HK1227106B - High-throughput and highly multiplexed imaging with programmable nucleic acid probes - Google Patents

Description

High-throughput and highly multiplexed imaging using programmable nucleic acid probes

Related applications

This application claims the benefit of U.S. Provisional Application No. 61/951,461, filed March 11, 2014, which is incorporated herein by reference in its entirety.

Field of the Invention

The present invention generally relates to the field of detecting and quantifying analytes (eg, targets).

Background of the Invention

Fluorescence microscopy is a powerful tool for exploring molecules in, for example, biological systems. However, the number of different species that can be visualised discriminatively and simultaneously (ie, multiplexing capability) is limited by the spectral overlap between fluorophores.

SUMMARY OF THE INVENTION

The present invention provides, inter alia, methods and compositions for detecting, imaging, and/or quantifying targets of interest (eg, biomolecules). Some of the methods provided herein include (1) contacting a sample to be analyzed (e.g., a sample suspected of containing one or more targets of interest) with moieties that specifically bind to these targets (each moiety is a binding partner for a given target), wherein each moiety is coupled to a nucleic acid (referred to herein as a docking strand) and wherein binding partners with different specificities are coupled to different docking strands, (2) optionally removing unbound binding partners, (3) contacting the sample with labeled (e.g., fluorescently labeled) nucleic acids having nucleotide sequences that are complementary to, and therefore specific for, a docking strand (such labeled nucleic acids are referred to herein as labeled imager strands), (4) optionally removing unbound imager strands, (5) imaging the sample in whole or in part to detect the location and number of bound imager strands, (6) quenching the signal from the labeled imager strands of the sample (e.g., by bleaching, including photobleaching), and (7) repeating steps (3)-(6), each time using an imager strand that has a unique nucleotide sequence relative to all other imager strands used in the method.

Imager strands can be identically labeled, including identically fluorescently labeled. In other embodiments, imager strands with an identical sequence can be identically labeled. The first approach may be more convenient because it requires a single excitation wavelength and detector.

In this way, two or more targets in a sample can be detected, imaged, and/or quantified regardless of their location in the sample, including whether or not their locations in the sample are so close that the signals from the two or more targets would be indistinguishable if observed simultaneously. Thus, the distance between the two or more targets can be less than the resolution distance of the imaging system used to detect the targets, and still, using the methods provided herein, the two or more targets can be distinguished from each other, thereby facilitating more accurate and robust detection and quantification of such targets. In some cases, the resolution distance can be about 50 nm, as an example.

It should be understood that, prior to performing the method, the "target content" of a sample may be known or suspected, or unknown and unsuspected. A binding partner contacting a sample may or may not bind to the sample, depending on whether the target is present (e.g., when the target is present, the binding partner may bind to the sample). An imager strand contacting a sample may or may not bind to the sample, depending on whether the target is present (e.g., when the target is present, the imager strand may bind to a corresponding docking strand that is bound to the target). "Bound to the sample" means that the binding partner or imager strand is bound to its corresponding target or docking strand.

The binding partner can be a naturally occurring protein, such as an antibody or antibody fragment. In the case where the binding partner is an antibody or antibody fragment, the docking chain can be coupled thereto at a constant region. The binding partner can be an antibody such as a monoclonal antibody, or it can be an antigen-binding antibody fragment, such as an antigen-binding fragment from a monoclonal antibody. In certain embodiments, the binding partner is a receptor.

The binding partner can be linked to the docking strand via an intermediate linker. In some embodiments, an intermediate linker comprises biotin and/or streptavidin.

Imager strands can be fluorescently labeled (i.e., coupled to a fluorophore). Fluorophores coupled to imager strands with different nucleotide sequences can be identical to one another, or they can have emission profiles that overlap or do not overlap with other fluorophores. Fluorescently labeled imager strands can include at least one fluorophore.

In some cases, fluorescently labeled imaging nucleic acids, such as imager strands, can include 1, 2, 3, or more fluorophores.

The sample can be a cell, a cell population, or a cell lysate from a cell or cell population.The target can be a protein.

It will thus be appreciated that the present invention provides a method for detecting an analyte by binding the analyte to its corresponding binding partner and sequentially determining the presence of such binding partner by repeatedly binding, detecting, and quenching (e.g., bleaching, such as photobleaching) imaging strands that are optionally identically labeled (e.g., identically fluorescently labeled).

Thus, the present disclosure provides a method comprising (1) contacting a sample to be tested for the presence of one or more targets with one or more target-specific binding partners, wherein each target-specific binding partner is linked to a docking strand, and wherein target-specific binding partners with different specificities are linked to different docking strands, (2) optionally removing unbound target-specific binding partners, (3) contacting the sample with a labeled imaging strand having a nucleotide sequence complementary to a docking strand, (4) optionally removing unbound imaging strands, (5) imaging the sample to detect the position and number of bound labeled imaging strands, (6) quenching the signal from the bound labeled imaging strands, and (7) repeating steps (3)-(6), each time using a labeled imaging strand having a unique nucleotide sequence relative to all other labeled imaging strands.

In some embodiments, the sample is contacted with more than one target-specific binding partner in step (1).

In some embodiments, the target-specific binding partner is an antibody or antibody fragment.

In some embodiments, the labeled imager strands are identically labeled. In some embodiments, the labeled imager strands each comprise a different label. In some embodiments, the labeled imager strands are fluorescently labeled imager strands.

In some embodiments, the one or more targets are proteins. In some embodiments, the sample is a cell, a cell lysate, or a tissue lysate.

In some embodiments, the sample is imaged in step (5) using confocal or epifluorescence microscopy.

In some embodiments, the quenching signal in step (6) comprises photobleaching.

The present disclosure further provides a composition comprising a sample bound to more than one target recognition moiety (such as a target-specific binding partner), each target recognition moiety bound to a docking nucleic acid (such as a docking strand), and at least one docking nucleic acid stably bound to a labeled imaging nucleic acid (such as an imaging strand).

The present disclosure further provides a composition comprising a sample bound to more than one target-specific binding partner, each binding partner bound to a docking strand; and at least one docking strand stably bound to a labeled imager strand.

The present disclosure further provides a method comprising (1) contacting a sample to be tested for the presence of one or more targets with one or more target recognition moieties (such as target-specific binding partners), wherein each target recognition moiety is linked to a docking nucleic acid (such as a docking strand), and wherein target recognition moieties with different specificities are linked to different docking nucleic acids, (2) optionally removing unbound target recognition moieties, (3) contacting the sample with a labeled imaging nucleic acid (such as an imaging strand) having a nucleotide sequence complementary to a docking nucleic acid, (4) optionally removing unbound labeled imaging nucleic acids, (5) imaging the sample to detect the location and number of bound labeled imaging nucleic acids, (6) removing bound labeled imaging nucleic acids from the docking nucleic acid by changing temperature and/or buffer conditions, and (7) repeating steps (3)-(6), each time using a labeled imaging nucleic acid having a unique nucleotide sequence relative to all other labeled imaging nucleic acids. Under such conditions, the imaging nucleic acid spontaneously dissociates from the docking nucleic acid.

The present disclosure further provides a method comprising (1) contacting a sample to be tested for the presence of one or more targets with one or more target-specific binding partners, wherein each target-specific binding partner is linked to a docking nucleic acid, and wherein target-specific binding partners with different specificities are linked to different docking nucleic acids, (2) optionally removing unbound target-specific binding partners, (3) contacting the sample with a labeled imaging nucleic acid having a nucleotide sequence complementary to a docking nucleic acid, (4) optionally removing unbound labeled imaging nucleic acids, (5) imaging the sample to detect the location and number of bound labeled imaging nucleic acids, (6) removing bound labeled imaging nucleic acids from the docking nucleic acid by changing temperature and/or buffer conditions, and (7) repeating steps (3)-(6), each time using a labeled imaging nucleic acid having a unique nucleotide sequence relative to all other labeled imaging nucleic acids. Under such conditions, the imaging nucleic acid spontaneously dissociates from the docking nucleic acid.

In some embodiments, the labeled imaging nucleic acid is removed from the docking nucleic acid by reducing the salt concentration, adding a denaturant, or increasing the temperature. In some embodiments, the salt is Mg++. In some embodiments, the denaturant is formamide, urea, or DMSO.

The present disclosure further provides a method comprising (1) contacting a sample to be tested for the presence of one or more targets with one or more target recognition moieties (such as target-specific binding partners), wherein each target recognition moiety is linked to a docking nucleic acid (such as a docking strand), and wherein target recognition moieties with different specificities are linked to different docking nucleic acids, (2) optionally removing unbound target recognition moieties, (3) contacting the sample with a labeled imaging nucleic acid (such as an imaging strand) having a nucleotide sequence complementary to a docking nucleic acid, (4) optionally removing unbound labeled imaging nucleic acids, (5) imaging the sample to detect the position and number of bound labeled imaging nucleic acids, (6) removing the bound labeled imaging nucleic acids from the docking nucleic acids, and (7) repeating steps (3)-(6), each time using a labeled imaging nucleic acid having a unique nucleotide sequence relative to all other labeled imaging nucleic acids.

The present disclosure further provides a method comprising (1) contacting a sample to be tested for the presence of one or more targets with one or more target-specific binding partners, wherein each target-specific binding partner is linked to a docking nucleic acid, and wherein target-specific binding partners with different specificities are linked to different docking nucleic acids, (2) optionally removing unbound target-specific binding partners, (3) contacting the sample with a labeled imaging nucleic acid having a nucleotide sequence complementary to a docking nucleic acid, (4) optionally removing unbound labeled imaging nucleic acids, (5) imaging the sample to detect the location and number of bound labeled imaging nucleic acids, (6) removing the bound labeled imaging nucleic acids from the docking nucleic acids, and (7) repeating steps (3)-(6), each time using a labeled imaging nucleic acid having a unique nucleotide sequence relative to all other labeled imaging nucleic acids.

In some embodiments, in step (6), the labeled imaging nucleic acid is not removed from the docking nucleic acid by strand displacement in the presence of a competitor nucleic acid.

In some embodiments, in step (6), the labeled imaging nucleic acid is removed from the docking nucleic acid by chemically, photochemically, or enzymatically cleaving, modifying, or degrading the labeled imaging nucleic acid.

In some embodiments, when the labeled imaging nucleic acid is bound to its corresponding docking nucleic acid, no single-stranded regions are present on the imaging nucleic acid or the docking nucleic acid. In some embodiments, the docking nucleic acid does not have a toehold sequence. In some embodiments, the imaging nucleic acid does not have a toehold sequence.

In some embodiments, the labeled imaging nucleic acid is not self-quenching.

The present disclosure further provides a method comprising (1) contacting a sample to be tested for the presence of one or more targets with one or more target recognition moieties (such as target-specific binding partners), wherein each target recognition moiety is linked to a docking nucleic acid (such as a docking strand), and wherein target recognition moieties with different specificities are linked to different docking nucleic acids, (2) optionally removing unbound target recognition moieties, (3) contacting the sample with a labeled imaging nucleic acid (such as an imaging strand) having a nucleotide sequence complementary to a docking nucleic acid, (4) optionally removing unbound labeled imaging nucleic acids, (5) imaging the sample to detect the location and number of bound labeled imaging nucleic acids, (6) inactivating the bound labeled imaging nucleic acids by removing or modifying their signaling moieties without completely removing the imaging nucleic acids, and (7) repeating steps (3)-(6), each time using a labeled imaging nucleic acid having a unique nucleotide sequence relative to all other labeled imaging nucleic acids.

