Improved methods for multiplex imaging using labeled nucleic acid imaging agents

Abstract

To provide certain advantageous ways of conducting multiplexed imaging.SOLUTION: Provided is a method to test a sample for the presence of one or more targets, comprising: contacting a sample being tested for the presence of one or more targets with one or more target-specific binding partners, each target- specific binding partner being linked to a nucleic acid strand, and target-specific binding partners of different specificity being linked to different nucleic acid strands; contacting the sample with a nonlinear amplifier strand having complementarity to a nucleic acid strand, the nucleic acid strand being either a docking strand or a primer strand; imaging the sample to detect bound labeled imager strands; and removing the bound labeled imager strands.SELECTED DRAWING: Figure 1D

Description

[Technical field]

[0001] The present application relates generally to the field of analyte (eg, target) detection and quantification. [Background technology]

[0002] Fluorescence microscopy is a powerful tool for investigating molecules, for example in biological systems. However, the number of different species that can be distinguished and simultaneously visualized (i.e., multiplexing magnification) is limited by the spectral overlap between fluorophores. Although some multiplexing imaging methods are known, they may not adequately emit a sufficiently strong signal or may require specific switching means between the targets being imaged. Therefore, there is a need for new and improved multiplexing imaging methods with improved amplification methods and additional switching means between the targets being imaged. Summary of the Invention

[0003] According to the present description, in some embodiments, a method for testing a sample for the presence of one or more targets comprises: (1) contacting a sample to be tested for the presence of one or more targets with one or more target-specific binding partners, each target-specific binding partner being bound to a nucleic acid strand, and target-specific binding partners of different specificities being bound to different nucleic acid strands; (2) optionally removing unbound target-specific binding partners; and (3) contacting the sample with a nonlinear amplifier strand having complementarity to the nucleic acid strand, the nucleic acid strand being either a docking strand or a primer strand; (4) optionally removing unbound nonlinear amplifier strands; (5) amplifying the docking strand by rolling circle amplification in either one or two steps and contacting the sample with a labeled imager strand that has complementarity to the docking strand or the amplified strand; (6) imaging the sample to detect bound labeled imager strands; and (7) removing the bound labeled imager strand; and (8) optionally repeating steps (1)-(8), or any subset thereof.

[0004] In some embodiments, a method for testing a sample for the presence of one or more targets comprises: (1) contacting the sample with one or more target-specific binding partners, each target-specific binding partner being bound, directly or indirectly, to a nucleic acid strand, and target-specific binding partners of different specificities being bound to different nucleic acid strands; (2) optionally removing unbound target-specific binding partners; and (3) The nucleic acid strand is a docking strand or a primer strand, and the nucleic acid is (a) if docking strands, optionally increasing the number of docking strands associated with each target-specific binding partner; or (b) if a primer strand, optionally associating two or more docking strands with the primer strand; (4) contacting the sample with a labeled imager strand capable of binding directly or indirectly to the docking strand; (5) optionally removing unbound labeled imager strands; (6) imaging the sample to detect bound labeled imager strands; and (7) optionally quenching the signal from the bound labeled imager strand; (8) optionally repeating steps (1) through (7), or any subset thereof.

[0005] In some embodiments, the composition comprises a sample bound to two or more target-specific binding partners, each binding partner bound to a nucleic acid strand and at least one docking strand stably bound directly or indirectly to a labeled imager strand, the nucleic acid strand being a docking strand or a primer strand, the nucleic acid being: (a) if a docking strand, increasing the number of docking strands associated with each target-specific binding partner; or (b) If it is a primer strand, two or more docking strands are associated with the primer strand.

[0006] In some embodiments, the composition comprises: (1) Signs; (2) a first nucleic acid domain, a second nucleic acid domain, and a third nucleic acid domain, each nucleic acid domain being 1 to 9 nucleotides in length; (3) a first binding moiety that links the first nucleic acid domain and the second nucleic acid domain; (4) a second binding moiety that links the second nucleic acid domain and the third nucleic acid domain; Both linking moieties are independently selected from (a) an abasic site with an intact phosphodiester backbone, (b) a linker that is cleavable by a nucleic acid glycosylase, (c) a non-naturally occurring nucleotide, or (d) a restriction site or a nicking site.

[0007] Additional objects and advantages will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice. The objects and advantages will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims.

[0008] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the scope of the claims.

[0009] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate one (or several) embodiment(s) and, together with the description, explain the principles described herein. [Brief description of the drawings]

