acid probe comprising a second detectable label and having a nucleotide sequence complementary to at least a portion of the second oligonucleotide primer or the second extender template; and (viii) a set of blocking probes, optionally, the set of blocking probes does not comprise a blocking probe having a sequence complementary to the first nucleic acid tag and / or the second nucleic acid tag.

58. The kit of claim 57, wherein the detectable label of the first labeled-nucleic acid probe and the second labeled-nucleic acid probe are distinguishable from each other.

59. The kit of claim 57, wherein the detectable label of the first labeled-nucleic acid probe and the second labeled-nucleic acid probe are the same.

60. The kit of any one of claims 52-59, wherein the kit further comprises one or more components for template dependent extension of the oligonucleotide primer with the polymerase.

61. The kit of any one of claims 52-60, wherein the kit further comprises dNTPs.

62. The kit of any of one of claims 52-61, wherein the kit further comprises a buffer for polymerization with the polymerase.

63. The kit of any one of claims 44-62, wherein the kit further comprises a buffer for digestion or cleavage with the nuclease.

64. The kit of any one of claims 44-63, wherein the kit further comprises means for detecting or imagining a signal produced by the detectable label.

65. A system for detecting targets in a sample, comprising: (i) a target-binding ligand conjugated with a nucleic acid tag; (ii) a labeled-nucleic acid probe, wherein the labeled- nucleic acid probe comprises a detectable label and has a nucleotide sequence complementary to at least a portion of the nucleic acid tag; and (iii) a nuclease.

66. A system comprising: (i) a first target-binding ligand conjugated with a first nucleic acid tag; (ii) a first labeled-nucleic acid probe, wherein the first labeled-nucleic acid probe comprises a detectable label and has a nucleotide sequence complementary to at least a portion of the first nucleic acid tag; (iii) a second target-binding ligand conjugated with a second nucleic acid tag, wherein the second target-binding ligand binds a target different from the first target-binding ligand; (iv) a second labeled-nucleic acid probe, wherein the second labeled- nucleic acid probe comprises a detectable label and has a nucleotide sequence complementary to at least a portion of the second nucleic acid tag; and (v) a nuclease.

67. The system of claim 66, wherein the detectable label of the first labeled-nucleic acid probe and the second labeled-nucleic acid probe are distinguishable from each other.

68. The system of claim 66, wherein the detectable label of the first labeled-nucleic acid probe and the second labeled-nucleic acid probe are the same.

69. A system comprising: (i) a target-binding ligand conjugated with a nucleic acid tag; (ii) a labeled-nucleic acid probe, wherein the labeled-nucleic acid probe comprises a detectable label and has a nucleotide sequence complementary to at least a portion of the nucleic acid tag; and (iii) a set of blocking probes, optionally, the set of blocking probes does not comprise a blocking probe having a sequence complementary to the nucleic acid tag conjugated to the target-binding ligand, and optionally, the system further comprises a nuclease.

70. A system comprising: (i) a first target-binding ligand conjugated with a first nucleic acid tag; (ii) a first labeled-nucleic acid probe, wherein the first labeled-nucleic acid probecomprises a detectable label and has a nucleotide sequence complementary to at least a portion of the first nucleic acid tag; (iii) a second target-binding ligand conjugated with a second nucleic acid tag, wherein the second target-binding ligand binds a target different from the first target-binding ligand; (iv) a second labeled-nucleic acid probe, wherein the second labeled- nucleic acid probe comprises a detectable label and has a nucleotide sequence complementary to at least a portion of the second nucleic acid tag; and (v) a set of blocking probes, optionally, the set of blocking probes does not comprise a blocking probe having a sequence complementary to the first nucleic acid tag and / or the second nucleic acid tag, and optionally, the system further comprises a nuclease.

71. The system of claim 70, wherein the detectable label of the first labeled-nucleic acid probe and the second labeled-nucleic acid probe are distinguishable from each other.

72. The system of claim 70, wherein the detectable label of the first labeled-nucleic acid probe and the second labeled-nucleic acid probe are the same.