The present disclosure further provides a method comprising (1) contacting a sample to be tested for the presence of one or more targets with one or more target-specific binding partners, wherein each target-specific binding partner is linked to a docking nucleic acid and wherein target-specific binding partners with different specificities are linked to different docking nucleic acids, (2) optionally removing unbound target-specific binding partners, (3) contacting the sample with a labeled imaging nucleic acid having a nucleotide sequence complementary to a docking nucleic acid, (4) optionally removing unbound labeled imaging nucleic acids, (5) imaging the sample to detect the location and number of bound labeled imaging nucleic acids, (6) inactivating the bound labeled imaging nucleic acids by removing or modifying their signal emitting moieties without completely removing the imaging nucleic acids, and (7) repeating steps (3)-(6), each time using a labeled imaging nucleic acid having a unique nucleotide sequence relative to all other labeled imaging nucleic acids.

Different embodiments are also applicable to the above method. These embodiments are as follows:

In some embodiments, the sample is contacted with more than one target-specific binding partner in step (1). In some embodiments, the target-specific binding partner is an antibody or antibody fragment. In some embodiments, the target-specific binding partner is a natural or engineered ligand, small molecule, aptamer, peptide, or oligonucleotide.

In some embodiments, the labeled imaging nucleic acids are identically labeled. In some embodiments, the labeled imaging nucleic acids each comprise a different label. In some embodiments, the labeled imaging nucleic acids are fluorescently labeled imaging nucleic acids.

In some embodiments, the one or more targets are proteins. In some embodiments, the sample is a cell, a cell lysate, or a tissue lysate.

In some embodiments, the sample is imaged in step (5) using confocal or epifluorescence microscopy.

In some embodiments, the unbound docking nucleic acid is partially double-stranded. In some embodiments, the unbound imaging nucleic acid is partially double-stranded.

In some embodiments, the imaging nucleic acid is a molecular beacon or comprises a hairpin secondary structure. In some embodiments, the imaging nucleic acid is a molecular beacon or comprises a self-quenching hairpin secondary structure. In some embodiments, the imaging nucleic acid is a half duplex. In some embodiments, the half duplex is self-quenching. In some embodiments, the imaging nucleic acid is bound to multiple signal emitting moieties via a dendritic structure or a polymeric structure. The imaging nucleic acid can be linear or branched.

In some embodiments, the docking nucleic acid comprises a hairpin secondary structure.

These and other embodiments are described in greater detail herein.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a schematic diagram of one embodiment of a high-throughput and inherently scalable multiplexed imaging method provided in the present disclosure. Cells are imaged after probe hybridization and then photobleached prior to subsequent rounds of imaging.

2 is a schematic diagram of one embodiment of a high-throughput and inherently scalable multiplexed imaging method based on buffer exchange using solutions with mildly denaturing characteristics, such as reduced salt concentration, increased formamide concentration, or higher temperature.

FIG3 is a schematic diagram of one embodiment of inactivating imager strands by removing the imager strands using methods provided in the present disclosure.

4 is a schematic diagram of one embodiment of inactivating an imager strand by inactivating the fluorophore without removing the nucleic acid portion of the imager strand.

FIG5 is a schematic diagram of one embodiment of a molecular beacon-like self-quenching imaging chain.

FIG6 is a schematic diagram of one embodiment of a half-duplex self-quenching imager strand.

FIG. 7 is a schematic diagram of one embodiment of a non-single-stranded docking chain.

FIG8 is a schematic diagram of one embodiment of an imaging chain that recruits multiple copies of signaling moieties to a docking chain.

FIG9 is a schematic diagram of one embodiment of a non-single-stranded imager strand.

FIG10 is a graph showing the predicted dissociation constants for 10 different oligonucleotides with corresponding reverse complements under 3 different conditions. Sequence a1: 5’-CATCTAAAGCC-3’ (SEQ ID NO: 1); sequence a2: 5’-GAATTTCCTCG-3’ (SEQ ID NO: 2); sequence a3: 5’-GTTTAATTGCG-3’ (SEQ ID NO: 3); sequence a4: 5’-ACAATTCTTCG-3’ (SEQ ID NO: 4); sequence a5: 5’-TTTCTTGCTTC-3’ (SEQ ID NO: 5); sequence a6: 5’-GCATTGTTACT-3’ (SEQ ID NO: 6); sequence a7: 5’-ATATACAAGCG-3’ (SEQ ID NO: 7); sequence a8: 5’-GCTGTCTATTG-3’ (SEQ ID NO: 8); sequence a9: 5’-TCTTTATGCTG-3’ (SEQ ID NO: 9); sequence a10: 5’-CAATCTCATCC-3’ (SEQ ID NO: 10).

Description of the Invention

The present invention particularly provides, for example, compositions and methods for multiplexed fluorescence imaging in a cellular environment using a nucleic acid-based imaging probe (e.g., a DNA-based imaging probe). The method provided herein is based in part on the programmability of nucleic acid docking chains and imaging chains. That is, for example, docking chains and imaging chains can be designed so that they bind to each other for a certain time under certain conditions. This programmability allows the imaging chain to stably bind to the docking chain, as provided herein. In general, the method provided herein relates to identifying one or more targets (e.g., one or more biomolecules such as proteins or nucleic acids) in a particular sample (e.g., a biological sample). In some cases, whether one or more targets are present in a sample is unknown. Therefore, the method disclosed herein can be used to determine whether the one or more targets are present in a sample suspected of containing one or more targets. In any one of the aspects and embodiments provided herein, a sample may contain or may be suspected of containing one or more targets.

Thus, the present invention provides methods for high-throughput and highly multiplexed imaging and analyte/target detection based on programmable nucleic acid (e.g., DNA) probes. These methods rely on a sequential imaging approach that employs orthogonal imaging strands that can be stably attached to a complementary docking strand immobilized on a binding partner (such as an antibody) ( FIG1 ). After hybridization and imaging with an imaging strand, a quenching step (such as a photobleaching step) is performed to eliminate and/or reduce fluorescence from the hybridized (bound) imaging strand.

In another embodiment, these methods exploit the weaker binding between the docking strand and the imager strand to remove the signal. For example, the hybridization conditions can be altered so that the melting point of the duplex formed between the docking strand or the imager strand is slightly above room temperature (e.g., 25°C) or the imaging temperature. The labeling step (i.e., the step in which the imager strand binds to its corresponding docking strand) and the imaging step are performed as described above. As an example, after imaging the first target, the sample is subjected to a denaturing condition. The denaturing condition can be provided in a buffer exchange step using a solution having, for example, a lower salt concentration, the presence or increase of a denaturant (such as formamide), or an increased temperature ( FIG. 2 ). The sample can alternatively or additionally be exposed to an increased temperature. The increase or decrease is relative to the conditions existing in the labeling step (i.e., when the imager strand is bound to the docking strand). In the case of a buffer exchange, the sample can be washed, the buffer exchange can be repeated, the sample can be washed again, and then the next imager strand can be added to the sample.

For multiplexing, different reservoirs of orthogonal imaging chains are sequentially applied to the same sample after each step of, for example, photobleaching or other methods for quenching the signal or inactivation or removal of the imaging chain, so as to potentially image an unlimited number of targets. Unlike traditional imaging methods where multiplexing is limited by spectral overlap between color channels, the method provided herein is limited only by the number of possible orthogonal nucleotide sequences (of the docking chain or alternatively the imaging chain). Since a larger number of orthogonal nucleotide sequences can be easily designed, this method has an essentially scalable multiplexing capability achieved by using only a single fluorophore. This method can be easily integrated with standard microscopy settings (e.g., confocal or epifluorescence microscopy), allowing the sample to be analyzed at high throughput.

These methods have the applicability in, for example, high-throughput screening assays (such as drug screening assays). This imaging method allows analyzing large cell populations (~1,000-10,000) or tissue samples in a super-multiplexed format while using standard confocal or epifluorescence microscopy imaging. Screening a large number of targets (such as proteins) from the same sample in a high-throughput manner will provide information about new drugs or regulators, while providing cell heterogeneity information. Large-scale screening of tissue samples with high-throughput and super-multiplexed imaging capabilities will be suitable for pathological analysis, for example, in hospitals or other service provider settings.

The methods provided herein can also be used to identify the absolute amount of a single target (such as, for example, a specific protein), or the amount of a single target relative to one or more other targets.

Additionally, the methods provided herein can be used to identify the location of a target in a sample or relative to other targets in a sample.

Thus, the present disclosure provides a method comprising (1) contacting a sample simultaneously with multiple sequence-tagged target recognition moieties, (2) introducing imaging nucleic acids (such as imaging strands) that recognize a subset of docking nucleic acids (such as docking strands) in the sequence-tagged target recognition moieties by sequence complementarity, (3) removing or inactivating the imaging nucleic acids or quenching the signal from the imaging nucleic acids, and (4) repeating step (2) and optionally step (3) at least once to image and detect one or more additional docking nucleic acids.

The method may optionally include labeling the plurality of target recognition moieties with a docking nucleic acid (such as a docking strand) to form sequence-tagged target recognition moieties.

The present disclosure provides a method comprising (1) contacting a sample to be tested for the presence of one or more targets with one or more target-specific binding partners, wherein each target-specific binding partner is linked to a docking strand, and wherein target-specific binding partners with different specificities are linked to different docking strands, (2) optionally removing unbound target-specific binding partners, (3) contacting the sample with a labeled imaging strand having a nucleotide sequence complementary to a docking strand, (4) optionally removing unbound imaging strands, (5) imaging the sample to detect the position and number of bound labeled imaging strands, (6) quenching the signal from the bound labeled imaging strands, and (7) repeating steps (3)-(6), each time using a labeled imaging strand having a unique nucleotide sequence relative to all other labeled imaging strands.

Steps (3)-(6) may be repeated one or more times. For example, steps (3)-(6) may be repeated 1-10 times or more. In some embodiments, steps (3)-(6) are repeated 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times.

The present disclosure further provides a method comprising (1) contacting a sample to be tested for the presence of one or more targets with one or more target recognition moieties (such as target-specific binding partners), wherein each target recognition moiety is linked to a docking nucleic acid, and wherein target recognition moieties with different specificities are linked to different docking nucleic acids, (2) optionally removing unbound target recognition moieties, (3) contacting the sample with a labeled imaging nucleic acid (such as an imaging strand) having a nucleotide sequence complementary to a docking nucleic acid, (4) optionally removing unbound labeled imaging nucleic acids, (5) imaging the sample to detect the position and number of bound labeled imaging nucleic acids, (6) removing the bound labeled imaging nucleic acids from the docking nucleic acids, and (7) repeating steps (3)-(6), each time using a labeled imaging nucleic acid having a unique nucleotide sequence relative to all other labeled imaging nucleic acids.

Steps (3)-(6) may be repeated one or more times. For example, steps (3)-(6) may be repeated 1-10 times or more. In some embodiments, steps (3)-(6) are repeated 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times.