[0010] [Figure 1A-C] A-E show schemes for attaching a docking strand to a target via a target recognition moiety. Specifically, FIG. 1A shows the attachment of a docking strand to a target recognition moiety without signal amplification. FIG. 1B shows the attachment of a docking strand to a target recognition moiety with signal amplification using a branched structure that can be made using a process such as HCR. FIG. 1C shows the attachment of a docking strand to a target recognition moiety with signal amplification using a linear structure that can be made using a process such as RCA. FIG. 1D shows a modified hybridization chain reaction (HCR) in which a docking site (domain b) is attached to one of the two hairpins of the HCR, allowing the introduction of multiple docking sites to one target recognition moiety. FIG. 1E shows rolling circle amplification to introduce multiple docking sites (domains c-d) to one target recognition moiety. The following reference numbers are used in this figure: 101: target. 102: target recognition moiety. 103: docking strand. 120: primer strand of the HCR reaction attached to a target recognition moiety. 121: One hairpin of HCR attached with a docking site. 122: Another hairpin of HCR. 123-126: Sequential hairpin assembly reactions. 127: Primer attached with a target recognition moiety. 128: Linear template that can be circularized by ligation. 129: Ligation reaction. 130: Primer extension with a DNA polymerase with strand displacement activity. 131: Multiple docking sites. [Figure 1D]A-E show schemes for attaching a docking strand to a target via a target recognition moiety. Specifically, FIG. 1A shows the attachment of a docking strand to a target recognition moiety without signal amplification. FIG. 1B shows the attachment of a docking strand to a target recognition moiety with signal amplification using a branched structure that can be made using a process such as HCR. FIG. 1C shows the attachment of a docking strand to a target recognition moiety with signal amplification using a linear structure that can be made using a process such as RCA. FIG. 1D shows a modified hybridization chain reaction (HCR) in which a docking site (domain b) is attached to one of the two hairpins of the HCR, allowing the introduction of multiple docking sites to one target recognition moiety. FIG. 1E shows rolling circle amplification to introduce multiple docking sites (domains c-d) to one target recognition moiety. The following reference numbers are used in this figure: 101: target. 102: target recognition moiety. 103: docking strand. 120: primer strand of the HCR reaction attached to a target recognition moiety. 121: One hairpin of HCR attached with a docking site. 122: Another hairpin of HCR. 123-126: Sequential hairpin assembly reactions. 127: Primer attached with a target recognition moiety. 128: Linear template that can be circularized by ligation. 129: Ligation reaction. 130: Primer extension with a DNA polymerase with strand displacement activity. 131: Multiple docking sites. [Figure 1E]A-E show schemes for attaching a docking strand to a target via a target recognition moiety. Specifically, FIG. 1A shows the attachment of a docking strand to a target recognition moiety without signal amplification. FIG. 1B shows the attachment of a docking strand to a target recognition moiety with signal amplification using a branched structure that can be made using a process such as HCR. FIG. 1C shows the attachment of a docking strand to a target recognition moiety with signal amplification using a linear structure that can be made using a process such as RCA. FIG. 1D shows a modified hybridization chain reaction (HCR) in which a docking site (domain b) is attached to one of the two hairpins of the HCR, allowing the introduction of multiple docking sites to one target recognition moiety. FIG. 1E shows rolling circle amplification to introduce multiple docking sites (domains c-d) to one target recognition moiety. The following reference numbers are used in this figure: 101: target. 102: target recognition moiety. 103: docking strand. 120: primer strand of the HCR reaction attached to a target recognition moiety. 121: One hairpin of HCR attached with a docking site. 122: Another hairpin of HCR. 123-126: Sequential hairpin assembly reactions. 127: Primer attached with a target recognition moiety. 128: Linear template that can be circularized by ligation. 129: Ligation reaction. 130: Primer extension with a DNA polymerase with strand displacement activity. 131: Multiple docking sites. [Diagram 2] Sequential amplification, polymerization, and dendrimerization for an amplified and removable signal are shown. [Diagram 3] Shown is simultaneous amplification, polymerization, and dendrimerization for an amplified and removable signal. [Figure 4A] Sequential imaging with successive amplification from an HRP-like enzyme is shown. [Figure 4B] Shown is simultaneous imaging with sequential amplification from an HRP-like enzyme. [Diagram 5]A-D show the removal of the imager strand using a nuclease. (A) General scheme. (B) An embodiment where there is a single deoxyuridine (dU) nucleotide in the docking strand recognition portion of the imager strand. (C) An embodiment where there are multiple dU nucleotides in the docking strand recognition portion of the imager strand. (D) An embodiment where a dU nucleotide is located in the linkage between the docking strand recognition portion of the imager strand and the signal generation portion. 104: Imager strand. 105: Signal generation portion of the imager strand. 106: Linkage between the target recognition portion and the docking strand. 107: Additional linkage to additional docking strands. 120: Attached hybridization chain reaction (HCR) primer strand. 201: dU as an example of a moiety that can be enzymatically degraded. 202: Enzymatic reaction to degrade dU. 203: Process where the remainder of the degradative reaction spontaneously dissociates from the docking strand. [Figure 6A-C]A-F show the removal of the imager strand using a polymerase enzyme. (A) A self-priming hairpin is placed at the 3' end of the imager strand, and the imager strand is removed using a polymerase with strand displacement activity (e.g., phi29). (B) A self-priming hairpin is placed at the 3' end of the imager strand that is linked to a signal generating moiety via nucleic acid hybridization, and the imager strand is removed using a polymerase with strand displacement activity. (C) A self-priming hairpin is placed at the 3' end of the docking strand, and the imager strand is removed using a polymerase with strand displacement activity. (D) A self-priming hairpin is placed at the 3' end of the imager strand, and the imager strand is removed using a polymerase with 5' to 3' exonuclease activity (e.g., DNA polymerase I). (E) A self-priming hairpin is placed at the 3' end of the docking strand, and the imager strand is removed using a polymerase with 5' to 3' exonuclease activity. (F) The self-priming hairpin is displaced by a hybridized duplex with an extendable 3' end. 301: Self-priming hairpin. 302: A reaction in which the self-priming hairpin or hybridized duplex with an extendable 3' end is extended by a DNA polymerase with strand displacement activity. 303: A short oligonucleotide that directs a signal generating moiety to an imager strand via hybridization. 304: A reaction in which the self-priming hairpin or hybridized duplex with an extendable 3' end is extended by a DNA polymerase with 5' to 3' exonuclease activity. 305: A hybridized duplex with an extendable 3' end. 306: A linkage between a target recognition moiety and a docking strand, the linkage comprising a covalent or non-covalent interaction. [Figure 6D-F]A-F show the removal of the imager strand using a polymerase enzyme. (A) A self-priming hairpin is placed at the 3' end of the imager strand, and the imager strand is removed using a polymerase with strand displacement activity (e.g., phi29). (B) A self-priming hairpin is placed at the 3' end of the imager strand that is linked to a signal generating moiety via nucleic acid hybridization, and the imager strand is removed using a polymerase with strand displacement activity. (C) A self-priming hairpin is placed at the 3' end of the docking strand, and the imager strand is removed using a polymerase with strand displacement activity. (D) A self-priming hairpin is placed at the 3' end of the imager strand, and the imager strand is removed using a polymerase with 5' to 3' exonuclease activity (e.g., DNA polymerase I). (E) A self-priming hairpin is placed at the 3' end of the docking strand, and the imager strand is removed using a polymerase with 5' to 3' exonuclease activity. (F) The self-priming hairpin is displaced by a hybridized duplex with an extendable 3' end. 301: Self-priming hairpin. 302: A reaction in which the self-priming hairpin or hybridized duplex with an extendable 3' end is extended by a DNA polymerase with strand displacement activity. 303: A short oligonucleotide that directs a signal generating moiety to an imager strand via hybridization. 304: A reaction in which the self-priming hairpin or hybridized duplex with an extendable 3' end is extended by a DNA polymerase with 5' to 3' exonuclease activity. 305: A hybridized duplex with an extendable 3' end. 306: A linkage between a target recognition moiety and a docking strand, the linkage comprising a covalent or non-covalent interaction. [Figure 7A]A-G provide images after amplification. Figure 7A provides a schematic diagram showing the switching process between fluorescent and bright-field imaging using DNA exchange and HRP-based amplification. Figure 7B provides the resulting fluorescent image of tonsil tissue cytokeratin labeled with antibody-DNA conjugate and the corresponding fluorescently labeled imager strand. Figure 7C provides the resulting bright-field immunohistochemistry image of cytokeratin in the same tonsil tissue after DNA exchange and HRP-based chromogenic amplification. Figures 7D and E show the fluorescent signal obtained from a cell sample stained for vimentin and DAPI without amplification (D, left) and with rolling circle amplification (E, right). Figure 7F provides the resulting fluorescent image of tonsil tissue cytokeratin labeled with antibody-DNA conjugate and the corresponding fluorescently labeled imager strand (F, left). Figure 7G, right, shows the fluorescent image from the same tissue section after HCR amplification of the docking strand and hybridization of the corresponding fluorescently labeled imager strand. In Figures 7D and 7E, the images show blue DAPI nuclear staining, which typically appears as roughly round or bean-shaped objects located in the central region of the cell. DAPI stains DNA inside the cell's nucleus and was included in the images as evidence that cells were present within the field of view. Vimentin staining (red) in Figure 7D was lower without amplification and brighter and more intense with amplification in Figure 7E. The red vimentin staining shows mainly filamentous or thin staining outside the cell nucleus. [Figure 7B-C]A-G provide images after amplification. Figure 7A provides a schematic diagram showing the switching process between fluorescent and bright-field imaging using DNA exchange and HRP-based amplification. Figure 7B provides the resulting fluorescent image of tonsil tissue cytokeratin labeled with antibody-DNA conjugate and the corresponding fluorescently labeled imager strand. Figure 7C provides the resulting bright-field immunohistochemistry image of cytokeratin in the same tonsil tissue after DNA exchange and HRP-based chromogenic amplification. Figures 7D and E show the fluorescent signal obtained from a cell sample stained for vimentin and DAPI without amplification (D, left) and with rolling circle amplification (E, right). Figure 7F provides the resulting fluorescent image of tonsil tissue cytokeratin labeled with antibody-DNA conjugate and the corresponding fluorescently labeled imager strand (F, left). Figure 7G, right, shows the fluorescent image from the same tissue section after HCR amplification of the docking strand and hybridization of the corresponding fluorescently labeled imager strand. In Figures 7D and 7E, the images show blue DAPI nuclear staining, which typically appears as roughly round or bean-shaped objects located in the central region of the cell. DAPI stains DNA inside the cell's nucleus and was included in the images as evidence that cells were present within the field of view. Vimentin staining (red) in Figure 7D was lower without amplification and brighter and more intense with amplification in Figure 7E. The red vimentin staining shows mainly filamentous or thin staining outside the cell nucleus. [Fig. 7D-G]A-G provide images after amplification. Figure 7A provides a schematic diagram showing the switching process between fluorescent and bright-field imaging using DNA exchange and HRP-based amplification. Figure 7B provides the resulting fluorescent image of tonsil tissue cytokeratin labeled with antibody-DNA conjugate and the corresponding fluorescently labeled imager strand. Figure 7C provides the resulting bright-field immunohistochemistry image of cytokeratin in the same tonsil tissue after DNA exchange and HRP-based chromogenic amplification. Figures 7D and E show the fluorescent signal obtained from a cell sample stained for vimentin and DAPI without amplification (D, left) and with rolling circle amplification (E, right). Figure 7F provides the resulting fluorescent image of tonsil tissue cytokeratin labeled with antibody-DNA conjugate and the corresponding fluorescently labeled imager strand (F, left). Figure 7G, right, shows the fluorescent image from the same tissue section after HCR amplification of the docking strand and hybridization of the corresponding fluorescently labeled imager strand. In Figures 7D and 7E, the images show blue DAPI nuclear staining, which typically appears as roughly round or bean-shaped objects located in the central region of the cell. DAPI stains DNA inside the cell's nucleus and was included in the images as evidence that cells were present within the field of view. Vimentin staining (red) in Figure 7D was lower without amplification and brighter and more intense with amplification in Figure 7E. The red vimentin staining shows mainly filamentous or thin staining outside the cell nucleus. [Figure 8] We demonstrate the use of a preformed nonlinear DNA strand that can be hybridized to a docking strand as an initiation point for rolling circle polymerization. [Figure 9A-G] A-F and H-K show a series of images acquired in the Cy5 channel during a swapped imaging experiment to re-search for targets in the sample. Figure 9G shows the average signal intensity of the images in Figure 9A-F. [Fig. 9H-K] A-F and H-K show a series of images acquired in the Cy5 channel during a swapped imaging experiment to re-search for targets in the sample. Figure 9G shows the average signal intensity of the images in Figure 9A-F. [Figure 10]Figures 10A-B show schematics and results of proximity detection with stable binding. Like Figures 7D-7E, Figures 10A-B show DAPI (blue) staining of the inner nucleus of the cells. Figure 10A also shows alpha-tubulin staining (green) mainly outside the nucleus of the cells. [Figure 11] A-C show cell staining in experiments designed to assess off-target cross-reactivity. These figures also use DAPI staining (blue) of the outer nucleus of the cells. Alpha-tubulin staining (red) is strongly present in Figure 11A, absent in Figure 11B, and weakly present in Figure 11C. [Figure 12] AD show that the signal from the target can be almost completely abolished using either USER or UDG. [Figure 13A-B] A-D show various embodiments of exchange imaging, some of which use a primer strand and an intermediate strand in addition to an imager strand and a docking strand. FIG. 13A shows DNA exchange imaging using an intermediate strand (401) to link an imager strand with a docking strand bound to a target through a target recognition moiety. FIG. 13B shows a primer strand (404) used to amplify the number of docking strands associated with a target binding complex, and the resulting amplification product (403) is attached to multiple docking sites (103) and can be imaged with an imager strand directly or indirectly through the intermediate strand as shown. FIG. 13C shows the amplification of the number of docking strands associated with a target using a primer strand to initiate a hybridization chain reaction, and imaging with the addition of an imager strand bound to a docking strand through the intermediate strand. FIG. 13D shows the amplification of the number of docking strands associated with a target using a primer strand as a template for ligation and rolling circle amplification, followed by the addition of an imager strand bound to a docking strand through the intermediate strand for imaging. [Figure 13C-D]A-D show various embodiments of exchange imaging, some of which use a primer strand and an intermediate strand in addition to an imager strand and a docking strand. FIG. 13A shows DNA exchange imaging using an intermediate strand (401) to link an imager strand with a docking strand bound to a target through a target recognition moiety. FIG. 13B shows a primer strand (404) used to amplify the number of docking strands associated with a target binding complex, and the resulting amplification product (403) is attached to multiple docking sites (103) and can be imaged with an imager strand directly or indirectly through the intermediate strand as shown. FIG. 13C shows the amplification of the number of docking strands associated with a target using a primer strand to initiate a hybridization chain reaction, and imaging with the addition of an imager strand bound to a docking strand through the intermediate strand. FIG. 13D shows the amplification of the number of docking strands associated with a target using a primer strand as a template for ligation and rolling circle amplification, followed by the addition of an imager strand bound to a docking strand through the intermediate strand for imaging. [Figure 14] A-D show that the efficiency of removal of the imager strand using nuclease varies depending on the sequence design. The imager strand was synthesized with (FIGS. 14B and D) and without (FIGS. 14A and C) abasic sites. Fluorescence images of CD3 in tonsil tissue labeled with antibody-DNA conjugates are shown before (FIGS. 14A-B, shown at 30,000 intensity scale) and after (FIGS. 14C-D, shown at 2,000 intensity scale) removal of the corresponding fluorescently labeled imager strand. [Figure 15] A-F show an embodiment of exchange imaging where four labels are imaged sequentially, two targets at a time, using four imager strands labeled with two different fluorophores. Thus, Figure 15 shows a combination of spectral and sequential multiplexing.

[0011] The following reference numbers are used throughout the drawings and this application: TIFF2025011077000002.tif255170TIFF2025011077000003.tif23170 DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] I. Methods of Testing a Sample for the Presence of One or More Targets The present application relates to improved methods and compositions for testing for the presence of one or more targets using one or more target-specific binding partners.

[0013] Exchange imaging is a method to achieve high multiplexing capabilities such that many targets can be imaged on the same sample. The main concept of exchange imaging involves the following steps: (1) attaching different decodable signaling molecules (called docking strands) to different target-specific binding partners (such as, but not limited to, antibodies that recognize the targets) (target-specific binding partners of different specificities (i.e., that bind to different targets) are attached to different docking strands) and optionally removing unbound target-specific binding partners; (2) labeling a subset of the docking strands using a set of molecules (called imager strands), each of which specifically recognizes a docking strand and carries an observable moiety, and imaging the corresponding subset of labels; (3) labeling a subset of the docking strands using a set of molecules (called imager strands), each of which specifically recognizes a docking strand and carries an observable moiety; and (4) imaging a subset of the corresponding labels using a set of molecules (called imager strands), each of which specifically recognizes a docking strand and carries an observable moiety. 3) removing the set of imager strands used in step 2 to eliminate the signal from the bound labeled imager strands, remove the observable moiety from the imager strands, or inactivate the observable moiety on such imager strands; (4) labeling another subset of docking strands using another set of imager strands, each of which specifically recognizes a docking strand and carries an observable moiety, and imaging the corresponding subset of labels; (5) optionally, steps 3 and 4 can be repeated to visualize multiple subsets of labels. An end user will easily understand that not all steps should be repeated in every experiment. For example, in the final round of imaging, there is no need to eliminate the signal from the bound labeled imager strands, since no additional imaging strands are applied.

[0014] One non-limiting example of exchange imaging is DNA exchange immunofluorescence, which uses an antibody as a target recognition molecule to image a target protein or other biomolecule, a DNA oligonucleotide as a docking strand, and a DNA oligonucleotide as an imager strand that is complementary to the docking strand and labeled with at least one observable moiety (such as a fluorophore). In step 3, eliminating the signal from the bound labeled imager strand includes some embodiments. In some embodiments, the imager strand may be removed, the label removed from the imager strand, and / or the label attached to the imager strand may be inactivated. These various methods may be used by using high temperature, low ionic strength buffers, denaturants (including, for example, formamide), DNA helicase, DNase, strand displacement, chemical cleavage, enzymatic cleavage, chemical bleaching, photobleaching, and / or photochemical bleaching. By bound labeled imager strand, we aim to distinguish the labeled imager strand that is bound to the docking strand at a given time from the excess labeled imager strand that did not bind to the docking strand. During the process of quenching the signal, the so-called bound labeled imager strand may or may not remain bound to the docking strand.