73. A system comprising: (i) a target-binding ligand conjugated with an oligonucleotide primer; (ii) a single-stranded extender template; (iii) a polymerase; (iv) a labeled-nucleic acid probe, wherein the labeled-nucleic acid probe comprises a detectable label and has a nucleotide sequence complementary to at least a portion of the oligonucleotide primer or the extender template; and (v) a nuclease.

74. A system comprising: (i) a first target-binding ligand conjugated with a first oligonucleotide primer; (ii) a first single-stranded extender template for template directed extension of the first oligonucleotide primer; (iii) a second target-binding ligand conjugated with a second oligonucleotide primer, wherein the second target-binding ligand binds a target different from the first target-binding ligand; (iv) a second single-stranded extender template for template directed extension of the second oligonucleotide primer; (v) a polymerase; (vi) a first labeled-nucleic acid probe comprising a first detectable label and having a nucleotide sequence complementary to at least a portion of the first oligonucleotide primer or the first extender template; (vii) a second labeled-nucleic acid probe comprising a second detectable label and having a nucleotide sequence complementary to at least a portion of the second oligonucleotide primer or the second extender template; and (viii) a nuclease.

75. The system of claim 74, wherein the detectable label of the first labeled-nucleic acid probe and the second labeled-nucleic acid probe are distinguishable from each other.

76. The system of claim 74, wherein the detectable label of the first labeled-nucleic acid probe and the second labeled-nucleic acid probe are the same.

77. A system comprising: (i) a first target-binding ligand conjugated with a first oligonucleotide primer; (ii) a first single-stranded extender template ; (iii) a polymerase; (iv) a labeled-nucleic acid probe, wherein the labeled-nucleic acid probe comprises a detectable label and has a nucleotide sequence complementary to at least a portion of the oligonucleotide primer or the extender template; and (v) a set of blocking probes, optionally, the set of blocking probes does not comprise a blocking probe having a sequence complementary to the nucleic acid tag conjugated to the target-binding ligand.

78. A system comprising: (i) a target-binding ligand conjugated with an oligonucleotide primer; ii) a first single-stranded extender template for template directed extension of the first oligonucleotide primer; (iii) a second target-binding ligand conjugated with a second oligonucleotide primer, wherein the second target-binding ligand binds a target different from the first target-binding ligand; (iv) a second single-stranded extender template for template directed extension of the second oligonucleotide primer; (v) a polymerase; (vi) a first labeled- nucleic acid probe comprising a first detectable label and having a nucleotide sequence complementary to at least a portion of the first oligonucleotide primer or the first extender template; (vii) a second labeled-nucleic acid probe comprising a second detectable label and having a nucleotide sequence complementary to at least a portion of the second oligonucleotide primer or the second extender template; and (viii) a set of blocking probes, optionally, the set of blocking probes does not comprise a blocking probe having a sequence complementary to the first nucleic acid tag and / or the second nucleic acid tag.

79. The system of claim 78, wherein the detectable label of the first labeled-nucleic acid probe and the second labeled-nucleic acid probe are distinguishable from each other.

80. The system of claim 78, wherein the detectable label of the first labeled-nucleic acid probe and the second labeled-nucleic acid probe are the same.

81. The system of any one of claims 73-80, wherein the system further comprises one or more components for template dependent extension of the oligonucleotide primer with the polymerase.

82. The system of any one of claims 73-81, wherein the system further comprises dNTPs.

83. The system of any of one of claims 73-82, wherein the system further comprises a buffer for polymerization with the polymerase.

84. The system of any one of claims 65-83, wherein the system further comprises a buffer for digestion or cleavage with the nuclease.

85. The system of any one of claims 65-84, wherein the system further comprises means for detecting or imagining a signal produced by the detectable label.

86. A reaction mixture comprising: (i) a target-binding ligand conjugated with a nucleic acid tag; (ii) a labeled-nucleic acid probe, wherein the labeled-nucleic acid probe comprises a detectable label and has a nucleotide sequence complementary to at least a portion of the nucleic acid tag; and (iii) a nuclease.