The present disclosure further provides a method comprising (1) contacting a sample to be tested for the presence of one or more targets with one or more target recognition moieties (such as target-specific binding partners), wherein each target recognition moiety is linked to a docking nucleic acid (such as a docking strand), and wherein target recognition moieties with different specificities are linked to different docking nucleic acids, (2) optionally removing unbound target recognition moieties, (3) contacting the sample with a labeled imaging nucleic acid (such as an imaging strand) having a nucleotide sequence complementary to a docking nucleic acid, (4) optionally removing unbound labeled imaging nucleic acids, (5) imaging the sample to detect the location and number of bound labeled imaging nucleic acids, (6) inactivating the bound labeled imaging nucleic acids by removing or modifying their signaling moieties without completely removing the imaging nucleic acids, and (7) repeating steps (3)-(6), each time using a labeled imaging nucleic acid having a unique nucleotide sequence relative to all other labeled imaging nucleic acids.

Steps (3)-(6) may be repeated one or more times. For example, steps (3)-(6) may be repeated 1-10 times or more. In some embodiments, steps (3)-(6) are repeated 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times.

In some embodiments, the methods provided herein include a step of removing an imaging nucleic acid (such as an imager strand) bound to a docking nucleic acid (such as a docking strand) using a method other than strand displacement.

In some embodiments, the methods provided herein include a step of removing an imaging nucleic acid (such as an imager strand) bound to a docking nucleic acid (such as a docking strand), wherein the imaging nucleic acid emits a signal before binding to the docking nucleic acid (i.e., the signal is not quenched).

In some embodiments, the methods provided herein include a step of removing imaging nucleic acids (such as imager strands) bound to docking nucleic acids (such as docking strands), wherein the imaging nucleic acids are removed using a nucleic acid that does not comprise a quencher.

In each of the foregoing methods, the docking nucleic acid (including the docking strand) can be a single-stranded docking nucleic acid or docking strand, or it can be a double-stranded docking nucleic acid or docking strand, or it can be a partially double-stranded docking nucleic acid or docking strand (e.g., containing one single-stranded and one double-stranded region).

In some embodiments, where multiple target recognition moieties (including multiple binding partners) are used, the multiple can be contacted with the sample and, therefore, the target of interest, simultaneously. Target recognition moieties (such as binding partners) need not be contacted with the sample sequentially, although they can be.

These different methods promote high throughput imaging with a spinning disk confocal microscope. It is estimated that a full-cell 3D imaging process of one color at a time will require an average of about 30 seconds. This method allows imaging of large areas (e.g., up to mm scale) with a compatible 10X or 20X objective lens. An imaging depth of about 30-50 microns can be achieved. The method provided here has been used to stain actin, Ki-67, clathrin, cytokeratin, etc. (data not shown).

Binding partner

These methods employ binding partners that are coupled to nucleic acids (e.g., docking nucleic acids such as docking strands). These may be referred to herein as binding partner-nucleic acid conjugates ("BP-NA conjugates"). They may also be referred to as target recognition moieties of sequence tags. As used herein, "binding partner-nucleic acid conjugates," or "BP-NA conjugates," refer to molecules that are connected (e.g., via an N-hydroxysuccinimide (NHS) linker) to a single-stranded nucleic acid (e.g., DNA) docking strand.

The binding partner of the conjugate can be any moiety (e.g., an antibody or aptamer) that has an affinity for a target of interest, such as a biomolecule (e.g., a protein or nucleic acid). In some embodiments, the binding partner is a protein. A BP-NA-conjugate comprising a protein (or peptide) attached to a docking chain may be referred to herein as a "protein-nucleic acid conjugate" or "protein-NA conjugate." Examples of proteins used in conjugates of the present invention include, but are not limited to, antibodies (e.g., monoclonal antibodies), antigen-binding antibody fragments (e.g., Fab fragments), receptors, peptides, and peptide aptamers. Other binding partners may be used in accordance with the present invention. For example, binding partners that bind to a target via electrostatic (e.g., electrostatic particles), hydrophobic, or magnetic (e.g., magnetic particles) interactions are contemplated herein.

As used herein, "antibody" includes full-length antibodies and any antigen-binding fragment (e.g., "antigen-binding portion") or single chains thereof. The term "antibody" includes, but is not limited to, a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or an antigen-binding portion thereof. Antibodies can be polyclonal or monoclonal; xenogeneic, allogeneic, or syngeneic; or modified forms thereof (e.g., humanized, chimeric).

As used herein, the "antigen-binding portion" of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. The antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed by the term "antigen-binding portion" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of _VH , _VL , _CL , and _CH1 domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments connected by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of _VH and _CH1 domains; (iv) an Fv fragment consisting of the _VH and _VL domains of a single arm of an antibody, (v) a dAb fragment consisting of a _VH domain (Ward et al., Nature 341:544-546, 1989); and (vi) an isolated complementarity-determining region (CDR), or (vii) a combination of two or more isolated CDRs, optionally linked by a synthetic linker. In addition, although the two domains _VH and _VL of the Fv fragment are encoded by independent genes, the two domains can be joined by a synthetic linker using recombinant methods, which enables them to be made into a single protein chain in which the _VH and _VL regions are paired to form a monovalent molecule (called single-chain Fv (scFv); see, for example, Bird et al., Science 242:423-426, 1988; and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883, 1988). Such single-chain antibodies are also encompassed within the scope of the term "antigen-binding portion" of an antibody. These antibody fragments are obtained using conventional techniques known to those skilled in the art and screened for usefulness in the same manner as intact antibodies.

As used herein, a "receptor" refers to a cellularly derived molecule (eg, a protein) that binds to a ligand such as, for example, a peptide or a small molecule (eg, an organic or inorganic compound of low molecular weight (<900 Daltons)).

As used herein, "peptide aptamer" refers to a molecule with a variable peptide sequence inserted into a constant scaffold protein (see, eg, Baines IC et al., Drug Discov. Today 11:334-341, 2006).

In some embodiments, the molecule of the BP-NA conjugate is a nucleic acid (such as, for example, a nucleic acid aptamer). As used herein, "nucleic acid aptamer" refers to a small RNA or DNA molecule that can form secondary and tertiary structures that can specifically bind to proteins or other cellular targets (see, for example, Ni X et al., Curr Med Chem 18(27):4206–4214, 2011). Therefore, in some embodiments, the BP-NA conjugate can be an aptamer-nucleic acid conjugate.

Some embodiments of the present invention use target recognition moieties to identify and label targets.Target recognition moieties are reagents that specifically identify targets of interest in samples.Examples of target recognition moieties include binding partners such as those listed herein.Target recognition moieties include antibodies, antibody fragments, and antibody derivatives, peptides, aptamers, and oligonucleotides such as single-chain antibodies, single-chain Fv domains, Fab domains, nanobodies, etc. (for example, to detect nucleic acids of interest in processes such as fluorescence in situ hybridization or FISH).

Docking nucleic acids such as docking strands

Certain embodiments of the present invention may relate to docking nucleic acids. Docking nucleic acids include docking chains as described herein. Docking nucleic acids are linear nucleic acids that can be bound to nucleic acids with complementary sequences (such as, imaging nucleic acids). Docking nucleic acids may include or may be composed of DNA, RNA, or nucleic acid-like structures having other phosphate-sugar backbones (such as 2'-O-methyl RNA, 2'-fluoro (2'-fluoral) RNA, LNA, XNA) or backbones (such as peptide nucleic acids and morpholinos) comprising non-phosphate-sugar moieties. Nucleobases may include naturally occurring nucleobases such as adenine, thymine, guanine, cytosine, inosine, and their derivatives, as well as non-naturally occurring nucleobases such as isoC, isoG, dP, and dZ. When not bound to its complementary imaging nucleic acid, the docking nucleic acid may be single-stranded without a stable secondary structure. Alternatively, the docking nucleic acid may include a secondary structure such as a hairpin loop (Fig. 7, top). The docking nucleic acid may be a part of a multi-stranded complex (Fig. 7, bottom).

As used herein, a "docking strand" refers to a single-stranded nucleic acid (e.g., DNA) that is capable of stably binding to its complementary imager strand. Stable binding may be a result of the length of the docking strand (and the opposing imager strand), or it may be a result of the specific conditions under which hybridization occurs (e.g., salt concentration, temperature, etc.). In some embodiments, the docking strand is about 20 to 60, or more, nucleotides in length. A docking strand may be capable of binding to one or more identical imager strands (having the same sequence and being identically labeled).

Imaging nucleic acids such as imager strands

Certain embodiments of the present invention may relate to imaging nucleic acids. Imaging nucleic acids include imaging strands as described herein. Imaging nucleic acids are nucleic acids that can (1) interact with docking nucleic acids via sequence-specific complementarity and (2) recruit a signaling moiety or multiple copies of a signaling moiety via covalent or non-covalent interactions. As described herein, imaging nucleic acids can be linear or branched. An imaging nucleic acid can recruit multiple copies of a signaling moiety via a polymeric structure ( FIG. 8 , top) or a dendritic structure ( FIG. 8 , bottom). For example, polymeric or dendritic structures can be chemically synthesized using methods such as those discussed in Nazemi A. et al., Chemistry of Bioconjugates: Synthesis, Characterization, and Biomedical Applications, published online: February 13, 2014, and references provided therein. Alternatively, polymeric or dendritic structures can be formed by DNA hybridization as shown, for example, in Dirks R. et al., Proc. Natl. Acad. Sci. USA, 2004; 1010(43): 15275-78; and in Um S.H. et al., Nat. Protocols 2006; 1: 995-1000 (each of which is incorporated herein by reference).

Imaging nucleic acids may comprise or may be composed of DNA, RNA, or nucleic acid-like structures having other phosphate-sugar backbones (e.g., 2'-O-methyl RNA, 2'-fluoro RNA, LNA, XNA) or backbones containing non-phosphate-sugar moieties (e.g., peptide nucleic acids and morpholinos). Nucleobases may include naturally occurring nucleobases such as adenine, thymine, guanine, cytosine, inosine, and their derivatives, as well as non-naturally occurring nucleobases such as isoC, isoG, dP, and dZ.

In some embodiments, the imaging nucleic acid is about 30 to about 60, or more, nucleotides in length, including 30, 35, 40, 45, 50, 55, or 60 nucleotides in length. In some embodiments, the imaging nucleic acid is 30 to 40, 30 to 50, 40 to 50, 40 to 60, or 50 to 60 nucleotides in length.

When not bound to its complementary docking nucleic acid, the imaging nucleic acid can be single-stranded and lack stable secondary structure. Alternatively, the imaging nucleic acid can include secondary structures such as hairpin loops (Figure 9, top). The imaging nucleic acid can be part of a multi-stranded complex (Figure 9, bottom).

In some embodiments, the imaging strands can be self-quenching, meaning that the unbound imaging nucleic acid can carry a quencher moiety in close proximity to a signal-emitting moiety (e.g., a fluorophore). To achieve this, the imaging nucleic acid can be designed to adopt a molecular beacon-like structure ( FIG5 ) or a half-duplex structure ( FIG6 ).

This self-quenching change can be used to simplify background and/or avoid wash steps. Additionally or alternatively, the binding and imaging buffer can contain additives conventionally used in FISH, Northern blotting, and Southern blotting (e.g., negatively charged polymers such as dextran sulfate and heparin) to reduce nonspecific binding.