[0015] In some embodiments, imaging the sample to detect bound, labeled imager strands detects the presence of bound, labeled imager strands. In some embodiments, imaging the sample to detect bound, labeled imager strands detects the presence, location, and / or number of bound, labeled imager strands.

[0016] Various types of imaging can be used with the method. For example, imaging can include any type of microscopy with an objective lens, illumination, and a sensor. In some embodiments, imaging is performed using optical microscopy, wide-field, confocal (line and point scanning, spinning disk), total internal reflection illumination (TIR), stimulated emission depletion (STED), light sheet illumination (including lattice light sheet illumination), fluorescence microscopy including structured illumination (SIM), expansion microscopy, and electron microscopy.

[0017] A. Amplification Methods In microscopy, signal amplification is desirable in many situations, such as when the abundance of the target is low, when the acceptable exposure time is short, and / or when the sensitivity of the imaging equipment is low. Signal amplification provides an advantage in DNA exchange immunofluorescence. In conventional singleplex immunofluorescence (where only one target is analyzed), often an unconjugated primary antibody and a fluorescently labeled secondary antibody are used. Since the secondary antibody is often polyclonal, multiple molecules of the secondary antibody can bind to one molecule of the primary antibody, resulting in signal amplification. However, in DNA exchange immunofluorescence, in some embodiments, users directly label the DNA docking strand to the primary antibody, thus eliminating such a signal amplification step obtained by using a polyclonal secondary antibody. As a result, in some cases, DNA exchange immunofluorescence may have a lower signal intensity compared to conventional immunofluorescence.

[0018] Therefore, in one embodiment, amplification is used to improve signal intensity in multiplexed DNA exchange immunofluorescence. Many methods exist for signal amplification in microscopy, but not all can be applied to DNA exchange immunofluorescence. One of the most well-known methods involves linking (covalently or non-covalently) a target recognition molecule (e.g., an antibody) to an enzyme that can convert an unobservable substrate into an observable product. Many enzymes have been used for these purposes, such as horseradish peroxidase (HRP), alkaline phosphatase (AP), glucose oxidase (GO), β-galactosidase (β-gal). Also, a number of chromogenic, fluorogenic, and chemiluminescent substrates for these enzymes have been developed, such as 3,3'-diaminobenzidine (DAB), nitroblue tetrazolium chloride (NBT), 5-bromo-4-chloro-3-indolyl phosphate (BCIP), and 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-Gal). Another strategy to utilize such enzymes in signal amplification is to create a covalent bond between the target (or other molecules in the vicinity of the target) and an observable reporter molecule. This strategy is exemplified by the tyramide signal amplification (TSA) technology, commercialized by Thermo Fisher and Perkin Elmer, among others. However, none of these signal amplification methods are compatible with exchange imaging, because the observable reporter, enzyme / primer, or substrate is brought to the vicinity of the target permanently or without a decodable docking strand. DNA-based (i.e., decodable) signal amplification methods have been reported in which the observable signal can be removed, at least in principle (e.g., Zimak, et al., Chembiochem 13(18):2722-8(2012)(PMID:23165916)). However, such methods involve multiple manipulations and the signal gain is small.

[0019] Another type of signal amplification involves linking (covalently or non-covalently) a target recognition molecule to a primer molecule of a polymerization or dendrimerization reaction. One example of such a polymerization reaction is rolling circle amplification (RCA), in which the primer of the RCA is linked to a target recognition molecule and converted into a long repetitive single-stranded DNA. Fluorescent molecules can either be directly incorporated into the RCA product via fluorescently labeled nucleotides or be attached to the RCA product as part of a fluorescently labeled oligonucleotide designed to hybridize to the RCA product. Other examples of such polymerization or dendrimerization reactions include branched DNA toehold-based strand displacement (Schweller et al. PMCID: PMC3517005), hybridization chain reaction (HCR) (Dirks et al., 2014, PMID: 15492210, 24712299), and similar DNA hairpin-based dendrimerization reactions (Yin et al., 2008, PMID 18202654) (herein referred to as HDR). Common applications of amplification methods such as RCA, HCR, and HDR do not include exchange imaging options, but can be adapted as demonstrated by the improvements described herein.

[0020] Herein, we consider embodiments that include signal amplification compatible with exchange imaging. We describe a series of embodiments that make signal amplification compatible with exchange imaging. These embodiments can be divided into two classes based on whether the amplification products are decodable. For example, if the amplification products contain docking strand components (e.g., single-stranded DNA), many (e.g., more than 5) antibodies against different targets can be programmed to generate such products of different docking strand sequences that can be subsequently decoded by complementary ssDNA molecules. Thus, such amplification products are considered to be decodable. In such cases, signal amplification for different targets can be performed simultaneously, followed by simultaneous and / or sequential imaging of the different amplification products. Simultaneous amplification performed for different targets can be considered as multiplexed amplification.

[0021] In contrast, for amplification products that are fluorophores or labels that are covalently attached to targets or non-covalently deposited near them but do not contain a docking strand that can interact with imager strands, these amplification products are considered to be non-decodable.For example, when the enzyme involved in signal amplification is HRP, the product is a chemical chromophore that does not allow many variations that can be specifically bound by many molecules that function as imager strands.In such cases, signal amplification for different targets can be carried out sequentially, and the enzyme that is bound to the target that has already been amplified can be removed from the sample.Simultaneous signal amplification of non-decodable amplification products is possible when orthogonal enzyme-substrate pairs can be used.

[0022] Thus, in some embodiments, the method includes: (1) contacting a sample to be tested for the presence of one or more targets with one or more target-specific binding partners, where each target-specific binding partner is attached to a nucleic acid strand, and where target-specific binding partners of different specificities are attached to different nucleic acid strands; (2) optionally removing unbound target-specific binding partners; and (3) where the nucleic acid strand is a docking strand or a primer strand, increasing the number of docking strands associated with each target-specific binding partner if the nucleic acid strand is (a) a docking strand, or (b) a primer strand, increasing the number of docking strands associated with each target-specific binding partner by two or more. (4) contacting the sample with a labeled imager strand capable of binding, directly or indirectly, to the docking strand; (5) optionally removing the unbound labeled imager strand; (6) imaging the sample to detect the bound labeled imager strand; (7) optionally removing the bound labeled imager strand from the docking strand; and (8) optionally repeating steps (1)-(6), or any subset thereof.

[0023] Here, and throughout this application, by docking strands associated with each target-specific binding partner, applicant does not intend that amplification occur on every single docking strand, but rather that amplification generally occurs on docking strands associated with various target-specific binding partners as desired by the user (including amplifying only a portion of the docking strands involved in the detection of targets A and B, while not amplifying the docking strand involved in the detection of target B). Amplification may also be incomplete, such as amplification occurring on only a portion, but not all, of the copies of the docking strand involved in the detection of a given target. In addition, amplification may replicate the entire docking strand, or may replicate only a portion of the docking strand sufficient to bind to the imager strand.

[0024] Additionally, in some embodiments, the method includes the steps of: (1) contacting a sample to be tested for the presence of one or more targets with one or more target-specific binding partners, where each target-specific binding partner is attached to a nucleic acid strand, and where target-specific binding partners of different specificities are attached to different nucleic acid strands; (2) optionally removing unbound target-specific binding partners; (3) contacting the sample with a labeled imager strand capable of binding, directly or indirectly, to a docking strand, where the nucleic acid strand of (1) is either a primer strand or a docking strand, and where if the nucleic acid strand is a primer strand, it is attached to a docking strand; and (4) contacting the sample with a labeled imager strand capable of binding, directly or indirectly, to a docking strand, where the nucleic acid strand of (1) is either a primer strand or a docking strand, and where if the nucleic acid strand is a primer strand, it is attached to a docking strand; (5) imaging the sample to detect bound labeled imager strands and determine if amplification (step (7)) is required; (6) optionally removing bound labeled imager strands from the docking strands; (7) optionally increasing the number of docking strands associated with each target-specific binding partner (e.g., amplifying the number of available docking strands by multiple means including, but not limited to, self-assembly of docking strand complexes, other assembly methods, branched and cyclic docking strands, etc.); and (8) optionally repeating steps (1)-(7), or any subset thereof.

[0025] 1. Amplification Decodable amplification products. Decodable amplification products include cases where the amplification product is a docking strand. In one embodiment, the docking strand does not contain an observable label. In one embodiment, the docking strand serves as a barcode (or imager strand) for an observable label. First, note that a docking strand or docking site can be introduced to the target during the signal amplification reaction (FIG. 1b-c), resulting in multiple docking strands attached to one target molecule. There are at least two strategies to achieve this, but are not limited to: (Strategy 1) creating multiple docking sites that are attached to a scaffold and then to a target recognition moiety (FIG. 1b), and (Strategy 2) creating multiple binding sites on one long single-stranded DNA attached to a target recognition moiety (FIG. 1c).

[0026] RCA, HCR, or HDR may be used to generate polymer or dendrimer products from primer molecules bound to antibodies. In some embodiments, the products may contain many (e.g., 2, 5, 10, 15, 20, 25, 50, 100, etc.) copies of a single-stranded DNA domain that can function as a docking strand and thus be recognized by an oligonucleotide that functions as an imager strand. Such a DNA domain may be sufficiently long (e.g., more than 7, 8, 9, 10, 11, 12, 13, 14, 15, 20 nucleotides or more, or may be capable of binding to its complementary strand with a Kd of more than 1 nM under imaging conditions). For RCA, this is usually achieved. For HCR and HDR, if necessary, a DNA domain that is not involved in the strand displacement cascade but constitutes part or all of the imager strand binding site may be included in the loop or tail of the substrate hairpin.

[0027] In one embodiment, a modified version of hybridization chain reaction (HCR) is used for signal amplification, where two hairpins (105 and 106 in FIG. 1d) are assembled on the primer strand (104 in FIG. 1d) in an alternating manner. One or both of the two hairpins can carry a docking site (domain b on hairpin 105 in FIG. 1d). Dozens to hundreds of hairpin units can be assembled on one primer strand, leading to tens to hundreds of docking sites to the target recognition moiety. Several pairs of hairpin sequences (without docking sites) have been demonstrated by the Pierce group to enable successful HCR reactions. Hairpin sequences with docking sites can be designed with the same principles, but care can be taken to ensure that the docking sites do not form undesired secondary structures with the remainder of the hairpin.

[0028] In another embodiment, signal amplification involves linking (covalently or non-covalently) a target recognition molecule to a primer molecule of a polymerization or dendrimerization reaction. One example of such a polymerization reaction is rolling circle amplification (RCA, FIG. 1e), in which the primer of RCA is linked to a target recognition molecule and converted into a long repetitive single-stranded DNA. Fluorescent molecules can either be directly incorporated into the RCA product via fluorescently labeled nucleotides, or be bound to the RCA product as part of a fluorescently labeled oligonucleotide designed to hybridize to the RCA product.