87. A reaction mixture comprising: (i) a first target-binding ligand conjugated with a first nucleic acid tag; (ii) a first labeled-nucleic acid probe, wherein the first labeled-nucleic acid probe comprises a detectable label and has a nucleotide sequence complementary to at least a portion of the first nucleic acid tag; (iii) a second target-binding ligand conjugated with a second nucleic acid tag, wherein the second target-binding ligand binds a target different from the first target-binding ligand; (iv) a second labeled-nucleic acid probe, wherein the second labeled-nucleic acid probe comprises a detectable label and has a nucleotide sequence complementary to at least a portion of the second nucleic acid tag; and (v) a nuclease, and wherein the detectable label of the first labeled-nucleic acid probe and the second labeled- nucleic acid probe are distinguishable from each other.

88. A reaction mixture comprising: (i) a target-binding ligand conjugated with a nucleic acid tag; (ii) a labeled-nucleic acid probe, wherein the labeled-nucleic acid probe comprises a detectable label and has a nucleotide sequence complementary to at least a portion of the nucleic acid tag; and (iii) a set of blocking probes, optionally, the set of blocking probes does not comprise a blocking probe having a sequence complementary to the nucleic acid tag conjugated to the target-binding ligand, and optionally, the reaction mixture further comprises a nuclease.

89. A reaction mixture comprising: (i) a first target-binding ligand conjugated with a first nucleic acid tag; (ii) a first labeled-nucleic acid probe, wherein the first labeled-nucleic acid probe comprises a detectable label and has a nucleotide sequence complementary to at least a portion of the first nucleic acid tag; (iii) a second target-binding ligand conjugated with a second nucleic acid tag, wherein the second target-binding ligand binds a target different from the first target-binding ligand; (iv) a second labeled-nucleic acid probe, wherein the second labeled-nucleic acid probe comprises a detectable label and has a nucleotide sequence complementary to at least a portion of the second nucleic acid tag; and (v) a set of blocking probes, optionally, the set of blocking probes does not comprise a blocking probe having a sequence complementary to the first nucleic acid tag and / or the second nucleic acid tag, wherein the detectable label of the first labeled-nucleic acid probe and the second labeled- nucleic acid probe are distinguishable from each other, and optionally, the reaction mixture further comprises a nuclease.

90. A reaction mixture comprising: (i) a target-binding ligand conjugated with an oligonucleotide primer; (ii) a single-stranded extender template; (iii) a polymerase; (iv) a labeled-nucleic acid probe, wherein the labeled-nucleic acid probe comprises a detectable label and has a nucleotide sequence complementary to at least a portion of the oligonucleotide primer or the extender template; and (v) a nuclease.

91. A reaction mixture comprising: (i) a first target-binding ligand conjugated with a first oligonucleotide primer; (ii) a first single-stranded extender template for template directed extension of the first oligonucleotide primer; (iii) a second target-binding ligand conjugated with a second oligonucleotide primer, wherein the second target-binding ligand binds a target different from the first target-binding ligand; (iv) a second single-stranded extender template for template directed extension of the second oligonucleotide primer; (v) a polymerase; (vi) a first labeled-nucleic acid probe comprising a first detectable label and having a nucleotide sequence complementary to at least a portion of the first oligonucleotide primer or the first extender template; (vii) a second labeled-nucleic acid probe comprising a second detectable label and having a nucleotide sequence complementary to at least a portion of the second oligonucleotide primer or the second extender template; and (viii) a nuclease, and wherein the detectable label of the first labeled-nucleic acid probe and the second labeled-nucleic acid probe are distinguishable from each other.

92. A reaction mixture comprising: (i) a first target-binding ligand conjugated with a first oligonucleotide primer; (ii) a first single-stranded extender template ; (iii) a polymerase; (iv) a labeled-nucleic acid probe, wherein the labeled-nucleic acid probe comprises a detectable label and has a nucleotide sequence complementary to at least a portion of the oligonucleotide primer or the extender template; and (v) a set of blocking probes, optionally, the set of blocking probes does not comprise a blocking probe having a sequence complementary to the nucleic acid tag conjugated to the target-binding ligand.