As used herein, a "signal emitting moiety" is a moiety that emits a detectable signal under certain conditions, such as photons, radiation, positrons, electromagnetic waves, and magnetic nuclear resonance.

As used herein, an "imager strand" is a single-stranded nucleic acid (e.g., DNA) of about 30 to about 60, or more, nucleotides in length. The imager strand of the present invention is complementary to and stably bound to the docking strand. Stable binding means that the imager strand and the docking strand remain bound to each other for the duration of the assay, or at least 30 minutes, or at least 60 minutes, or at least 2 hours, or longer. This binding may or may not be reversible or irreversible.

In some embodiments, a docking nucleic acid is considered stably bound to an imaging nucleic acid (such as an imager strand) if the nucleic acids remain bound to each other for (or at least) 30, 35, 40, 45, 50, 55, or 60 minutes (min). In some embodiments, a docking nucleic acid is considered stably bound to an imaging nucleic acid if the nucleic acids remain bound to each other for (or at least) 30 to 60 min, 30 to 120 min, 40 to 60 min, 40 to 120 min, or 60 to 120 min. This binding may or may not be reversible or irreversible.

As used herein, "binding" refers to an association between at least two molecules due to, for example, electrostatic, hydrophobic, ionic and/or hydrogen bonding interactions, optionally under physiological conditions.

Two nucleic acids or nucleic acid domains are "complementary" to each other if they base pair or bind to each other to form a double-stranded nucleic acid via Watson-Crick interactions.

In some embodiments, nucleic acids of the invention (such as docking nucleic acids and imaging nucleic acids) bind to each other with "perfect complementarity," which refers to 100% complementarity (e.g., 5'-ATTCGC-3' is perfectly complementary to 5'GCGAAT-3').

The imager strands of the present invention can be labeled with a detectable label (e.g., a fluorescent label, and thus considered “fluorescently labeled”). For example, in some embodiments, an imager strand can comprise at least one (ie, one or more) fluorophores. Examples of fluorophores for use in accordance with the present invention include, but are not limited to, xanthene derivatives (e.g., fluorescein, rhodamine, Oregon green, eosin, and Texas red), cyanine derivatives (e.g., cyanine, indocarbocyanine, oxacarbocyanine, thiocarbocyanine, and merocyanine), naphthalene derivatives (e.g., dansyl and prodan derivatives), coumarin derivatives, oxadiazole derivatives (e.g., pyridyloxazole, nitrobenzoxadiazole, and benzoxadiazole), pyrene derivatives (e.g., cascade blue), oxazine derivatives (e.g., Nile Red, Nile Blue, cresyl violet, and oxazine 170), acridine derivatives (e.g., proflavine, acridine orange, and acridine yellow), arylmethine derivatives (e.g., auramine, crystal violet, and malachite green), and tetrapyrrole derivatives (e.g., porphine, phthalocyanine, and bilirubin).

Imaging nucleic acids comprising imager strands can be covalently labeled with one detectable label such as those listed herein or known in the art. In some cases, imaging nucleic acids comprising imager strands can include 2, 3, 4, or more detectable labels (such as fluorophores).

Orthogonal imaging nucleic acids comprising an imager strand can contain one different label (eg, a red fluorophore, a blue fluorophore, or a green fluorophore), or they can all contain the same label (eg, a red fluorophore) even if their nucleotide sequences differ.

Sequence-tagged target recognition moieties such as binding partners and docking strand conjugates

In some embodiments, the BP-NA conjugates (e.g., protein-nucleic acid conjugates) of the present invention may comprise an intermediate linker (e.g., covalently or non-covalently) that connects the binding partner to the docking chain. The intermediate linker may comprise biotin and/or streptavidin. For example, in some embodiments, an antibody and a docking chain may each be biotinylated (i.e., linked to at least one biotin molecule) and connected to each other via biotin binding to an intermediate streptavidin molecule. Other intermediate linkers may be used according to the present invention. In some embodiments, such as those in which the molecule is a nucleic acid, an intermediate linker may not be required. For example, the docking chain of the BP-NA conjugate may be an extension (e.g., 5' or 3' extension) of a nucleic acid molecule (such as, for example, a nucleic acid aptamer). Similar methods can be used to produce target recognition moieties of sequence tags as provided herein.

Provided herein are a plurality of BP-NA conjugates (e.g., protein-nucleic acid conjugates) and imager strands. The plurality can be of the same species or a population of different species. A plurality of BP-NA conjugates of the same species can include conjugates that all bind to the same target (e.g., biomolecule) (e.g., the same epitope or region/domain). Conversely, a plurality of BP-NA conjugates of different species can include conjugates, or subsets of conjugates, each conjugate or subset of conjugates binding to a different epitope on the same target or to different targets. A plurality of imager strands of the same species can include imager strands having the same nucleotide sequence and the same fluorescent label (e.g., Cy2, Cy3, or Cy4). Conversely, a plurality of imager strands of different species can include imager strands having different nucleotide sequences (e.g., DNA sequences) and different fluorescent labels (e.g., Cy2, Cy3, or Cy4), or having different nucleotide sequences and the same fluorescent label (e.g., all Cy2). The number of different species in a given plurality of BP-NA conjugates is limited by the number of binding partners (e.g., antibodies) and the number of docking strands (and therefore complementary imaging strands) having different nucleotide sequences. In some embodiments, the plurality of BP-NA conjugates (e.g., protein-nucleic acid conjugates) comprises at least 10, 50, 100, 500, 1000, 2000, 3000, 4000, 5000, ^{10 4} , 50000, 10 ⁵ , 10 ⁵ , 10 ⁶ , 10 ⁷ , 10 ⁸ , 10 ⁹ , 10 ¹⁰ , 10 ¹¹ BP-NA conjugates. Similarly, in some embodiments, the plurality of fluorescently labeled imager strands comprises at least 10, 50, 100, 500, 1000, 2000, 3000, 4000, 5000, ^{10 4} , 50000, 10 ⁵ , 10 ⁵ , 10 ⁶ , 10 ⁷ , 10 ⁸ , 10 ⁹ , 10 ¹⁰ , 10 ¹¹ fluorescently labeled imager strands. In some embodiments, the plurality can contain 1 to about 200 or more different species of BP-NA conjugates and/or imager strands. For example, a plurality can contain at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, or more different species. In some embodiments, a plurality can contain less than about 5 to about 200 different species of BP-NA conjugates and/or imager strands. For example, a plurality can contain less than 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, or 200 different species. These embodiments are applicable to target recognition moieties of sequence tags as provided herein.

Inactivation of signaling or imaging nucleic acids

To achieve inactivation of the imaging nucleic acid in some of the methods provided herein, the imaging nucleic acid (including the imager strand) can be removed from the target recognition moiety (including the binding partner) by means such as, but not limited to, increasing the temperature; reducing the concentration of counterions (e.g., free Mg++); introducing or increasing the concentration of denaturing agents (e.g., formamide, urea, DMSO, etc.); and chemically, photochemically, or enzymatically cleaving, modifying, or degrading the imager strand, or any combination thereof ( FIG. 3 ).

To achieve inactivation of imaging nucleic acids in some of the methods provided herein, the imaging nucleic acids (including the imager strand) can be inactivated by removing and/or modifying the signaling moiety without removing the entire nucleic acid portion of the imaging nucleic acid from the docking strand. ( FIG. 4 .)

As an example, removal of imaging nucleic acids can be facilitated by cleaving the imager strand into multiple moieties. In some embodiments, the imaging nucleic acid comprises a chemically cleavable moiety that can be cleaved by introducing a compound that acts on the cleavable moiety. Examples of such chemically cleavable moieties include, but are not limited to, allyl groups that can be cleaved by certain Pd-based reagents (Ju J. et al., Proc. Natl. Acad. Sci. USA, Dec. 26, 2006; 103(52): 19635-40, which reference is incorporated herein by reference); azido groups that can be cleaved by certain phosphorus-based reagents such as TCEP (Guo J. et al., Proc. Natl. Acad. Sci. USA, Jul. 8, 2008; 105(27): 9145-50, which reference is incorporated herein by reference); bridged phosphorothioates that can be cleaved by silver-based reagents (Mag M. et al., Nucleic Acids Res., Apr. 11, 1991; 19(7): 1437-41, which reference is incorporated herein by reference); disulfide bonds that can be cleaved by reducing agents such as DTT and TCEP; and ribose groups that can be cleaved by various nucleophiles such as hydroxide and imidazole.

In some embodiments, the imaging nucleic acid comprises a photocleavable linker that can be cleaved photochemically (e.g., by UV exposure). In some embodiments, the imaging nucleic acid contains a moiety that can be cleaved by an enzyme. Examples of such enzymatically cleavable moieties include, but are not limited to, ribonucleotides that can be cleaved by various ribonucleases; deoxyuridine that can be cleaved by an enzyme combination such as USER (New England Biolabs); and restriction enzyme sites that can be cleaved by sequence-specific nickases or restriction endonucleases. In some embodiments, the restriction endonuclease can cleave both the imaging nucleic acid and the docking nucleic acid. In still other embodiments, removal of the imaging nucleic acid can be facilitated by modifying the imaging nucleic acid into a form that allows the docking nucleic acid to bind so as to form a duplex with reduced stability (or reduced melting temperature).

As an example, the imaging nucleic acid comprises azobenzene that can photoisomerize, wherein different isomers affect the binding strength of the imaging nucleic acid to the docking nucleic acid differently (Asanuma H. et al., Angew Chem Int Ed Engl, 2001 Jul 16;40(14):2671-2673, which is incorporated herein by reference). In some embodiments, the imaging nucleic acid comprises a deoxyuridine, wherein the uracil group is cleavable by uracil-DNA glycosylase. After removal of the uracil, the binding strength of the imaging strand is weakened.

Alternatively, removal of the signaling moiety can be achieved by cleaving the linker between the imaging nucleic acid and the signaling moiety, if such a linker is present. The chemistries described herein can also be used for this purpose.

Inactivation of the signaling moiety can be achieved by chemically or photochemically modifying the signaling moiety. For example, when the signaling moiety is a fluorophore, it can be bleached by a chemical agent (such as, for example, hydrogen peroxide, Gerdes M. et al., Proc. Nat. Acad. Sci. USA 2013 Jul 16; 110(29): 11982-87, which reference is incorporated herein by reference) or photobleached (e.g., using soft multi-wavelength excitation as described in Schubert W. et al., Nat. Biotech. 2006; 24: 1270-78, which reference is incorporated herein by reference).

As will be understood in the art, "photobleaching" refers to the photochemical change of a dye or fluorophore molecule so that it cannot fluoresce. This is caused by cleavage of covalent bonds between the fluorophore and surrounding molecules or nonspecific reactions. In some embodiments, the loss of activity caused by photobleaching can be controlled by reducing the intensity or time interval of the exposure, by increasing the concentration of the fluorophore, by reducing the frequency of the input light and thus reducing its photon energy, or by using more robust fluorophores that are less prone to bleaching (such as Alexa Fluors or DyLight Fluors). See, for example, Ghauharali R. et al., Journal of Microscopy 2001; 198: 88-100; and Eggeling C. et al., Analytical Chemistry 1998; 70: 2651-59.