[0029] There are many ways to perform RCA, one of them is to first ensure that the oligonucleotide conjugated to the target recognition moiety (herein referred to as "primer", 111 in Fig. 1e) has an extendable 3' end. A linear template strand (112 in Fig. 1e) can then be introduced that can hybridize to the primer in a circular fashion, and the primer brings together the two ends of the template, which allows the two ends to be ligated. A ligase (e.g., T4 DNA ligase or CircLigase™ ssLigase, etc.) is then used to ligate the two ends to form a circle. After ligation, the primer is hybridized to the circular template. Next, a DNA polymerase with strand displacement activity (e.g., phi29, Bst, Vent (exo)) is used to ligate the two ends to form a circle. - )) can extend the primer multiple times along the circular template to create a concatenated repeat. A portion of the entire repeat unit (domains c-d or 115 in Figure 1e) can serve as a docking site (or docking strand) for the imager strand.

[0030] An alternative method of RCA involves the use of a nonlinear amplifier or template strand, in which an oligonucleotide (such as a docking strand) conjugated to a target recognition moiety is hybridized to a circular DNA template (the amplifier strand), followed by extension of the docking strand by a DNA polymerase to create a stranded repeat of the reverse complement of the amplifier strand (i.e., the amplified strand or RCA product). Hybridization of the amplifier strand to the oligonucleotide-conjugated target recognition moiety can occur before (pre-assembly or pre-hybridization) or after the oligonucleotide-conjugated target recognition moiety is contacted with the sample.

[0031] Thus, in some embodiments, at least one oligonucleotide-conjugated target recognition moiety is hybridized to the nonlinear amplifier strand before being introduced into the sample. If the user chooses to preassemble the antibody-DNA conjugate with the amplifier, the method for testing the sample for the presence of one or more targets includes: (1) contacting the sample to be tested for the presence of one or more targets with one or more target-specific binding partners, each target-specific binding partner being bound to a nucleic acid strand, and target-specific binding partners of different specificities being bound to different nucleic acid strands, and at least one nucleic acid strand being hybridized to the nonlinear amplifier strand; (2) optionally removing unbound target-specific binding partners; (3) amplifying the docking strand by rolling circle amplification (i.e., increasing the number of docking strands or introducing multiple docking strands) to generate rolling circle amplification products, (4) contacting the sample with a labeled imager strand having complementarity to the docking strand or the rolling circle amplification products, (5) imaging the sample to detect the bound labeled imager strand, (6) optionally removing the bound labeled imager strand, and (7) optionally repeating steps (1)-(7) or any subset thereof, in which the rolling circle amplification products comprise a stranded repeat of the reverse complement of the amplifier strand.

[0032] In another embodiment, the imager strand may be hybridized to the RCA product during the RCA reaction (e.g., a stranded repeat of the reverse complement of the amplifier strand linked to a target recognition moiety). Thus, in some embodiments, amplification occurs using rolling circle amplification while in the presence of a labeled imager strand that has complementarity to the amplified strand. For example, the sample may be contacted with an oligonucleotide conjugated to a target recognition moiety that is either pre-hybridized to the amplifier strand or to which the amplifier strand may be hybridized in a later step. All additional components for the RCA reaction, including proteins (e.g., DNA polymerase, optionally BSA), nucleotides, buffer solutions, salts, and the imager strand, may then be added in one step. In some embodiments, a user may wish to prevent the imager strand from amplifying. This may be accomplished by several means, including but not limited to using a 3'-modified imager strand that has a modification on the 3' end. For example, 3' modifications on the imager strand can include a label (such as a fluorophore), a modified base, a stop code or terminator, a 3'-O-modification, dideoxy-C, dideoxy-G, dideoxy-A, dideoxy-T, an inverted nucleotide, any modification that eliminates the presence of a 3' hydroxyl group, or a single-stranded extension at the 3' end that is not complementary to the amplifier strand.

[0033] In addition to HCR and RCA, other examples of such polymerization or dendrimerization reactions include DNA hairpin-based dendrimerization reactions (HDR) (Yin et al., 2008, PMID 18202654), and toehold-mediated strand displacement.

[0034] DNA strand displacement is a method for isothermal and dynamic exchange of DNA complexes. Strand displacement can be designed and deliberately controlled based on an understanding of DNA hybridization interactions and thermodynamics, and can be facilitated by introducing engineered handles known as "toehold domains." The ability to modulate binding interactions and exchange hybridization partners opens up a range of potential signal amplification applications.

[0035] In another embodiment, a codable tyramide-based signal amplification product is described. The method includes: (1) contacting a sample to be tested for the presence of one or more targets with one or more target-specific binding partners, where each target-specific binding partner is bound to a nucleic acid strand, and where target-specific binding partners of different specificities are bound to different nucleic acid strands; (2) optionally removing unbound target-specific binding partners; and (3) contacting the sample with an enzyme-labeled strand capable of binding to a docking strand, where the nucleic acid strand of (1) is either a primer strand or a docking strand, and the nucleic acid strand is either a primer strand or a docking strand. If the enzyme-labeled strand is a primer strand, it is attached to the docking strand; (5) optionally removing the unbound enzyme-labeled strand; (4) contacting the sample with the tyramide-linked docking strand; (5) enzymatically converting the tyramide moiety to an activated state, which activates the tyramide moiety to provide covalent linkage to the enzyme-labeled label site of the tyramide-linked docking strand; (6) optionally quenching the enzymatic reaction; (7) removing the enzyme-labeled strand; and (8) optionally repeating a subset of steps 3-8. In one embodiment, the enzyme-linked strand is an HRP-linked strand.

[0036] In another embodiment, the method includes (1) contacting a sample to be tested for the presence of one or more targets with one target-specific binding partner, where the target-specific binding partner is linked to an enzyme, (2) optionally removing unbound target-specific binding partner, (3) contacting the sample with a tyramide-linked docking strand, (4) enzymatically converting the tyramide moiety to an activated state, where the activated state results in covalent linkage of the tyramide-linked docking strand to an enzyme-labeled target moiety, (5) quenching the enzymatic reaction, and (6) optionally repeating a subset of steps 1-8, where a target-specific binding partner of different specificity is introduced. In one embodiment, the enzyme-linked target-specific binding partner comprises HRP.

[0037] Amplification of multiple targets can be performed sequentially, as shown in Figure 2. Alternatively, amplification of multiple targets can be performed simultaneously (Figure 3). An imaging step can be performed between rounds of amplification or after every round of amplification.

[0038] Non-decodable amplification products. Non-decodable amplification products include cases where the amplification product is an observable label that does not have specific affinity for the imager strand. In one embodiment, the non-decodable amplification product can be a fluorophore, a chromogenic dye, or a nanoparticle.

[0039] In one embodiment, a method for generating non-decodable amplification products includes: (1) contacting a sample to be tested for the presence of one or more targets with one or more target-specific binding partners, where each target-specific binding partner is attached to a nucleic acid strand, and where target-specific binding partners of different specificities are attached to different nucleic acid strands; (2) optionally removing unbound target-specific binding partners; (3) contacting the sample with an enzyme-labeled strand capable of binding to a docking strand, where the nucleic acid strand of (1) is either a primer strand or a docking strand, and if the nucleic acid strand is a primer strand, it is attached to a docking strand; (5) optionally removing unbound enzyme-labeled strand; (4) contacting the sample with a group for an enzyme; (5) allowing the enzymatic reaction to generate an amplification product; (6) quenching the enzymatic reaction; (7) imaging the sample to detect the presence or absence of one or more targets; (8) removing the amplification product; and (9) repeating a subset of steps 1-9. Examples of enzymes that can be used include HRP, AP, GO, β-gal. When an enzyme is attached to the imager strand (i.e., a strand capable of binding to the docking strand), sequential amplification and imaging can be performed (Figure 4). Figure 4a shows a method for sequential amplification and sequential imaging. Here, a method is used to remove or inactivate the amplification products between each imaging round. Removing or inactivating the amplification products can be done by carefully selecting the substrate. For example, a chromogenic substrate of HRP that is soluble in an organic solution suitable for the sample can be used (e.g., 3-amino-9-ethylcarbazole, PMID 19365090, which is alcohol soluble). In this case, after staining the docking strand-conjugated antibody (Figure 4a, step 1), introducing the imager strand-conjugated HRP and substrate for one target (Figure 4, step 2), and imaging the sample, alcohol (e.g., methanol) can be used to remove the HRP product and remove the imager strand using any of the methods described herein or a combination thereof. An imager chain-conjugated HRP for another target can then be introduced and the process repeated.

[0040] Also, TSA amplification methods can be used in which the fluorophore can be easily bleached. For example, many cyanine fluorophores and Alexa fluorophores can be easily bleached by hydrogen peroxide in acidic or basic conditions (PMID: 26399630). Alternatively, TSA dyes can be synthesized that contain a cleavable bond between the tyramide and the fluorophore. In this case, the fluorophore can be inactivated by cleaving this bond and washing.

[0041] Figure 4b shows a method for sequential amplification and simultaneous imaging, where after staining the docking strand-conjugated antibody (Figure 4b, step 1) and introducing the imager strand-conjugated enzyme and substrate for one target (Figure 4, step 2, e.g., using HRP and TSA) to generate an amplification product, the imager strand-conjugated enzyme can be removed without removing the amplification product, and multiple rounds of amplification can be repeated for multiple targets before imaging the sample in a single imaging step.

[0042] Also, RCA, HCR, and HDR can be used to achieve signal amplification without decoding the amplification product. For example, after staining of multiplexed antibodies, a reagent (circular template for RCA, and substrate hairpin for HCR and HDR) can be added that only supports polymerization / dendrimerization of one subset of targets and direct incorporation of fluorescent dye into amplification products (e.g., via fluorescently labeled nucleotides for RCA, and via fluorophore-labeled hairpin substrates for HCR and HDR). After imaging of this subset of targets and inactivation of the dye by bleaching or cleavage as described above, a reagent can be introduced that supports polymerization / dendrimerization of another subset of targets and direct incorporation of fluorescent dye into amplification products. This process can then be repeated.

[0043] Multiple types of signal amplification can also be used in combination. For example, Gusev et al. report a combination of rolling circle amplification and HRP-based signal amplification (PMID: 11438455).

[0044] The fluorophores (directed to the target via DNA complexes or other amplification methods) can be replaced by other molecules or moieties that can be observed directly or indirectly, including but not limited to metal particles, plasmonic enhancers, and proteins.

[0045] 2. Nonlinear Amplification The nonlinear DNA template can be used for signal amplification as a circular amplification strand. A circular oligo with complementarity to the docking strand can be generated separately from the amplification method. For example, in situ ligation can be performed on the template DNA strand to form a circular strand of DNA. The circular strand can be hybridized to the docking strand attached to the target-specific binding partner before contacting the sample. Alternatively, the target-specific binding partner can be used first to stain the sample, and then the circular strand can be subsequently introduced into the sample to hybridize with the docking strand on the target-specific binding partner. After the formation of the complex in which the circular strand is attached to the docking strand linked to the target-specific binding partner, rolling circle amplification (RCA) can be performed. This method offers certain advantages, as it can be used to avoid problems with inefficient in situ ligation steps.

[0046] In some situations, an amplifier strand may be used. For example, in some embodiments, the method includes: (1) contacting a sample to be tested for the presence of one or more targets with one or more target-specific binding partners, where each target-specific binding partner is bound to a nucleic acid strand, and where target-specific binding partners of different specificities are bound to different nucleic acid strands; (2) optionally removing unbound target-specific binding partners; (3) contacting the sample with a nonlinear amplifier strand having complementarity to the nucleic acid strand, where the nucleic acid strand of (1) is either a primer strand or a docking strand; (4) optionally removing the nonlinear amplifier strand; (5) amplifying the docking strand by rolling circle amplification (i.e., increasing the number of docking strands or introducing multiple docking strands); (6) contacting the sample with a labeled imager strand capable of binding to the docking strand either directly or indirectly; (7) imaging the sample to detect the bound labeled imager strand; (8) removing the bound labeled imager strand; and (9) optionally repeating steps (1)-(8), or any subset thereof.