93. A reaction mixture comprising: (i) a target-binding ligand conjugated with an oligonucleotide primer; ii) a first single-stranded extender template for template directed extension of the first oligonucleotide primer; (iii) a second target-binding ligand conjugated with a second oligonucleotide primer, wherein the second target-binding ligand binds a target different from the first target-binding ligand; (iv) a second single-stranded extender template for template directed extension of the second oligonucleotide primer; (v) a polymerase; (vi) a first labeled-nucleic acid probe comprising a first detectable label and having a nucleotide sequence complementary to at least a portion of the first oligonucleotide primer or the first extender template; (vii) a second labeled-nucleic acid probe comprising a second detectablelabel and having a nucleotide sequence complementary to at least a portion of the second oligonucleotide primer or the second extender template; and (viii) a set of blocking probes, wherein the detectable label of the first labeled-nucleic acid probe and the second labeled- nucleic acid probe are distinguishable from each other, and optionally, the set of blocking probes does not comprise a blocking probe having a sequence complementary to the first nucleic acid tag and / or the second nucleic acid tag.

94. The reaction mixture of any one of claims 90-93, wherein the reaction mixture further comprises one or more components for template dependent extension of the oligonucleotide primer with the polymerase.

95. The reaction mixture of any one of claims 90-94, wherein the reaction mixture further comprises dNTPs.

96. The reaction mixture of any of one of claims 90-95, wherein the reaction mixture further comprises a buffer for polymerization with the polymerase.

97. The reaction mixture of any one of claims 86-96, wherein the reaction mixture further comprises a buffer for digestion or cleavage with the nuclease.

98. The reaction mixture of any one of claims 86-97, wherein the target-binding ligand is bound to the target.

99. The reaction mixture of any one of claims 86-98, wherein the labeled-nucleic acid probe and the nucleic acid tag are bound or hybridized with each other.

100. A composition comprising: (i) a target-binding ligand conjugated with a nucleic acid tag; (ii) a labeled-nucleic acid probe, wherein the labeled-nucleic acid probe comprises a detectable label and has a nucleotide sequence complementary to at least a portion of the nucleic acid tag; and (iii) a nuclease.

101. A composition comprising: (i) a first target-binding ligand conjugated with a first nucleic acid tag; (ii) a first labeled-nucleic acid probe, wherein the first labeled-nucleic acid probe comprises a detectable label and has a nucleotide sequence complementary to at least a portion of the first nucleic acid tag; (iii) a second target-binding ligand conjugated with a second nucleic acid tag, wherein the second target-binding ligand binds a target different from the first target-binding ligand; (iv) a second labeled-nucleic acid probe, wherein the second labeled-nucleic acid probe comprises a detectable label and has a nucleotide sequence complementary to at least a portion of the second nucleic acid tag; and (v) a nuclease, and wherein the detectable label of the first labeled-nucleic acid probe and the second labeled- nucleic acid probe are distinguishable from each other.

102. A composition comprising: (i) a target-binding ligand conjugated with a nucleic acid tag; (ii) a labeled-nucleic acid probe, wherein the labeled-nucleic acid probe comprises a detectable label and has a nucleotide sequence complementary to at least a portion of the nucleic acid tag; and (iii) a set of blocking probes, optionally, the set of blocking probes does not comprise a blocking probe having a sequence complementary to the nucleic acid tag conjugated to the target-binding ligand, and optionally, the composition further comprises a nuclease.

103. A composition comprising: (i) a first target-binding ligand conjugated with a first nucleic acid tag; (ii) a first labeled-nucleic acid probe, wherein the first labeled-nucleic acid probe comprises a detectable label and has a nucleotide sequence complementary to at least a portion of the first nucleic acid tag; (iii) a second target-binding ligand conjugated with a second nucleic acid tag, wherein the second target-binding ligand binds a target different from the first target-binding ligand; (iv) a second labeled-nucleic acid probe, wherein the second labeled-nucleic acid probe comprises a detectable label and has a nucleotide sequence complementary to at least a portion of the second nucleic acid tag; and (v) a set of blocking probes, optionally, the set of blocking probes does not comprise a blocking probe having a sequence complementary to the first nucleic acid tag and / or the second nucleic acid tag, wherein the detectable label of the first labeled-nucleic acid probe and the second labeled- nucleic acid probe are distinguishable from each other, and optionally, the composition further comprises a nuclease.