Thus, photobleaching can be used to remove, modify, or in some cases eliminate the signal from the signal-emitting moiety. Photobleaching can be performed by exposing the fluorophore to a wavelength of light having a suitable wavelength, energy, and duration to permanently and irreversibly eliminate the fluorophore's ability to further emit a signal. Photobleaching techniques are known in the art.

It was also found that, unlike antibodies, which are generally able to bind to their targets over a wide range of temperatures below physiological temperature (i.e., 0°C to 37°C) and can tolerate slight changes in salt concentration (i.e., monovalent cation concentrations from 10mM to 1M; divalent cation concentrations from 0 to 10mM), the affinity of short nucleic acid hybrids depends on temperature and salt concentration. For example, at 23°C, with 500mM [Na+] and 10mM [Mg++] concentrations, the predicted dissociation constant between ssDNA 5'-CATCTAAAGCC-3' and its reverse complement 5'-GGCTTTAGATG-3' (using the parameter set summarized in reference PMID 15139820) is ∼90pM. In other words, under these conditions, the binding is very strong. At 37°C, with 150mM [Na+] and 0mM [Mg++], the predicted dissociation constant for this ssDNA pair is as high as ∼500nM. In other words, under these conditions, this combination is quite weak. The dissociation constant of these two conditions changes nearly 4 orders of magnitude, although it is expected that most antibodies all brute-forcely bind to their targets under two conditions. Similar trends (Figure 10) are observed for other DNA sequences. As an additional example, imaging conditions can be 23 ℃, with 500mM [Na + ] and 10mM [Mg ++ ], and dye inactivation conditions can be 37 ℃, with 150mM [Na + ] and 0mM [Mg ++ ].

In some embodiments, the sample being analyzed is a cultured cell, a tissue section, or other sample from a living organism.

In some embodiments, the sample is dissociated cells fixed (including individually fixed) on a solid surface (e.g., a glass slide or coverslip). For example, the sample can be cells in blood. For example, the sample can contain cancer cells circulating in the blood (also known as circulating tumor cells, or CTCs). The sample can be cells grown in suspension. The sample can be cells disseminated from a solid tissue.

sample

"Sample" may include cells (or a cell), tissue, or body fluids such as blood (serum and/or plasma), urine, semen, lymph, cerebrospinal fluid, or amniotic fluid. The sample may be obtained from (or derived from) any source, including but not limited to humans, animals, bacteria, viruses, microorganisms, and plants. In some embodiments, the sample is a cell lysate or a tissue lysate. The sample may also contain a mixture of materials from one source or different sources. The sample may be a spatial region or volume (e.g., a grid on an array, or a well in a plate or dish). In some embodiments, the sample includes one or more targets, one or more BP-NA conjugates, and one or more imaging chains. The cells may be disseminated (or dissociated) cells.

target

A "target" is any moiety for which one wishes to observe or quantify and for which a binding partner exists. In some embodiments, a target may be non-naturally occurring. In some embodiments, a target may be a biomolecule. As used herein, a "biomolecule" is any molecule that can be produced by a living organism, including large macromolecules such as proteins, polysaccharides, lipids, and nucleic acids (e.g., DNA and RNA (such as mRNA)), as well as small molecules such as primary metabolites, secondary metabolites, and natural products. Examples of biomolecules include, but are not limited to, DNA, RNA, cDNA, or the DNA product of RNA subjected to reverse transcription, A23187 (calcimycin, calcium ionophore), avermectin, 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, alamethicone, alanine, albumin, aldosterone, aleurone, α-muscarin, allantoin, allethrin, α-amanitin, amino acids, amylase, anabolic steroids, 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, strychnine, cadaverine, caffeine, calciferol (vitamin D), calcitonin, calmodulin, calreticulin, camphor-(C10H16O), cannabinol, capsaicin, carbohydrase, carbohydrates, carnitine, carrageenan, casein, caspase, cellulase, cellulose-(C6H10O5), cerulenin, cetrimide-C19H 42BrN, chelerythrine, chromomycin A3, chaperone, chitin, α-chloralose, chlorophyll, cholecystokinin (CCK), cholesterol, choline, chondroitin sulfate, cinnamaldehyde, citral, citric acid, citrinin, citronellal, citronellol, citrulline, cobalamin (vitamin B12), coenzyme Q, colchicine, collagen, coniine, corticosteroids, corticosterone, corticotropin-releasing hormone (CRH), cortisol , creatine, creatine kinase, crystallin, α-cyclodextrin, cyclodextrin glycosyltransferase, cyclopamine, cyclopiazonic acid, cysteine, cystine, cytosine, cytochalasin, cytochalasin E, cytochrome, cytochrome c, cytochrome c oxidase, cytochrome c peroxidase, cytokine, cytosine – C4H5N3O, deoxycholic acid, DON (deoxynivalenol), deoxyribofuranose, deoxyribose, deoxyribonucleic acid (DNA), dextran, dextrin, DNA, dopamine, enzyme, ephedrine, epinephrine – C9H13NO3, erucic acid – CH3(CH2)7CH＝CH(CH2)11COOH, erythritol, erythropoietin (EPO), estradiol, eugenol, fatty acids, fibrin, fibronectin, folic acid (vitamin M), follicle-stimulating hormone (FSH), formaldehyde, formic acid, formnoci, fructose, fumonisin B1, gamma globulin, galactose, gamma globulin, gamma-aminobutyric acid, gamma-butyrolactone, gamma-hydroxybutyrate (GHB), gastrin, gelatin, geraniol, globulin, glucagon, glucosamine, glucose – C6H12O6, glucose oxidase, gluten, glutamic acid, glutamine, glutathione, gluten, glycerol (glycerol), glycine, glycogen, glycolic acid, glycoprotein, gonadotropin-releasing hormone (GnR) H), granzyme, green fluorescent protein, growth hormone, growth hormone-releasing hormone (GHRH), GTPase, guanine, guanosine, guanosine triphosphate (+GTP), haptoglobin, hematoxylin, heme, earthworm hemoglobin, 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, ionomycin, ionone, isoleucine, iron-sulfur cluster, K252a, K252b, KT5720, KT582 3. Keratin, kinase, lactase, lactic acid, lactose, lanolin, lauric acid, leptin, leuprocin B, leucine, lignin, limonene, linalool, linoleic acid, linolenic acid, lipase, lipids, lipid-anchored protein, lipoamide, lipoprotein, low-density lipoprotein LDL, luteinizing hormone (LH), lycopene, lysine, lysozyme, malic acid, maltose, melatonin, membrane protein, metalloprotein, metallothionein, methionine, containing Mimosine, Mithramycin A, Mitomycin C, Monomer, Mycophenolic Acid, Myoglobin, Myosin, Natural Phenols, Nucleic Acids, Ochratoxin A, Estrogen, Oligopeptides, Oligomycin, Lichenol, Olexin, Ornithine, Oxalic Acid, Oxidase, Oxytocin, p53, PABA, Paclitaxel, Palmitic Acid, Pantothenic Acid (Vitamin B5), Parathyroid Hormone (PTH), Paraprotein, Mosesin, Parthenolide, Patulin, Mycopenicillin, Penicillin Acid, Penicillin, Penicillin A, Peptidase, Pepsin, Peptide, Epimycin, Peripheral Membrane Protein, Perosamine, Phenylethylamine, Phenylalanine, Creatine Phosphate, Phosphatase, Phospholipid, Phenylalanine, Phytic Acid, Plant Hormone, Peptide, Polyphenol, Polysaccharide, Porphyrin, Prion, Progesterone, Prolactin (PRL), Proline, Propionic Acid, Protamine, Protease, Protein, Proteinoid, Putrescine, Pyrethrin, Pyridoxine or Pyridoxamine (Vitamin Vitamin B6), pyrrolysine, pyruvate, quinone, radicicol, raffinose, renin, retinal, retinol (vitamin A), rhodopsin (rhodopsin), riboflavin (vitamin B2), ribofuranoside, ribose, ribozyme, ricin, RNA-ribonucleic acid, RuBisCO, safrole, salicylaldehyde, salicylic acid, salvinorin-A–C23H28O8, saponin, secretin, selenocysteine Acid, selenomethionine, selenoproteins, serine, serine kinase, serotonin, skatole, signal recognition particles, somatostatin, sorbic acid, squalene, staurosporine, stearic acid, sterols, strychnine, sucrose (sugar), carbohydrates (generally), superoxide, T2 toxin, tannic acid, tannic acid, tartaric acid, taurine, tetrodotoxin, thaumatin, topoisomerase, tyrosine kinase, taurine, testosterone, tetrahydrocannabinol (THC), tetrodotoxin, thapsigargin, thiamin (vitamin B1) – C₁₂H₁₇ClN₄OS·HCl, threonine, thrombopoietin, thymidine, thymine, triazocine C, thyroid-stimulating hormone (TSH), thyrotropin-releasing hormone (TRH), thyroxine (T4), tocopherol (vitamin E), topoisomerase, triiodothyronine (T3) , transmembrane receptors, trichostatin A, tropin, trypsin, tryptophan, tubulin, tunicamycin, tyrosine, ubiquitin, uracil, urea, urease, uric acid – C5H4N4O3, uridine, valine, valinomycin, vanadium binding proteins (Vanabins), vasopressin, verruculogen, vitamins (in general), vitamin A (retinol), vitamin B, vitamin B1 (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.

In some embodiments, a target can be a protein target, such as, for example, a protein of the cellular environment (e.g., an intracellular or membrane protein). Examples of proteins include, but are not limited to, fibrous proteins, such as cytoskeletal proteins (e.g., actin, arp2/3, coronin, dystrophin, FtsZ, keratin, myosin, connexin, 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); amyloid P component and serum albumin), coagulation factors (e.g., complement proteins, C1-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; heme proteins; cell adhesion proteins (e.g., cadherins, ependymin, integrins, Ncam, and selectins); transmembrane trafficking proteins (e.g., CF TR, glycophorin D, and scramblase), such as ion channels (e.g., ligand-gated ion channels, such as nicotinic acetylcholine receptors and GABAa receptors, and voltage-gated ion channels, such as potassium, calcium, and sodium channels), symporter/antiporter proteins (e.g., glucose transporters); 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 and steroid hormones (such as androgens, estrogens, and progesterone); receptors, such as transmembrane receptors (e.g., G protein-coupled receptors, rhodopsin) and intracellular receptors (e.g., estrogen receptor); DNA binding proteins (e.g., histones, protamines, CI proteins); transcriptional 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.

In certain embodiments, the target can be a nucleic acid target, such as, for example, a nucleic acid of a cellular environment. As used herein with respect to a target, a docking strand, and an imaging strand, "nucleic acid" refers to a polymeric form of nucleotides of any length, such as deoxyribonucleotides or ribonucleotides, or their analogs. For example, the nucleic acid can be a DNA, RNA, or a DNA product of an RNA subjected to reverse transcription. Non-limiting examples of nucleic acids include: coding or non-coding regions of a gene or gene fragment, loci defined by linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant nucleic acids, branched nucleic acids, plasmids, vectors, isolated DNA with any sequence, isolated RNA with any sequence, nucleic acid probes, and primers. Other examples of nucleic acids include, but are not limited to, cDNA, aptamers, peptide nucleic acids ("PNA"), 2'-5' DNA (a synthetic material with a shortened backbone having base spacing that matches the A conformation of DNA; 2'-5' DNA will not typically hybridize with DNA in the B form, but it will readily hybridize with RNA), locked nucleic acids ("LNA"), and nucleic acids with modified backbones (e.g., base-modified or sugar-modified forms of naturally occurring nucleic acids). Nucleic acids may contain modified nucleotides, such as methylated nucleotides and nucleotide analogs ("analog" forms of purines and pyrimidines are well known in the art). If modifications to the nucleotide structure are present, they are performed before or after assembly of the polymer. The nucleic acid can be a single-stranded, double-stranded, partially single-stranded, or partially double-stranded DNA or RNA.