[0047] In some embodiments, a polymerase may be used for RCA. In some cases, the labeled imager strand is linear. In some cases, the nonlinear amplifier strand is a circular strand. In some cases, the nonlinear amplifier strand is a branched strand. In some cases, the nonlinear amplifier strand becomes circular after ligation.

[0048] In some embodiments, the amplification products may include geometric shapes such as triangles, quadrilaterals, pentagons, hexagons, and the like.

[0049] B. Method Step Variations There are various ways to approach multiplexed imaging, including the option to use amplification steps at different time points to amplify the signal and repeat the steps to allow imaging of multiple targets or re-searching of a single target. The method may also optionally include erasing the signaling images at various time points.

[0050] 1. Amplifying the signal before applying the imager strand and, optionally, quenching the signal from the bound, labeled imager strand. In some embodiments, a method for testing a sample for the presence of one or more targets includes: (1) contacting the sample with one or more target-specific binding partners, where each target-specific binding partner is bound to a nucleic acid strand, and where target-specific binding partners of different specificities are bound to different nucleic acid strands; (2) optionally removing unbound target-specific binding partners; (3) where the nucleic acid strand is a docking strand or a primer strand, and where the nucleic acid strand is (a) a docking strand, increasing the number of docking strands associated with each target-specific binding partner, or (b) a primer strand, associating two or more docking strands with the primer strand; (4) contacting the sample with a labeled imager strand capable of binding to the docking strand, either directly or indirectly; (5) optionally removing unbound labeled imager strand; (6) imaging the sample to detect bound labeled imager strands; and (7) optionally dissipating signal from the bound labeled imager strands. In some cases, after step (4) and optionally after performing step (5), the method further comprises increasing the number of docking strands associated with each target-specific binding partner. In some embodiments, the method further comprises removing unbound labeled imager strands after increasing the number of docking strands. Thus, in some formats, amplifying the docking strands with rolling circle amplification occurs separately from contacting the sample with a labeled imager strand that has complementarity to the amplified strand. By amplified strand is meant the product of amplification (which may also be referred to as an amplified product or RCA product when rolling circle amplification is used).

[0051] In some cases, the sample is placed on an optically transparent support. In some embodiments, increasing the number of docking strands associated with each target-specific binding partner is achieved using enzymes. For example, the enzyme approach described in section IA above can be used.

[0052] 1. Amplifying the signal in the presence of the imager strand, and optionally quenching the signal from the bound, labeled imager strand. In some embodiments, a method for testing a sample for the presence of one or more targets includes: (1) contacting the sample with one or more target-specific binding partners, where each target-specific binding partner is bound to a nucleic acid strand, and where target-specific binding partners of different specificities are bound to different nucleic acid strands; (2) optionally removing unbound target-specific binding partners; (3) contacting the sample with a labeled imager strand capable of binding to a docking strand, either directly or indirectly; (4) where the nucleic acid strand is a docking strand or a primer strand, and where the nucleic acid strand is (a) a docking strand, increasing the number of docking strands associated with each target-specific binding partner, or (b) a primer strand, associating two or more docking strands with the primer strand (amplification occurs in the presence of the imager strand); (5) optionally removing unbound labeled imager strand; (6) imaging the sample to detect bound labeled imager strands; and (7) optionally dissipating signal from the bound labeled imager strands. In some cases, after step (4), and optionally after performing step (5), the method further comprises increasing the number of docking strands associated with each target-specific binding partner. In some embodiments, the method further comprises removing unbound labeled imager strands after increasing the number of docking strands.

[0053] In some cases, the sample is placed on an optically transparent support. In some embodiments, increasing the number of docking strands associated with each target-specific binding partner is achieved using enzymes. For example, the enzyme approach described in section IA above can be used.

[0054] The imager strand may have complementarity to the docking strand. The imager strand may be a circular imager strand for rolling circle amplification. The imager strand may be an imager strand that circularizes in the presence of the docking strand and a ligase. In some embodiments, the imager strand may include at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 regions that are complementary to the docking strand.

[0055] 2. Method for testing samples mounted on optically transparent supports In some embodiments, a method for testing a sample mounted on an optically transparent support for the presence of one or more targets includes (1) contacting the sample with one or more target-specific binding partners, where each target-specific binding partner is bound to a nucleic acid strand, and where target-specific binding partners of different specificities are bound to different nucleic acid strands; (2) optionally removing unbound target-specific binding partners; and (3) where the nucleic acid strand is a docking strand or a primer strand, optionally where the nucleic acid strand is (a) a docking strand, increasing the number of docking strands associated with each target-specific binding partner, or (b) if it is a primer strand, associating two or more docking strands with the primer strand; (4) contacting the sample with a labeled imager strand capable of binding to the docking strand either directly or indirectly, where the labeled imager strand is provided in a liquid medium or buffer solution; (5) optionally removing unbound labeled imager strands; (6) optionally removing the liquid to create a liquid-free sample; (7) attaching a second optically transparent material parallel to the first support; and (8) imaging the sample to detect bound labeled imager strands.

[0056] In some embodiments, the second optically transparent material is glass or plastic. In some cases, the second optically transparent material is about 5 microns to 5 mm, 50 microns to 500 microns, or 500 microns to 5 mm from the first support. In some cases, imaging is performed using an upright microscope.

[0057] In some embodiments, optionally removing liquid to create a liquid-free sample comprises preparing the sample for storage, such as long-term storage of at least 4 hours, 1 day, 3 days, 1 week, 2 weeks, or 1 month. In some embodiments, optionally removing liquid to create a liquid-free sample increases the convenience of handling the sample since the user does not need to keep the sample hydrated.

[0058] Optionally removing liquid to create a liquid-free sample means removing at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the liquid in the sample (i.e., liquid already present in the sample and associated with other sample components such as the sample itself, docking strands, imager strands, etc.). In some embodiments, the mounting medium comprises air. In other embodiments, the mounting medium comprises a mounting medium in a gel formulation. In some embodiments, the mounting medium comprises a formulation that starts as a liquid but changes to a gel or solid over time (such as a hardening material, glue, cement, or other optically clear and similarly functioning material).

[0059] In other embodiments, the liquid in the sample may be replaced by a liquid mounting medium of a saline-based buffer solution (such as PBS).

[0060] Mounting media may be used to hold the specimen in place, prevent the sample from drying out, more closely match the refractive index of the intended use, prevent photobleaching (if undesirable), and preserve the sample for long-term storage. The choice of mounting medium depends on the sample type, the imaging strategy in which the observable portion will be used, and the user's goals (whether the user wants to hydrate the sample or whether the user wants to store the sample).

[0061] In some embodiments, a method for testing a fixed sample mounted on an optically transparent support for the presence of one or more targets includes (1) contacting the sample with one or more target-specific binding partners, where each target-specific binding partner is bound to a nucleic acid strand, and where target-specific binding partners of different specificities are bound to different nucleic acid strands; (2) optionally removing unbound target-specific binding partners; and (3) where the nucleic acid strand is a docking strand or a primer strand, and where the nucleic acid is (a) a docking strand, a nucleic acid associated with each target-specific binding partner. (b) increasing the number of docking strands attached to the first support, or (c) associating two or more docking strands with the primer strand if the docking strand is a primer strand; (4) contacting the sample with a labeled imager strand capable of binding to the docking strand either directly or indirectly; (5) optionally removing unbound labeled imager strands; (6) optionally removing the liquid to create a liquid-free sample; (7) attaching a second optically transparent material parallel to the first support; and (8) imaging the sample to detect bound labeled imager strands.

[0062] In various embodiments, the optically transparent support and the second optically transparent material parallel to the first support may comprise a flow cell. In various embodiments, parallel includes perfectly parallel geometric arrangements as well as arrangements that deviate from parallel by up to 1°, 2°, 3°, 4°, 5°, 6°, 7°, 8°, 9°, or 10°.

[0063] 3. Sample re-search In some embodiments, a user may wish to re-screen a sample for the same target multiple times. If re-screening is desired, multiplex imaging is performed by performing another round of imaging with the same imager strand. Thus, if a user wishes to image a different target, the imager strand has a unique nucleotide sequence relative to all other imager strands. If a user wishes to image the same target multiple times, the repeated steps do not have a unique nucleotide sequence relative to all other labeled imager strands, but instead use an imager strand that has the same sequence as the imager strand already used.

[0064] Thus, in some embodiments, the method includes: (1) contacting a sample to be tested for the presence of one or more targets with one or more target-specific binding partners, where each target-specific binding partner is bound to a nucleic acid strand, and where target-specific binding partners of different specificities are bound to different nucleic acid strands; (2) optionally removing unbound target-specific binding partners; and (3) where the nucleic acid strand is a docking strand or a primer strand, optionally removing unbound target-specific binding partners by: (a) increasing the number of docking strands associated with each target-specific binding partner if the nucleic acid strand is a docking strand, or (b) increasing the number of docking strands associated with two or more target-specific binding partners if the nucleic acid strand is a primer strand. (4) contacting the sample with a labeled imager strand capable of binding to the docking strand either directly or indirectly; (5) optionally removing unbound labeled imager strands; (6) imaging the sample to detect the presence, location, and number of bound labeled imager strands; (7) eliminating signal from the bound labeled imager strands; and (8) repeating steps (3)-(6) or (3)-(7), where the labeled imager strand optionally has a unique nucleotide sequence relative to every other labeled imager strand.

[0065] 4. Spectral and Sequential Multiplexing There are two main ways to create multiplexing in exchange imaging: spectral multiplexing and serial multiplexing. Spectral multiplexing refers to the ability to use different labels (such as different fluorophores) in a single imaging run. Spectral multiplexing does not require the signal to be extinguished from the first label before viewing the second label. For example, in the case of fluorophores, different wavelengths of excitation of light can be used to excite the different fluorophores individually. This does not require separate rounds of imaging. Serial multiplexing refers to the ability to use the same label (such as the same fluorophore) in multiple rounds of imaging by extinguishing the signal from the first round of imaging before the second round of imaging. Spectral multiplexing and serial multiplexing can be used either alone or in conjunction with each other. However, using two or more techniques of multiplexing can significantly increase the number of labels a user can visualize during a particular experiment.

[0066] In some embodiments, multiple rounds of imaging are performed with at least some of the same fluorophores. For example, in the first round of imaging, target A can be imaged with label X, target B can be imaged with label Y, and target C can be imaged with label Z. As a next step, the signals from these labels can be extinguished. Then, in the second round of imaging, target D can be imaged with label X, target E can be imaged with label Y, and target F can be imaged with label Z. In some embodiments, at least two targets are imaged using at least two labels, the signal is extinguished, and then at least one more target is imaged using at least one of the same labels, and the imaging steps can be performed in any order. This means that the order of the steps can be reversed, whereby the first imaging step includes imaging at least one target and extinguishing the signal, and the second imaging step includes imaging at least two targets.

[0067] Combining both spectral and sequential multiplexing can increase the overall convenience of performing imaging for the user and reduce destruction of the sample being imaged.