104. A composition comprising: (i) a target-binding ligand conjugated with an oligonucleotide primer; (ii) a single-stranded extender template; (iii) a polymerase; (iv) a labeled-nucleic acid probe, wherein the labeled-nucleic acid probe comprises a detectable label and has a nucleotide sequence complementary to at least a portion of the oligonucleotide primer or the extender template; and (v) a nuclease.

105. A composition comprising: (i) a first target-binding ligand conjugated with a first oligonucleotide primer; (ii) a first single-stranded extender template for template directed extension of the first oligonucleotide primer; (iii) a second target-binding ligand conjugated with a second oligonucleotide primer, wherein the second target-binding ligand binds a target different from the first target-binding ligand; (iv) a second single-stranded extender template for template directed extension of the second oligonucleotide primer; (v) a polymerase; (vi) a first labeled-nucleic acid probe comprising a first detectable label and having a nucleotide sequence complementary to at least a portion of the first oligonucleotide primer or the first extender template; (vii) a second labeled-nucleic acid probe comprising a second detectablelabel and having a nucleotide sequence complementary to at least a portion of the second oligonucleotide primer or the second extender template; and (viii) a nuclease, and wherein the detectable label of the first labeled-nucleic acid probe and the second labeled-nucleic acid probe are distinguishable from each other.

106. A composition comprising: (i) a first target-binding ligand conjugated with a first oligonucleotide primer; (ii) a first single-stranded extender template ; (iii) a polymerase; (iv) a labeled-nucleic acid probe, wherein the labeled-nucleic acid probe comprises a detectable label and has a nucleotide sequence complementary to at least a portion of the oligonucleotide primer or the extender template; and (v) a set of blocking probes, optionally, the set of blocking probes does not comprise a blocking probe having a sequence complementary to the nucleic acid tag conjugated to the target-binding ligand.

107. A composition comprising: (i) a target-binding ligand conjugated with an oligonucleotide primer; ii) a first single-stranded extender template for template directed extension of the first oligonucleotide primer; (iii) a second target-binding ligand conjugated with a second oligonucleotide primer, wherein the second target-binding ligand binds a target different from the first target-binding ligand; (iv) a second single-stranded extender template for template directed extension of the second oligonucleotide primer; (v) a polymerase; (vi) a first labeled-nucleic acid probe comprising a first detectable label and having a nucleotide sequence complementary to at least a portion of the first oligonucleotide primer or the first extender template; (vii) a second labeled-nucleic acid probe comprising a second detectable label and having a nucleotide sequence complementary to at least a portion of the second oligonucleotide primer or the second extender template; and (viii) a set of blocking probes, wherein the detectable label of the first labeled-nucleic acid probe and the second labeled- nucleic acid probe are distinguishable from each other, and optionally, the set of blocking probes does not comprise a blocking probe having a sequence complementary to the first nucleic acid tag and / or the second nucleic acid tag.

108. The composition of any one of claims 104-107, wherein the composition further comprises one or more components for template dependent extension of the oligonucleotide primer with the polymerase.

109. The composition of any one of claims 104-108, wherein the composition further comprises dNTPs.

110. The composition of any of one of claims 104-109, wherein the composition further comprises a buffer for polymerization with the polymerase.

111. The composition of any one of claims 100-110, wherein the composition further comprises a buffer for digestion or cleavage with the nuclease.

112. The composition of any one of claims 100-111, wherein the target-binding ligand is bound to the target.

113. The composition of any one of claims 100-112, wherein the labeled-nucleic acid probe and the nucleic acid tag are bound or hybridized with each other.

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