In certain embodiments, nucleic acid (e.g., nucleic acid target) is naturally occurring. As used herein, "naturally occurring" refers to a nucleic acid present in an organism or virus existing in nature in the absence of human intervention. In certain embodiments, nucleic acid is naturally present in an organism or virus. In certain embodiments, nucleic acid is genomic DNA, messenger RNA, ribosomal RNA, microRNA, pre-microRNA (pre-micro-RNA), pro-microRNA (pro-micro-RNA), viral DNA, viral RNA or piwi-RNA. In certain embodiments, nucleic acid target is not a synthetic DNA nanostructure (e.g., comprising two-dimensional (2-D) or three-dimensional (3-D) DNA nanostructures of two or more nucleic acids that hybridize to form a 2-D or 3-D nanostructure by Watson-Crick interactions).

The nucleic acid docking strands and imager strands described herein can be any of the nucleic acids described above (eg, DNA, RNA, modified nucleic acids, nucleic acid analogs, naturally occurring nucleic acids, synthetic nucleic acids).

Composition

Compositions provided herein include at least one or at least two (e.g., multiple) BP-NA conjugates (e.g., protein-nucleic acid conjugates) of the present invention. BP-NA conjugates can bind to a target of interest (e.g., a biomolecule) and/or stably bind to a complementary fluorescently labeled imager strand. Compositions can include multiple BP-NA conjugates of the same species or different species. In some embodiments, a composition can include at least 10, 50, 100, 500, 1000, 2000, 3000, 4000, 5000, ¹⁰⁴ , 50000, ¹⁰⁵ , ¹⁰⁵ , ¹⁰⁶ , ¹⁰⁷ , ¹⁰⁸ , ¹⁰⁹ , ¹⁰¹⁰ , or ¹⁰¹¹ BP-NA conjugates. In some embodiments, a composition may comprise at least 10, 50, 100, 500, 1000, 2000, 3000, 4000, 5000, ^{10 4} , 50000, ^{10 5} , 10 ⁵ , 10 6 , ^{10 7} , 10 ⁸ , 10 ⁹ , 10 ¹⁰ , 10 ¹¹ complementary fluorescently labeled imager strands. In some embodiments, a composition may contain 1 to about 200 or more BP-NA conjugates and/or imager strands of different species. For example, a composition can contain at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, or more different species. In some embodiments, a composition can contain less than about 5 to about 200 different species of BP-NA conjugates and/or imager strands. For example, a composition can contain less than 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, or 200 different species.

It should be understood that the number of complementary fluorescently labeled imager strands in the composition can be less than, equal to, or greater than the number of BP-NA conjugates in the composition.

Reagent test kit

The present invention further provides kits comprising one or more components of the present invention. The kits can include, for example, BP-NA conjugates and/or fluorescently labeled imager strands. The kits can also include components for producing BP-NA conjugates or for labeling imager strands. For example, the kits can include binding partners (e.g., antibodies), docking strands and intermediate linkers (such as, for example, biotin and streptavidin molecules), and/or imager strands. The kits can be used for purposes apparent to those skilled in the art, including those described above.

The kit may include other reagents, and, for example, buffers for performing hybridization reactions. The kit may also include instructions for using the kit components, and/or for making and/or using BP-NA conjugates and/or labeled imager strands.

application

The BP-NA conjugates (eg, protein-nucleic acid conjugates or antibody-nucleic acid conjugates) of the present invention can be used in, inter alia, any assay in which existing target detection technology is used.

Typically, the assay includes the following detection assays: including diagnostic assays, prognostic assays, patient monitoring assays, screening assays, biowarfare assays, forensic analysis assays, prenatal genetic diagnostic assays, etc. The assay can be an in vitro assay or an in vivo assay. The present invention provides the following advantages: many different targets can be analyzed simultaneously from a single sample using the methods of the present invention, even when such targets are spatially indistinguishable (and therefore spatially difficult to identify) using prior art imaging methods. This allows, for example, several diagnostic tests to be performed on a single sample.

BP-NA conjugates can also be used to simply visualize a region or area.

The methods of the present invention can be applied to analyze samples obtained or derived from a patient to determine whether a diseased cell type is present in the sample and/or to stage the disease. For example, a blood sample can be assayed according to any of the methods described herein to determine the presence and/or amount of a marker for a cancerous cell type in the sample, thereby diagnosing or staging the cancer.

Alternatively, the methods described herein can be used to diagnose pathogen infections, e.g., infections caused by intracellular bacteria and viruses, by determining the presence and/or amount of bacterial or viral markers, respectively, in a sample. Thus, the target detected using the compositions and methods of the invention can be a patient marker (such as a cancer marker) or a marker of infection by an external agent, such as a bacterial or viral marker.

The quantitative imaging methods of the present invention can be used, for example, to quantify targets (eg, target biomolecules) whose abundance is indicative of a physiological state or disease condition (eg, a blood marker that is upregulated or downregulated as a result of a disease state).

In addition, the compositions and methods of the present invention can be used to provide prognostic information that helps determine a patient's course of treatment. For example, the amount of a specific marker for a tumor can be accurately quantified from even a small sample from a patient. For certain diseases (like breast cancer), overexpression of certain proteins (such as Her2-neu) indicates that a more aggressive course of treatment will be needed.

The methods of the present invention can also be used to determine the effects of perturbations (including compounds, mutations, temperature changes, growth hormones, growth factors, diseases, or changes in culture conditions) on different targets, thereby identifying targets whose presence, absence, or levels are indicative of a specific biological state. In some embodiments, the present invention is used to elucidate and discover components and pathways of disease states. For example, comparison of the amount of a target present in a diseased tissue versus a "normal" tissue allows for the identification of important targets involved in the disease, thereby identifying targets for discovery/screening of novel drug candidates that can be used to treat the disease.

The sample analyzed can be a biological sample, such as blood, saliva, lymph, mucus, feces, urine, etc. The sample can be an environmental sample, such as a water sample, an air sample, a food sample, etc. The assay can be performed using one or more fixed components of the binding reaction. Therefore, the target or BP-NA conjugate can be fixed. The assay can be performed using one or more non-fixed components of the binding reaction. In view of the multiplexing potential provided by the BP-NA conjugate of the present invention and the fluorescently labeled imaging chain, these assays can involve substantially simultaneous detection of multiple targets in a sample. As an example, a determination can be used to detect a specific cell type (e.g., based on a specific cell surface receptor) and a specific genetic mutation in the specific cell type. In this way, as an example, the end user can determine how many cells of a specific type carry a mutation of interest.

Different embodiments

The present disclosure provides multiple embodiments including but not limited to the following numbered embodiments:

1. A method comprising

(1) contacting a sample to be tested for the presence of one or more targets with one or more target-specific binding partners, wherein each target-specific binding partner is linked to a docking strand, and wherein target-specific binding partners with different specificities are linked to different docking strands,

(2) optionally removing unbound target-specific binding partner,

(3) contacting the sample with a labeled imaging strand having a nucleotide sequence complementary to a docking strand,

(4) optionally removing unbound labeled imager strands,

(5) imaging the sample to detect the location and number of bound labeled imager strands,

(6) quenching the signal from the bound labeled imaging strand, and

(7) Repeat steps (3)-(6), each time using one labeled imager strand that has a unique nucleotide sequence relative to all other labeled imager strands.

2. The method of embodiment 1, wherein the sample is contacted with more than one target-specific binding partner in step (1).

3. The method of embodiment 1 or 2, wherein the target-specific binding partner is an antibody or antibody fragment.

4. The method of any one of embodiments 1-3, wherein the labeled imager strands are identically labeled.

5. The method of any one of embodiments 1-3, wherein each of the labeled imager strands comprises a different label.

6. The method of any one of embodiments 1-5, wherein the labeled imager strands are fluorescently labeled imager strands.

7. The method of any one of embodiments 1-6, wherein the one or more targets are proteins.

8. The method of any one of embodiments 1-7, wherein the sample is a cell, a cell lysate, or a tissue lysate.

9. The method of any one of embodiments 1-8, wherein the sample is imaged in step (5) using confocal or epifluorescence microscopy.

10. The method of any one of embodiments 1-9, wherein the quenching signal in step (6) comprises photobleaching.

11. A composition comprising:

a sample bound to more than one target-specific binding partner, each binding partner bound to a docking strand, and

At least one docking strand stably bound to one labeled imager strand.

12. A method comprising

(1) contacting a sample to be tested for the presence of one or more targets with one or more target-specific binding partners, wherein each target-specific binding partner is linked to a docking nucleic acid, and wherein target-specific binding partners with different specificities are linked to different docking nucleic acids,

(2) optionally removing unbound target-specific binding partner,

(3) contacting the sample with a labeled imaging nucleic acid having a nucleotide sequence complementary to a docking nucleic acid,

(4) optionally removing unbound labeled imaging nucleic acid,

(5) imaging the sample to detect the location and number of bound labeled imaging nucleic acids,

(6) removing the bound labeled imaging nucleic acid from the docking nucleic acid, and

(7) Repeat steps (3)-(6), each time using a labeled imaging nucleic acid having a unique nucleotide sequence relative to all other labeled imaging nucleic acids.

13. The method of embodiment 12, wherein the sample is contacted with more than one target-specific binding partner in step (1).

14. The method of embodiment 12 or 13, wherein the target-specific binding partner is an antibody or antibody fragment.

15. The method of embodiment 12 or 13, wherein the target-specific binding partner is a natural or engineered ligand, small molecule, aptamer, peptide, or oligonucleotide.

16. The method of any one of embodiments 12-15, wherein the labeled imaging nucleic acids are identically labeled.

17. The method of any one of embodiments 12-15, wherein the labeled imaging nucleic acids each comprise a different label.

18. The method of any one of embodiments 12-17, wherein the labeled imaging nucleic acids are fluorescently labeled imaging nucleic acids.

19. The method of any one of embodiments 12-18, wherein the one or more targets are proteins.

20. The method of any one of embodiments 12-19, wherein the sample is a cell, a cell lysate, or a tissue lysate.

21. The method of any one of embodiments 12-20, wherein the sample is imaged in step (5) using confocal or epifluorescence microscopy.

22. The method of any one of embodiments 12-21, wherein the labeled imaging nucleic acids are removed from the docking nucleic acid by reducing salt concentration, adding a denaturant, or increasing temperature.

23. The method of embodiment 22, wherein the salt is Mg++.

24. The method of embodiment 22, wherein the denaturing agent is formamide, urea, or DMSO.

25. The method of any one of embodiments 12-21, wherein the labeled imaging nucleic acid is not removed from the docking nucleic acid by strand displacement in the presence of a competitor nucleic acid.