[0068] C. Stable Bonds In some embodiments, at least a portion of the complementary agents used in imaging are stably bound to each other, and in other embodiments, all of the complementary agents used in imaging are stably bound to each other. In some embodiments, the docking strand is stably bound to the imager strand. In some embodiments, the amplifier strand is stably bound to the docking strand. In some embodiments, the amplifier strand is stably bound to the imager strand.

[0069] In some embodiments, the intermediate strand is stably attached to the docking strand. In some embodiments, the intermediate strand is stably attached to the amplifier strand. In some embodiments, the intermediate strand is stably attached to the imager strand. In some embodiments, the amplifier strand is stably attached to the imager strand. In some embodiments, the primer strand is stably attached to the docking strand.

[0070] In some embodiments, the composition comprises a sample bound to two or more target-specific binding partners, each binding partner bound to a docking strand, and at least one docking strand stably bound to a labeled imager strand.

[0071] In some embodiments, stable binding includes at least the percentage of binding over the time period listed in Table 2. For example, Table 2 includes 90% binding for at least 30 minutes. This means that of those bound at t=0, 90% remain bound for at least 30 minutes. This also means, for example, that an equilibrium is reached that has at least 90% of the docking strands bound to the imager strands at any one time, and no more than 10% unbound. Stable binding can include unbinding of some molecules and rebinding of others to achieve a population binding percentage, and does not require that at least 90% are permanently bound, only that at least 90% are bound at any one time. Stable binding can also include the case where V is a variable amount of time corresponding to the length required for the user's experiment, and x means that the percentage bound over that length of time is considered stable binding. TIFF2025011077000004.tif150170

[0072] For example, stably bound can include 70%, 80%, or 90% binding in about 5 minutes to 7 days, 10 minutes to 60 minutes, 20 minutes to 3 hours, 30 minutes to 24 hours, 3 hours to 24 hours, or 24 hours to 7 days.

[0073] The values ​​in Table 2 apply to all types of stable bonds described above. Such measurements can be performed under imaging conditions (in some circumstances, room temperature, neutral pH, and physiological buffer conditions) after non-reactive buffer wash(s) or after non-enzymatic buffer wash(s).

[0074] In some embodiments, the docking strand, primer strand, or intermediate strand involved in stable binding comprises 30 or fewer nucleotides, such as about 8-30 nucleotides, 10-25 nucleotides, or 10-20 nucleotides. In some embodiments, the imager strand involved in stable binding comprises 30 or fewer nucleotides, such as about 8-30 nucleotides, 10-25 nucleotides, or 10-20 nucleotides. In some embodiments, the amplifier strand is about 30-60 nucleotides, 20-80 nucleotides, 30-70 nucleotides, 30-60 nucleotides, or more than 60 nucleotides. The type of amplification reaction can also affect the length of the amplifier strand that is desired, for example, longer lengths are desired for hybridization chain reactions (HCR). One of skill in the art will recognize that different base compositions in the docking strand, imager strand, and / or amplifier strand will affect the number of nucleotides that are desired in each strand due to differences in affinity. Temperature will also affect the number of nucleotides that are desired.

[0075] 1. Proximity imaging with stable binding In some embodiments, the user may use proximity imaging with stable binding. In some embodiments, the composition comprises at least one first binding partner-oligonucleotide conjugate comprising a binding partner attached to an oligonucleotide comprising a half docking domain, an optional stable domain, and optionally a spacer domain, and at least one second binding partner-oligonucleotide conjugate comprising a binding partner attached to an oligonucleotide comprising a half docking domain, an optional stable domain, and optionally a spacer domain, (the stable domains of (a) and (b) are complementary to each other, and the ... The half docking domains can be linearly combined to form a full docking domain), and (c) at least one labeled imager strand or intermediate strand, comprising a 5' domain, a 3' domain, and a linker domain located between the 5' and 3' domains (the 5' domain is complementary to the half docking domain of (a) and the 3' domain is complementary to the half docking domain of (b), the labeled imager strand or intermediate strand can stably bind to the full docking domain and, if an intermediate strand is used, also provides a labeled imager strand). Alternatively, an intermediate strand can be used and can be complementary to both the docking strand and the imager strand. Amplification can also be used. The stability criteria set forth in Table 2 also apply in this regard.

[0076] In some embodiments, the user may use proximity imaging with stable binding when using a primer. In some embodiments, the composition includes at least one first binding partner-oligonucleotide conjugate, comprising a binding partner attached to an oligonucleotide comprising a half primer domain, an optional stable domain, and an optional spacer domain; at least one second binding partner-oligonucleotide conjugate, comprising a binding partner attached to an oligonucleotide comprising a half primer domain, an optional stable domain, and an optional spacer domain (the stable domains of (a) and (b) are complementary to each other, and the half primer domains of (a) and (b) can be linearly combined to form a complete primer domain); (c) at least one docking strand capable of stably binding to the complete primer domain; and (d) at least one labeled imager strand capable of stably binding to the docking strand or an intermediate strand capable of stably binding to the docking strand, and a labeled imager strand capable of stably binding to the intermediate strand. Docking strands capable of stably binding to the complete primer domain include complementary binding (non-covalent binding) and extension (covalent binding) from the same nucleic acid strand. In embodiments where the docking strand is non-covalently bound to the complete primer domain and has a complementary nucleic acid sequence, the docking strand may include a 5' domain, a 3' domain, and a linker domain located between the 5' domain and the 3' domain, where the 5' domain is complementary to the half primer domain of (a) and the 3' domain is complementary to the half primer domain of (b). In embodiments where the docking strand is covalently bound, it may be extended from the complete primer domain by nucleic acid synthesis. Additionally or alternatively, an intermediate strand may be used and may be complementary to both the docking strand and the imager strand. Amplification may also be used. The stability criteria set forth in Table 2 also apply in this regard.

[0077] D. Control experiments and background subtraction Control experiments and background subtraction can be used to further improve the results of methods that test samples for the presence of one or more targets. Neither of these aspects is necessary for a useful experiment, but both improve the quality of multiplexing and can be used alone or in conjunction with each other.

[0078] 1. Control Experiment Control measurements can be added at multiple points in the multiplexing process. In some embodiments, a method for testing a fixed sample mounted on an optically clear support for the presence of one or more targets includes: (1) contacting the sample with one or more target-specific binding partners, where each target-specific binding partner is bound to a nucleic acid strand, and where target-specific binding partners of different specificities are bound to different nucleic acid strands; (2) optionally removing unbound target-specific binding partners; (3) where the nucleic acid strand is a docking strand or a primer strand, optionally removing unbound target-specific binding partners; (a) if the nucleic acid is a docking strand, increasing the number of docking strands associated with each target-specific binding partner, or (b) if the nucleic acid is a primer strand, associating two or more docking strands with the primer strand; and (4) contacting the sample with the docking strands, either directly or indirectly, such as through an intermediate strand. (5) optionally removing unbound labeled imager strands; (6) imaging the sample to detect the bound labeled imager strands; (7) dissipating signal from the bound labeled imager strands; (8) performing a control step by contacting the sample with a labeled imager strand having a nucleotide sequence that is not complementary to the docking strand or intermediate strand (i.e., not a hypothetical intermediate strand, but one that is actually present in the method, if used); (9) optionally removing unbound labeled imager strands; (10) imaging the sample to detect the bound labeled imager strands; and (11) optionally dissipating signal from the bound labeled imager strands.

[0079] In some embodiments, a method for testing a fixed sample mounted on an optically clear support for the presence of one or more targets includes: (1) contacting the sample with one or more target-specific binding partners, where each target-specific binding partner is bound to a nucleic acid strand, and where target-specific binding partners of different specificities are bound to different nucleic acid strands; (2) optionally removing unbound target-specific binding partners; (3) where the nucleic acid strand is a docking strand or a primer strand, optionally removing unbound target-specific binding partners; (4) removing the nucleic acid strand from the docking strand or intermediate strand (i.e., rather than a virtual intermediate strand, if used in the method); (5) optionally removing unbound target-specific binding partners; (6) optionally removing unbound target-specific binding partners; (7) optionally removing unbound target-specific binding partners; (8) optionally removing unbound target-specific binding partners; (9) optionally removing unbound target-specific binding partners; (10) optionally removing unbound target-specific binding partners; (11) optionally removing unbound target-specific binding partners; (12) optionally removing unbound target-specific binding partners; (13) optionally removing unbound target-specific binding partners; (5) optionally removing unbound labeled imager strands; (6) imaging the sample to detect the presence, location, and / or number of bound labeled imager strands; (7) dissipating signal from the bound labeled imager strands; (8) contacting the sample with a labeled imager strand capable of binding to the docking strand either directly or indirectly, such as through an intermediate strand; (9) optionally removing unbound labeled imager strands; (10) imaging the sample to detect the presence, location, and / or number of bound labeled imager strands; and (11) optionally dissipating signal from the bound labeled imager strands.

[0080] In either of the above two embodiments, optionally the method further comprises repeating steps (4)-(11). In some embodiments, the user does not repeat the control step. In other embodiments, the user repeats the control step to assess non-complementary binding.

[0081] In some of these embodiments, the labeled imager strand complementary to the docking strand used in the repeated steps optionally has a unique nucleotide sequence relative to at least one other labeled imager strand.

[0082] In some embodiments, control experiments are used to assess cross-linking of non-complementary strands to ensure that an imager strand (imager A) intended to bind to a docking strand (docking A) attached to a target specific binding partner for target A (anti-A) does not interact with, or to measure the extent of, that docking strand (imager B) attached to a target specific binding partner for B, in an example evaluating targets A, B, and C (for example). In such embodiments, the iterative process may assess the interaction of docking strand A with imager C, and so on. In these embodiments, the complementary strand (imager A binding to docking A) is not necessarily iterated.

[0083] 2. Background Removal In any of the embodiments discussed throughout this application, the method may employ background subtraction. In some embodiments, the method includes imaging the sample to detect and / or measure a background signal, and subtracting the background signal from the image of the sample to detect the bound, labeled imager strand. Such background signal may include autofluorescence and / or residual fluorescence associated with incomplete loss of signal from the bound, labeled imager strand. In some aspects, the background signal is measured prior to imaging the sample to detect the bound, labeled imager strand. In other aspects, the background signal is measured after imaging the sample to detect the bound, labeled imager strand.

[0084] E. Methods for quenching signal from bound labeled imager strands A variety of methods can be used to quench the signal from the attached labeled imager strands, which may be desirable so that the same type of detectable moiety (such as a fluorophore) can be used on multiple imager strands, thereby preventing the experiment from being spectrally limited.

[0085] In other words, removing a set of imager strands or inactivating an observable moiety on the imager strand allows for spectrally unlimited multiplex imaging. Some conventional methods for multiplex imaging are limited by the number of colors of fluorophores or other imaging agents available. Removing an imager strand, removing a label from the imager strand, or inactivating an observable moiety allows for reuse of the same color of fluorophore in a single experiment. In some embodiments, ideally, as much signal as possible should be removed to ensure the lowest possible background for sequential imaging. In some embodiments, 100% of the previous signal generating moiety is removed or destroyed, while in some embodiments, at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the previous signal generating moiety is removed or destroyed.

[0086] Thus, eliminating the signal from the labeled imager strand includes any method for directly or indirectly removing the imager strand from binding to the docking strand, removing the label from the imager strand, or inactivating the label on the imager strand.

[0087] All methods for eliminating signal from the imager strand can also be applied to the docking strand. In some embodiments, eliminating signal from the bound labeled imager strand involves breaking the bond between the docking strand (or primer strand) and the target recognition moiety. In some embodiments, the docking strand comprises a photocleavable linker that can be cleaved photochemically (e.g., by UV exposure, visible light, infrared, near infrared, X-rays, microwaves, radio waves, or gamma rays). In some embodiments, the docking strand (or primer strand) itself contains a moiety that can be cleaved by an enzyme. Examples of such enzyme-cleavable moieties include, but are not limited to, ribonucleotides that can be cleaved by various RNases, deoxyuridines that can be cleaved by combinations of enzymes such as USER (New England Biolabs), and restriction sites that can be cleaved by sequence-specific nicking enzymes or restriction enzymes. In some embodiments, the docking nucleic acid comprises deoxyuridine and the uracil group can be cleaved by uracil-DNA glycosylase. In some embodiments, the docking nucleic acid comprises an abasic site that can be cleaved by an endonuclease.

[0088] One non-limiting example of exchange imaging is DNA exchange immunofluorescence, which uses an antibody as a target recognition molecule to image a target protein or other biomolecule, a DNA oligonucleotide as a docking strand, and a DNA oligonucleotide complementary to the docking strand and labeled with a fluorophore as the imager strand. The user can eliminate the signal from the labeled imager strand by using high temperature, denaturants, DNA helicase, DNase, and / or strand displacement, or remove the fluorophore on the imager strand by chemical cleavage, enzymatic cleavage, chemical bleaching, photobleaching, and / or photochemical bleaching.

[0089] 1. Nucleolytic enzyme Many enzymes can break covalent bonds within nucleic acid molecules. For example, some glycosylases can remove bases from the sugar moiety of nucleotides, endonucleases can cleave bonds within phosphodiester bridges inside nucleic acid molecules, while exonucleases can sequentially break phosphodiester bridges at the 5' or 3' ends of nucleic acid molecules as well. Another example includes DNAzymes or deoxyribozymes, catalytic oligonucleotides capable of cleaving phosphodiester bonds in nucleic acid molecules. All these types of enzymes can be engineered for imager strand removal (Figure 5), resulting in enzymatic cleavage, modification, or degradation of the labeled imager strand nucleic acid.

[0090] Glycosylase. If a glycosylase can specifically remove a base involved in base pairing between the docking strand and the imager strand, it can reduce the strength of the interaction between the two strands. For example, deoxyuridine (dU) can be used to replace deoxythymidine (dT) in the imager strand. dU can pair with dA in the docking strand similarly to dT, but can be specifically removed by uracil-DNA glycosylase (UDG, commercially available from New England Biolabs, catalog number M0280S). This reaction results in abasic site(s) on the imager strand. Such abasic site can be further cleaved by endonuclease VIII. This further promotes dissociation between the remainder of the imager strand and the docking strand. Enzyme blends containing both UDG and endonuclease VIII are also commercially available (e.g., under the trade name USER, catalog number M5505S, from New England Biolabs). About 1-20, 1-15, 1-10, or 1-5 dU nucleotides may be placed in the imager strand. In USER, the dUs may be placed in such a way that after removal of the U, the residues are short enough (e.g., about 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleotides or less) that they dissociate spontaneously and rapidly. If only UDG (i.e., no endonuclease VIII) is used, removal of the dU unit may destabilize the strand enough to facilitate removal. The total number of base pairs between the imager strand and the docking strand after dU removal may be 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleotides or less. Thus, in some embodiments, the imager strand or intermediate strand may contain at least one U that is capable of cleavage by USER.

[0091] The sequence of the residue as well as the temperature affect how short the residue should be to spontaneously dissociate.For example, a sequence with high GC content may have more binding affinity at a short length than another sequence with a longer length.Thus, in some cases, a 9-mer may be sufficient for stable binding, and in other cases, a 9-mer may be sufficient for dissociation.Those skilled in the art can evaluate the sequence, temperature, and affinity in the cleavage of non-natural nucleotides discussed here and below.

[0092] Restriction endonucleases and nicking endonucleases. Restriction sites in the docking strand:imager strand duplex can be engineered. This allows the use of a corresponding restriction endonuclease to cleave such restriction sites, which disrupts the linkage between the target and the signal-generating portion of the imager strand. As an example, Cas9 (CRISPR-associated protein 9) is an RNA-guided endonuclease that can be used to specifically cleave the docking:imager strand duplex by engineering a specific recognition site in the corresponding sequence. As a result, both strands are cleaved, making it impossible to re-search for the corresponding target. To solve this problem, a nicking endonuclease that cleaves only one strand can be used. As an example, Cas9 nickase is a Cas9 enzyme engineered to contain only one active cleavage site, resulting in a single strand cleavage while retaining the high specificity of Cas9. The restriction sites can be designed in such a way that only the imager strand is cleaved and the remainder of the imager strand, carrying the signal generating moiety, is short enough (e.g., less than 7 nucleotides) to spontaneously and rapidly dissociate from the docking strand. Other examples of endonucleases with site-specific activity include, but are not limited to, zinc finger nucleases, transcription activator-like effector nucleases (TALENs), and deoxyribozymes.

[0093] Rnase. Some or all of the nucleotides in the imager strand may be made RNA nucleotides (also called ribonucleotides) instead of DNA nucleotides (also called deoxynucleotides). Such RNA nucleotides may be removed by Rnase. If the docking strand is made of DNA nucleotides and the imager strand contains RNA nucleotides, such RNA nucleotides in the DNA:RNA heteroduplex may be removed by Rnase H.

[0094] Polymerase. The imager strand can also be removed by using a polymerase with strand displacement activity or 5' to 3' exonuclease activity. For example, a hairpin structure at the 3' end of the docking strand made from DNA can be engineered. When a DNA polymerase with strand displacement activity (e.g., Phi29, Bst, Vent) is introduced and a suitable buffer and dNTPs are supplied, the 3' of the docking strand can be extended while the imager strand is displaced (Figure 6c). A self-priming hairpin can also be engineered on the imager strand (Figure 6a-b), for which a signal generating moiety can be attached directly to the imager strand (Figure 6a) or via DNA hybridization (Figure 6b). When a DNA polymerase with 5' to 3' exonuclease activity (e.g., DNA polymerase I, Taq) is introduced, a suitable buffer and dNTPs are provided, and a self-priming hairpin is engineered at the 3' end of the docking strand, the 3' can be extended while the imager strand is degraded (Figure 6e). A similar effect can be achieved if a self-priming hairpin is engineered at the 3' end of the imager strand (Figure 6d). Note that the self-priming hairpin can also be replaced by a stable duplex (e.g., Figure 6f).

[0095] Cleavage of non-natural nucleotides. Non-natural nucleotides that function as substrates for certain enzymes can be used. For example, 8-oxoguanine can be cleaved by DNA glycosylase OGG1. Abasic sites can also be incorporated into DNA strands, such as the imager strand, that can be cleaved by endonucleases. For example, 1',2'-dideoxyribose, dSpacer, apurinic / apyrimidinic, tetrahydrofuran, or abasic furan can be cleaved by endonuclease VIII. Thus, in some embodiments, the imager strand or intermediate strand can contain at least one abasic site that can be cleaved by endonuclease VIII. In some embodiments, the imager strand or intermediate strand can contain at least one deoxyuridine and at least one abasic site that can be cleaved by USER, UDG, or endonuclease VIII. Photocleavable spacers or RNA abasic sites, such as ribospacers (rSpacers) or abasic II modifications, can also be used. Other pairs of non-natural nucleotides and their paired enzymes can be used.

[0096] Thus, in some embodiments, the composition includes: (1) a label; (2) a first nucleic acid domain, a second nucleic acid domain, and a third nucleic acid domain, each nucleic acid domain being about 1-9 nucleotides in length (e.g., about 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleotide); (3) a first linking moiety linking the first nucleic acid domain and the second nucleic acid domain; and (4) a second linking moiety linking the second nucleic acid domain and the third nucleic acid domain, both linking moieties being independently selected from (a) an abasic site with an intact phosphodiester backbone, (b) a linker cleavable by a nucleic acid glycosylase, or (c) a restriction site or a nicking site. In some embodiments, the additional nucleic acid domains are linked by additional linking moieties. In some embodiments, at least one linking moiety is an abasic site (apyrimidinic) with an intact phosphodiester backbone. In some embodiments, at least one linking moiety is susceptible to cleavage from endonuclease VIII. In some embodiments, the nucleic acid domain comprises DNA, and in some embodiments, the nucleic acid domain comprises RNA. In some aspects, at least one binding moiety comprises at least one non-natural nucleotide. In some aspects, at least one binding moiety comprises 8-oxoguanine.

[0097] In some embodiments, the method of removing the imager strand may be combined with an amplification step. In some embodiments, the method for testing a sample for the presence of one or more targets includes (1) contacting the sample with one or more target-specific binding partners, where each target-specific binding partner is directly or indirectly bound to a nucleic acid strand, and where target-specific binding partners of different specificities are bound to different nucleic acid strands; (2) optionally removing unbound target-specific binding partners; (3) if the nucleic acid strand is a docking strand or a primer strand, and the nucleic acid is (a) increasing the number of docking strands associated with each target-specific binding partner, or (b) if the nucleic acid is a primer strand, associating two or more docking strands with the primer strand; (4) contacting the sample with a labeled imager strand capable of directly or indirectly binding to the docking strand, where the labeled imager strand comprises a composition described immediately above; (5) optionally removing the unbound labeled imager strand; (6) imaging the sample to detect the bound labeled imager strand; (7) removing the bound labeled imager strand from the docking strand, where the labeled imager strand is removed from the docking strand by enzymatically cleaving, modifying, or degrading the labeled imager nucleic acid; and (8) optionally repeating steps (1)-(7), or any subset thereof.

[0098] In some embodiments, the labeled imager nucleic acid is removed by enzymatic cleavage of the labeled imager strand.

[0099] F. Sample Description 1. Sample Type Various types of samples can be imaged using these methods. In some embodiments, the sample is a fixed sample. In some embodiments, the sample is a cell, a cell lysate, a tissue, a tissue lysate, and / or a whole organism. In some embodiments, the sample is a cell or tissue sample, a cell or tissue lysate, or a bodily fluid. In some embodiments, the sample is a tissue and imaging involves multiplexing in the tissue for immunostaining.

[0100] The sample may be provided in a liquid medium or a buffer solution.

[0101] 2. Antigen Retrieval In some embodiments, staining a sample with a target-specific binding partner requires specific conditions, and not all target-specific binding partners bind to their antigen under the same conditions, which may be because their target antigen is not available under the same conditions. Thus, in some embodiments, a method for testing a sample for the presence of one or more targets includes (1) treating the sample to expose one or more previously unavailable targets; (2) contacting the sample with one or more target-specific binding partners, where each target-specific binding partner is bound to a nucleic acid strand, and where target-specific binding partners of different specificities are bound to different nucleic acid strands; (3) optionally removing unbound target-specific binding partners; and (4) optionally removing unbound target-specific binding partners, where the nucleic acid strand is a docking strand or a primer strand, and optionally the nucleic acid (a) increases the number of docking strands associated with each target-specific binding partner if it is a docking strand, or (b) increases the number of docking strands associated with two or more target-specific binding partners if it is a primer strand. (5) contacting the sample with a labeled imager strand capable of binding directly or indirectly to the docking strand; (6) optionally removing unbound labeled imager strand; (7) imaging the sample to detect the bound labeled imager strand; (8) dissipating signal from the bound labeled imager strand; and (9) optionally repeating steps (1)-(8), each time (a) exposing a different set of previously unavailable targets; (b) using one or more different target-specific binding partners; and (c) using a labeled imager strand having a unique nucleotide sequence relative to at least one other labeled imager strand.