26. The method of any one of embodiments 12-21, wherein the labeled imaging nucleic acid is removed from the docking nucleic acid by chemically, photochemically, or enzymatically cleaving, modifying, or degrading the labeled imaging nucleic acid.

27. The method of any one of embodiments 12-21, wherein when the labeled imaging nucleic acid is bound to its corresponding docking nucleic acid, no single-stranded regions are present on the imaging nucleic acid or the docking nucleic acid.

28. The method of any one of embodiments 12-21, wherein the labeled imaging nucleic acid is not self-quenching.

29. The method of any one of embodiments 12-28, wherein the unbound docking nucleic acid is partially double-stranded.

30. The method of any one of embodiments 12-28, wherein the unbound imaging nucleic acid is partially double-stranded.

31. The method of any one of embodiments 12-28, wherein the imaging nucleic acid is a molecular beacon or comprises a hairpin secondary structure.

32. The method of any one of embodiments 12-27 and 29-31, wherein the imaging nucleic acid is a molecular beacon or comprises a self-quenching hairpin secondary structure.

33. The method of any one of embodiments 12-28, wherein the imaging nucleic acid is a half-duplex.

34. The method of embodiment 33, wherein the half-duplex is self-quenching.

35. The method of any one of embodiments 12-34, wherein the docking nucleic acid comprises a hairpin secondary structure.

36. The method of any one of embodiments 12-35, wherein the imaging nucleic acid is bound to signaling moieties via a dendritic structure or a polymeric structure.

34. A method comprising

(1) contacting a sample to be tested for the presence of one or more targets with one or more target-specific binding partners, wherein each target-specific binding partner is linked to a docking nucleic acid, and wherein target-specific binding partners with different specificities are linked to different docking nucleic acids,

(2) optionally removing unbound target-specific binding partner,

(3) contacting the sample with a labeled imaging nucleic acid having a nucleotide sequence complementary to a docking nucleic acid,

(4) optionally removing unbound labeled imaging nucleic acid,

(5) imaging the sample to detect the location and number of bound labeled imaging nucleic acids,

(6) inactivating the bound labeled imaging nucleic acids by removing or modifying their signaling moieties without completely removing the imaging nucleic acids, and

(7) Repeat steps (3)-(6), each time using a labeled imaging nucleic acid having a unique nucleotide sequence relative to all other labeled imaging nucleic acids.

35. The method of embodiment 34, wherein the sample is contacted with more than one target-specific binding partner in step (1).

36. The method of embodiment 34 or 35, wherein the target-specific binding partner is an antibody or antibody fragment.

37. The method of embodiment 34 or 35, wherein the target-specific binding partner is a natural or engineered ligand, small molecule, aptamer, peptide, or oligonucleotide.

38. The method of any one of embodiments 34-37, wherein the labeled imaging nucleic acids are identically labeled.

39. The method of any one of embodiments 34-37, wherein the labeled imaging nucleic acids each comprise a different label.

40. The method of any one of embodiments 34-39, wherein the labeled imaging nucleic acids are fluorescently labeled imaging nucleic acids.

41. The method of any one of embodiments 34-40, wherein the one or more targets are proteins.

42. The method of any one of claims 34-41, wherein the sample is a cell, a cell lysate, or a tissue lysate.

43. The method of any one of embodiments 34-42, wherein the sample is imaged in step (5) using confocal or epifluorescence microscopy.

44. The method of any one of embodiments 34-43, wherein the unbound docking nucleic acid is partially double-stranded.

45. The method of any one of embodiments 34-43, wherein the unbound imaging nucleic acid is partially double-stranded.

46. The method of any one of embodiments 34-45, wherein the imaging nucleic acid is a molecular beacon or comprises a hairpin secondary structure.

47. The method of any one of embodiments 34-45, wherein the imaging nucleic acid is a molecular beacon or comprises a self-quenching hairpin secondary structure.

48. The method of any one of embodiments 34-45, wherein the imaging nucleic acid is a half-duplex.

49. The method of embodiment 48, wherein the half-duplex is self-quenching.

50. The method of any one of embodiments 34-49, wherein the docking nucleic acid comprises a hairpin secondary structure.

51. The method of any one of embodiments 34-50, wherein the imaging nucleic acid is bound to multiple signaling moieties via a dendritic structure or a polymeric structure.

Equivalent form

Although several inventive embodiments have been described and shown herein, a person of ordinary skill in the art will readily imagine a variety of other devices and/or structures for performing the functions and/or obtaining the results and/or one or more of the advantages described herein, and each of such variations and/or modifications is considered to be within the scope of the inventive embodiments described herein. More generally, a person of ordinary skill in the art will readily understand that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary, and that actual parameters, dimensions, materials, and/or configurations will depend on one or more specific applications for which the invention is taught. A person of ordinary skill in the art will recognize or be able to confirm many equivalents to the specific inventive embodiments described herein using only routine experimentation. Therefore, it should be understood that the foregoing embodiments are presented by way of example only, and within the scope of the appended claims and their equivalents, the inventive embodiments may be practiced in a manner different from that specifically described and required. The inventive embodiments of the present disclosure relate to each individual feature, system, article, material, kit, and/or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits and/or methods is included within the scope of the invention of the present disclosure if such features, systems, articles, materials, kits and/or methods are not mutually inconsistent. All definitions, as defined and used herein, should be understood to supersede dictionary definitions, definitions in documents incorporated by reference, and/or ordinary meanings of the defined terms.

All references, patents, and patent applications disclosed herein are incorporated by reference with respect to each cited subject matter, which in some cases may include the entire contents of the documents.

Unless clearly indicated otherwise, the indefinite articles "a" and "an" as used herein should be understood to mean "at least one."

In the specification and in the claims, the phrase "and/or" as used herein should be understood to mean "any one or both" of the elements so conjoined, i.e., the elements are present jointly in some cases and separately in other cases. Multiple elements listed with "and/or" should be interpreted in the same manner, i.e., "one or more" of the elements so conjoined. In addition to the elements specifically identified by the "and/or" clause, other elements may optionally be present, whether related or unrelated to those specifically identified. Thus, as a non-limiting example, when used in conjunction with open language such as "comprising," a reference to "A and/or B" may, in one embodiment, refer to only A (optionally including elements other than B); in another embodiment, to only B (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); and so on.

In the specification and in the claims, "or" as used herein should be understood to have the same meaning as "and/or" as defined above. For example, when separating items in a list, "or" or "and/or" should be interpreted as inclusive, that is, including at least one of a plurality of elements or a list of elements, but also including more than one thereof, and optionally, additional unlisted items. Only terms that clearly indicate the contrary, such as "only one of..." or "exactly one of...", or when used in the claims, "consisting of..." will mean including exactly one element of some or a list of elements. In general, the term "or" as used herein, when preceded by an exclusive term, such as "any one of," "one of...", "only one of..." or "only one of...", should be interpreted only to indicate exclusive alternatives (i.e., "one or the other, but not both"). "Consisting essentially of...", when used in the claims, should have its ordinary meaning as used in the art of patent law.

As used herein, the phrase "at least one" in relation to a list of one or more elements should be understood to mean at least one element selected from any one or more elements in the list of elements, but not necessarily including at least one of each element specifically listed in the list of elements, and not excluding any combination of multiple elements in the list of elements. This definition also allows that elements other than the elements specifically identified in the list of elements to which the phrase "at least one" refers may optionally be present, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, "at least one of A and B" (or equivalently, "at least one of A or B," or equivalently, "at least one of A and/or B") may, in one embodiment, mean at least one, optionally including more than one A, without B (and optionally including elements in addition to B); in another embodiment, mean at least one, optionally including more than one B, without A (and optionally including elements in addition to A); in yet another embodiment, mean at least one, optionally including more than one A, and at least one, optionally including more than one B (and optionally including other elements); and so on.

It should also be understood that, unless explicitly stated to the contrary, in any method claimed herein that includes more than one step or action, the order of the method steps or actions is not necessarily limited to the order in which the method steps or actions are listed.

In the claims, as well as in the foregoing description, all transitional phrases such as "comprising," "including," "carrying," "having," "containing," "involving," "having," "consisting of," and the like are to be construed as open-ended, i.e., meaning including but not limited to. Only the transitional phrases "consisting of" and "consisting essentially of" are to be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03.

Claims (121)

1. A method, comprising (1) A sample to be tested for the presence of one or more targets is brought into contact with one or more target-specific binding couplers, wherein each target-specific binding coupler is connected to a docking chain, and wherein target-specific binding couplers with different specificities are connected to different docking chains. (2) Optional removal of unbound target-specific binding couplers, (3) The sample is brought into contact with a labeled imaging strand having a nucleotide sequence complementary to the docking strand to produce a labeled imaging strand that is stably bound to the docking strand. (4) Optionally remove unbound labeled imaging chains, (5) Image the sample to detect the location and number of labeled imaging chains that are stably bound to the docking chain. (6) Quenching the signal from the bound labeled imaging chain, or removing the bound labeled imaging chain from the docking chain by enzymatic cleavage, modification, or degradation of the labeled imaging chain, and (7) Repeat steps (3)-(6), each time using a labeled imaging strand with a unique nucleotide sequence relative to all other labeled imaging strands. 2. The method of claim 1, wherein the sample is in contact with more than one target-specific binding partner in step (1). 3. The method of claim 1, wherein the target-specific binding partner is an antibody or antibody fragment. 4. The method of claim 1, wherein the labeled imaging chains are labeled identically. 5. The method of claim 1, wherein each of the labeled imaging chains contains a different label. 6. The method of claim 1, wherein the labeled imaging chains are fluorescently labeled imaging chains. 7. The method of claim 1, wherein the one or more targets are proteins. 8. The method of claim 1, wherein the sample is a cell or tissue sample, cell lysate or tissue lysate or body fluid. 9. The method of claim 1, wherein the sample is imaged in step (5) using confocal or epifluorescence microscopy. 10. The method of claim 1, wherein the quenching signal in step (6) includes photobleaching. 11. A method comprising (1) A sample to be tested for the presence of one or more targets is brought into contact with one or more target-specific binding couplers, wherein each target-specific binding coupler is connected to a docking chain, and wherein target-specific binding couplers with different specificities are connected to different docking chains. (2) Optional removal of unbound target-specific binding couplers, (3) The sample is brought into contact with a labeled imaging strand having a nucleotide sequence complementary to the docking strand to produce a labeled imaging strand that is stably bound to the docking strand. (4) Optionally remove unbound labeled imaging chains, (5) Image the sample to detect the location and number of labeled imaging chains that are stably bound to the docking chain. (6) Remove the bound, labeled imaging chain from the docking chain by changing the temperature and/or buffer conditions, and (7) Repeat steps (3)-(6), each time using a labeled imaging strand with a unique nucleotide sequence relative to all other labeled imaging strands. 12. The method of claim 11, wherein the sample is in contact with more than one target-specific binding partner in step (1). 13. The method of claim 11, wherein the target-specific binding partner is an antibody or antibody fragment. 14. The method of claim 11, wherein the target-specific binding ligand is a natural or engineered ligand. 15. The method of claim 11, wherein the target-specific binding partner is a small molecule. 16. The method of claim 11, wherein the target-specific binding partner is a peptide. 17. The method of claim 11, wherein the target-specific binding partner is an oligonucleotide. 18. The method of claim 11, wherein the labeled imaging chains are labeled identically. 19. The method of claim 11, wherein each of the labeled imaging chains contains a different label. 20. The method of claim 11, wherein the labeled imaging chains are fluorescently labeled imaging chains. 21. The method of claim 11, wherein the one or more targets are proteins. 22. The method of claim 11, wherein the sample is a cell, cell lysate, or tissue lysate. 23. The method of claim 11, wherein the sample is imaged in step (5) using confocal or epifluorescence microscopy. 24. The method of claim 11, wherein the labeled imaging chains are removed from the docking chains by reducing the salt concentration, adding a denaturing agent, or increasing the temperature. 25. The method of claim 24, wherein the salt is ^Mg²⁺ . 26. The method of claim 24, wherein the denaturant is formamide, urea, or DMSO. 27. The method of claim 11, wherein, in the presence of a competing nucleic acid, the labeled imaging strands are removed from the docking strands without strand substitution. 28. The method of claim 1, wherein the unbonded docking strands are partially bistranded. 29. The method of claim 1, wherein the unbound imaging strand is partially bistranded. 30. The method of claim 1, wherein the imaging chain is a molecular beacon. 31. The method of claim 1, wherein the imaging chain includes a hairpin secondary structure. 32. The method of claim 1, wherein the imaging chain includes a self-quenching hairpin secondary structure. 33. The method of claim 1, wherein the imaging chain is a semi-double chain. 34. The method of claim 33, wherein the semi-bichain is self-quenched. 35. The method of claim 1, wherein the docking chain includes a hairpin secondary structure. 36. The method of claim 1, wherein the imaging chain is combined to a plurality of signal transmitting parts by a dendritic structure or a polymeric structure. 37. A method comprising (1) A sample to be tested for the presence of one or more targets is brought into contact with one or more target-specific binding couplers, wherein each target-specific binding coupler is connected to a docking chain, and wherein target-specific binding couplers with different specificities are connected to different docking chains. (2) Optional removal of unbound target-specific binding couplers, (3) The sample is brought into contact with a labeled imaging strand having a nucleotide sequence complementary to the docking strand to produce a labeled imaging strand that is stably bound to the docking strand. (4) Optionally remove unbound labeled imaging chains, (5) Image the sample to detect the location and number of labeled imaging chains that are stably bound to the docking chain. (6) These bound labeled imaging chains are inactivated by removing or modifying their signal emission portions without completely removing the imaging chain, and (7) Repeat steps (3)-(6), each time using a labeled imaging strand with a unique nucleotide sequence relative to all other labeled imaging strands. 38. The method of claim 37, wherein the sample is contacted with more than one target-specific binding partner in step (1). 39. The method of claim 37, wherein the target-specific binding coupler is an antibody or antibody fragment. 40. The method of claim 37, wherein the target-specific binding ligand is a natural or engineered ligand. 41. The method of claim 37, wherein the target-specific binding partner is a small molecule. 42. The method of claim 37, wherein the target-specific binding partner is a peptide. 43. The method of claim 37, wherein the target-specific binding coupler is an oligonucleotide. 44. The method of claim 37, wherein the labeled imaging chains are labeled identically. 45. The method of claim 37, wherein each of the labeled imaging chains contains a different label. 46. The method of claim 37, wherein the labeled imaging chains are fluorescently labeled imaging chains. 47. The method of claim 37, wherein the one or more targets are proteins. 48. The method of claim 37, wherein the sample is a cell, cell lysate, or tissue lysate. 49. The method of claim 37, wherein the sample is imaged in step (5) using confocal or epifluorescence microscopy. 50. The method of claim 37, wherein the unbonded docking strands are partially bistranded. 51. The method of claim 37, wherein the unbound imaging strand is partially double-stranded. 52. The method of claim 37, wherein the imaging chain is a molecular beacon. 53. The method of claim 37, wherein the imaging chain includes a hairpin secondary structure. 54. The method of claim 37, wherein the imaging chain includes a self-quenching hairpin secondary structure. 55. The method of claim 37, wherein the imaging chain is a semi-double chain. 56. The method of claim 55, wherein the semi-bichain is self-quenched. 57. The method of claim 37, wherein the docking chain includes a hairpin secondary structure. 58. The method of claim 37, wherein the imaging chain is combined to a plurality of signal transmitting sections via a dendritic structure or a polymeric structure. 59. The method of claim 1, wherein the quenching step removes the signal from the combined labeled imaging chain. 60. The method of claim 1, wherein the quenching step reduces the signal from the combined labeled imaging chain. 61. The method of claim 1, wherein the detection includes detecting the number of bound labeled imaging chains. 62. The method of claim 1, wherein the detection includes detecting the location of the combined labeled imaging chain. 63. The method of claim 1, wherein the labeled imaging chain is labeled with signal emitting molecules and a plurality of copies of the signal emitting molecules are recruited by combining the imaging chain with the docking chain. 64. The method of claim 63, wherein the imaging chain is a branched imaging chain. 65. The method of claim 1, wherein the docking chain may be part of a multi-chain complex. 66. The method of claim 1, wherein the imaging chain and/or docking chain comprises a secondary structure. 67. The method of claim 1, wherein at least one docking chain is stably bonded to the labeled imaging chain. 68. The method of claim 1, wherein all imaging chains are labeled with the same fluorophore. 69. The method of claim 68, wherein the fluorophore is a red fluorophore. 70. The method of claim 1, wherein the imaging chain is incorporated into a plurality of signal-emitting molecules. 71. The method of claim 1, wherein at least some of the labeled imaging chains are identically labeled, and at least some of the labeled imaging chains contain different markers. 72. The method of claim 1, wherein step (1) comprises contacting the sample with a plurality of different target-specific binding partners attached to the docking chain. 73. The method of claim 1, wherein the imaging strand and the docking strand are complementary oligonucleotides. 74. The method of claim 1, wherein step (6) comprises removing the bound labeled imaging chain from the docking chain, wherein the labeled imaging chain is removed from the docking chain by enzymatic cleavage, modification or degradation of the labeled imaging chain. 75. The method of any one of claims 1, 11, 37 or 74, wherein the imaging chain and the docking chain remain joined together for at least 30 minutes. 76. The method of any one of claims 1, 11, 37 or 74, wherein the imaging chain and the docking chain remain joined together for at least 35 minutes. 77. The method of any one of claims 1, 11, 37 or 74, wherein the imaging chain and the docking chain remain joined together for at least 40 minutes. 78. The method of any one of claims 1, 11, 37 or 74, wherein the imaging chain and the docking chain remain joined together for at least 45 minutes. 79. The method of any one of claims 1, 11, 37 or 74, wherein the imaging chain and the docking chain remain joined together for at least 50 minutes. 80. The method of any one of claims 1, 11, 37 or 74, wherein the imaging chain and the docking chain remain joined together for at least 55 minutes. 81. The method of any one of claims 1, 11, 37 or 74, wherein the imaging chain and the docking chain remain joined together for at least 60 minutes. 82. The method of any one of claims 1, 11, 37 or 74, wherein the imaging chain and the docking chain remain bonded to each other for at least 2 hours. 83. The method of any one of claims 1, 11, 37 or 74, wherein the imaging chain and the docking chain remain joined together for 30 to 60 minutes. 84. The method of any one of claims 1, 11, 37 or 74, wherein the imaging chain and the docking chain remain joined together for 30 to 120 minutes. 85. The method of any one of claims 1, 11, 37 or 74, wherein the imaging chain and the docking chain remain joined together for 40 to 50 minutes. 86. The method of any one of claims 1, 11, 37 or 74, wherein the imaging chain and the docking chain remain joined together for 40 to 120 minutes. 87. The method of any one of claims 1, 11, 37 or 74, wherein the imaging chain and the docking chain remain joined together for 60 to 120 minutes. 88. The method of claim 74, wherein the labeled imaging strand is enzymatically removed. 89. The method of claim 88, wherein the labeled imaging strand is a labeled imaging nucleic acid strand and the docking strand is a docking nucleic acid strand, and wherein the labeled imaging nucleic acid strand and the docking nucleic acid strand are complementary nucleic acids. 90. The method of claim 89, wherein the labeled imaging strand is cleaved by a nucleic acid glycosylation enzyme, and wherein the nucleic acid glycosylation enzyme is a uracil-DNA glycosylation enzyme. 91. The method of claim 89, wherein the labeled imaging strand is removed by at least one nicking enzyme or restriction endonuclease. 92. The method of claim 89, wherein the labeled imaging strand is removed by cleavage at one or more ribonucleotides using RNase. 93. The method of claim 74, wherein the marked imaging chain includes a light-cuttable portion that is exposed by UV light. 94. The method of claim 89, wherein the labeled imaging strand is replaced by a competitive nucleic acid. 95. The method of claim 89, wherein the labeled imaging chain is removed by cleavage at one or more deoxyuridine bases. 96. The method of claim 95, wherein cleavage at one or more deoxyuridine bases is performed by the USER enzyme. 97. The method of claim 96, wherein in step (1), the sample is contacted with more than one target-specific binding partner. 98. The method of claim 96, wherein the target-specific binding partner is an antibody or an antibody fragment. 99. The method of claim 96, wherein the target-specific binding mate is a natural or engineered ligand. 100. The method of claim 96, wherein the target-specific binding partner is a small molecule. 101. The method of claim 96, wherein the target-specific binding partner is a peptide. 102. The method of claim 96, wherein the target-specific binding partner is an oligonucleotide. 103. The method of claim 96, wherein the labeled imaging chains are identically labeled. 104. The method of claim 96, wherein the labeled imaging chain comprises different markers. 105. The method of claim 96, wherein the labeled imaging chain is a fluorescently labeled imaging chain. 106. The method of claim 96, wherein one or more targets are proteins and/or the sample is a cell or tissue sample, cell lysate or tissue lysate or body fluid. 107. The method of claim 96, wherein the sample is imaged in step (5) using confocal or epifluorescence microscopy. 108. The method of claim 74, wherein the labeled imaging chain and/or docking chain have a foothold sequence. 109. The method of claim 108, wherein the imaging chain has a foothold sequence. 110. The method of claim 109, wherein in step (1), the sample is contacted with more than one target-specific binding partner. 111. The method of claim 109, wherein the target-specific binding partner is an antibody or an antibody fragment. 112. The method of claim 109, wherein the target-specific binding mate is a natural or engineered ligand. 113. The method of claim 109, wherein the target-specific binding partner is a small molecule. 114. The method of claim 109, wherein the target-specific binding partner is a peptide. 115. The method of claim 109, wherein the target-specific binding partner is an oligonucleotide. 116. The method of claim 109, wherein the labeled imaging chains are identically labeled to each other. 117. The method of claim 109, wherein each of the labeled imaging chains comprises a different marker. 118. The method of claim 109, wherein the labeled imaging chain is a fluorescently labeled imaging chain. 119. The method of claim 109, wherein one or more targets are proteins and/or the sample is a cell or tissue sample, cell lysate or tissue lysate or body fluid. 120. The method of claim 109, wherein the sample is imaged in step (5) using confocal or epifluorescence microscopy. 121. The method of claim 1, 11 or 37, wherein the target-specific binding mate is an aptamer.