[0102] 3. Use in target description and biomarker identification In some embodiments, the method is useful for identifying biomarkers. In some cases, samples are imaged and data analysis is performed on the samples. In some embodiments, multiple targets are tested using corresponding target-specific binding partners for each target. In some cases, the relationship between different targets can be evaluated, for example, a user may want to determine the relationship of multiple markers to a disease state and conclude that diseased samples have elevated levels of A, reduced levels of B, and levels of C within a certain range compared to healthy tissue that does not have that biomarker distribution.

[0103] In some embodiments, at least 10, 96, 100, 384, or 500 samples are imaged and data analysis is performed on those samples.

[0104] In some embodiments, at least 5, 10, 15, 25, 30, 50, 75, or 100 or more targets are tested using a corresponding target-specific binding partner for each target.

[0105] G. Equipment and Software 1. Imaging chamber as flow cell In some embodiments, an imaging chamber may be used. In some cases, the imaging chamber is a fixed chamber with no inlet or outlet. In some embodiments, the imaging chamber has a single inlet / outlet combination. In other cases, the imaging chamber allows for flow and is designated a flow cell. The flow cell may consist of a first optically transparent support in combination with a second optically transparent material (such as a glass or plastic coverslip) to provide the flow cell with top and bottom surfaces and fluid flow therebetween. When a first and second optically transparent material are used, they may be arranged parallel to one another. Parallel includes geometric arrangements that are completely parallel, as well as arrangements that deviate from parallel by up to 1°, 2°, 3°, 4°, 5°, 6°, 7°, 8°, 9°, or 10°. In some embodiments, the second optically transparent material is in close proximity to the first optically transparent material, such as about 5 microns to 5 mm, 50 microns to 500 microns, or 500 microns to 5 mm.

[0106] The imaging chamber may also consist of a first optically transparent support and a gasket (also called an isolator or spacer). The gasket may be open to air on a top surface or may have a closed, optically transparent top surface. The gasket may have a combined inlet / outlet or may have both an inlet and an outlet. The gasket may also have no outlet. The gasket may be plastic, rubber, adhesive. The gasket may include CoverWell Chamber Gasket (Thermo Fisher), Ultra-Thin Sealing Chamber for Upright or Inverted Microscopes (Bioscience Tools), or an incubation chamber (Grace Bio-Labs, including HybriSlip™ Hybridization Covers, HybriWell™ Sealing Systems, CoverWell™ Incubation Chambers, Imaging Spacers, SecureSeal™ Hybridization Chambers, FlexWell™ Incubation Chambers, FastWells™ Reagent Barriers, and Silicone Isolators™).

[0107] In some cases, the gasket may be used in conjunction with a coverslip that forms the top surface of the imaging chamber or flow cell.

[0108] The imaging chamber, such as, but not limited to, the flow cell, may be reusable or disposable.

[0109] Thus, in some embodiments, a method for testing a fixed sample mounted on a first optically transparent support for the presence of one or more targets includes (1) contacting the sample with one or more target-specific binding partners, where each target-specific binding partner is bound to a nucleic acid strand, and where target-specific binding partners of different specificities are bound to different nucleic acid strands; (2) optionally removing unbound target-specific binding partners; and (3) optionally removing the unbound target-specific binding partners, where the nucleic acid strands are docking strands or primer strands, and optionally removing the unbound target-specific binding partners, where the nucleic acid strands are (a) docking strands, increasing the number of docking strands associated with each target-specific binding partner, or (b) primer strands, increasing the number of docking strands associated with each target-specific binding partner, where the nucleic acid strands are primer strands. (4) contacting the sample in an imaging chamber (such as a flow cell) with a labeled imager strand capable of binding directly or indirectly to the docking strand; (5) optionally removing unbound labeled imager strand; (6) imaging the sample to detect the presence, location, and number of bound labeled imager strands and optionally removing the imaging chamber; (7) dissipating signal from the bound labeled imager strands; and (8) repeating steps (4)-(6) or (4)-(7), at least once where the labeled imager strand optionally has a unique nucleotide sequence relative to at least one other labeled imager strand.

[0110] A method for imaging a fixed sample mounted on an optically transparent support includes: (1) contacting the sample with one or more target-specific binding partners, where each target-specific binding partner is bound to a nucleic acid strand, and where target-specific binding partners of different specificities are bound to different nucleic acid strands; (2) optionally removing unbound target-specific binding partners; and (3) where the nucleic acid strands are docking strands or primer strands, the nucleic acid (a) increases the number of docking strands associated with each target-specific binding partner if the nucleic acid is a docking strand, or (b) increases the number of docking strands associated with two or more target-specific binding partners if the nucleic acid is a primer strand. The method includes associating the docking strand with the primer strand; (4) contacting the sample with a labeled imager strand capable of binding, either directly or indirectly, to the docking strand; (5) optionally removing unbound labeled imager strand; (6) optionally removing the liquid to create a liquid-free sample; (7) attaching a second optically transparent material in close proximity (i.e., about 5 microns to 5 mm, 50 microns to 500 microns, or 500 microns to 5 mm) and parallel to the first support; and (8) imaging the sample to detect bound labeled imager strand.

[0111] 2. Software for Control of Fluidic Steps In some embodiments, all fluid exchange steps are performed using a fluidic system including electronic, and / or pneumatic, and / or hydraulic, and / or electro-fluidic actuators and systems. In certain circumstances, the fluidic system is controlled by software. In some embodiments, the fluidic system is automatically controlled by software that synchronizes steps (1 (contacting the sample with the target-specific binding partner), and / or 2 (optionally removing unbound target-specific binding partner), and / or 3 (increasing or amplifying the number of docking strands), and / or 4 (contacting the sample with a labeled imager strand capable of directly or indirectly binding to the docking strand), and / or 5 (optionally removing unbound labeled imager strand), and / or 7 (optionally removing bound labeled imager strand from the docking strand) with the imaging step (6).

[0112] In some embodiments, the fluidic system is controlled by software that synchronizes steps (1, and / or 2, and / or 3, and / or 4, and / or 5, and / or 7) with the imaging step (6) by communicating with the imaging software with reference to the step numbers listed in the previous paragraph.

[0113] In some embodiments, all fluidic steps are performed while the sample is on the imaging device. In some embodiments, steps 4, 5, and 7 are performed while the sample is on the imaging device, with reference to the step numbers in the previous two paragraphs.

[0114] The sample may be fixed in a disposable imaging chamber (such as a flow cell) or a reusable imaging chamber (such as a flow cell).

[0115] H.Kit In some embodiments, the composition includes (1) one or more reagent(s) including, but not limited to, a target-specific binding partner bound to a docking strand (wherein target-specific binding partners of different specificities are bound to different docking strands), a labeled imager strand, a buffer, an amplification reagent, and / or a reagent for removing the bound imager strand, (2) a fluidic system for performing all fluid exchange steps, software for controlling the fluidic system and time and / or synchronizing the fluidic steps with the imaging steps, and (3) an imaging chamber (such as a flow cell) for attaching at least one optically transparent side to a sample of interest to allow imaging of the sample. In some embodiments, the imaging chamber (such as a flow cell) is disposable.

[0116] II. Method Components A. Target-specific Binding Partners Target recognition moieties refer to antibodies and antibody-like molecules that can be used to detect target molecules. Antibodies refer to any immunoglobulin from any species that can specifically recognize a target molecule. Antibody-like molecules include (Class A) Fab, Fab', F(ab') 2 Class B) any engineered mutation or fragment of an antibody (antigen-binding fragment of an antibody), such as a single heavy chain, diabody, etc.; (Class B) any known binding partner of a target molecule and engineered variants of such binding partners; (Class C) any binding partner of a target molecule that has been engineered via directed evolution (e.g., peptides and aptamers); and (Class D) any molecule that selectively forms a covalent bond(s) with a target (e.g., a suicide substrate for an enzyme of interest).

[0117] The target-specific binding partner can be provided in a liquid medium or buffer solution.

[0118] Table 3 provides a representative list of targets and corresponding target recognition moieties. TIFF2025011077000005.tif221170

[0119] Table 4 provides a list of additional targets. Antibodies and other known binding partners of these targets can be used as target recognition moieties. TIFF2025011077000006.tif175170

[0120] B. Docking strand In some embodiments, the docking moiety or docking strand is a nucleic acid, a protein, a peptide, or a chemical compound. Many proteins and protein domains are known to interact with other proteins, domains, or peptides. Some of the most well-known domains include the SH2, SH3, and WD40 domains. In many cases, the binding partners of these proteins and domains are known and can be engineered to have the desired affinity. For example, biotin and avidin / streptavidin interact with sufficient specificity. Many other chemical compounds, such as digoxigenin, fluorescein, tacrolimus, and rapamycin, also have known binding partners.

[0121] In some embodiments, the docking strand comprises a nucleic acid. In some embodiments, the nucleic acid is a single-stranded nucleic acid, such as single-stranded DNA, RNA, or a nucleic acid analog. The nucleic acid analog (also known as a non-natural nucleic acid) may include a modified phosphate backbone, a modified pentose sugar, and / or a modified nucleobase. The nucleic acid analog may include, but is not limited to, 2'-O-methyl ribonucleic acid, 2'-fluoro ribonucleic acid, peptide nucleic acid, morpholino and locked nucleic acid, glycol nucleic acid, and threose nucleic acid.

[0122] In some embodiments, the docking strand is covalently, and in other embodiments non-covalently, attached to the imager strand.

[0123] In some embodiments, the docking strand comprises a single stranded nucleic acid and may be about 5-20 nucleotides in length, about 8-15, or about 10-12 nucleotides in length, in some embodiments, the docking strand is about 5, 8, 9, 10, 11, 12, 13, 14, 15, 18, or 20 nucleotides in length.

[0124] The docking chain can be an independent element or can be a part of the target recognition part.For example, when the target recognition part is an antibody, the part of the Fc domain of the antibody can be the docking chain, and the peptide or protein that binds to the Fc domain, such as protein A or protein G, can be used.

[0125] The docking strand may be provided in a liquid medium or buffer solution.

[0126] C. Imager Chain In some embodiments, the docking strand may be a nucleic acid strand. In such cases, the observable moiety or label may be conjugated to an imager moiety, which may be a nucleic acid strand that is complementary to the docking strand. In other words, the imager strand specifically binds to the docking strand. In such cases, the label may be conjugated to an imager moiety, which may be about 5-20 nucleotides in length, about 8-15, or about 10-12 nucleotides in length. In some embodiments, the imager moiety is about 5, 8, 9, 10, 11, 12, 13, 14, 15, 18, or 20 nucleotides in length.

[0127] In some embodiments, the imager strand is longer, such as 20-80 nucleotides in length, e.g., 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, or 30 nucleotides in length or less. In embodiments that use a hairpin structure for the imager strand, the length of the imager strand can be longer than if a hairpin structure is not used.

[0128] In some embodiments, the complementary portion between the imager portion and the docking strand may be about 5-20 nucleotides in length, about 8-15, or about 10-12 nucleotides in length. In some embodiments, the complementary portion between the imager portion and the docking strand may be about 5, 8, 9, 10, 11, 12, 13, 14, 15, 18, or 20 nucleotides in length.

[0129] In some embodiments, the nucleic acid imager strand comprises a single-stranded nucleic acid, such as a single-stranded DNA, RNA, or a nucleic acid analog. The nucleic acid analog (also known as a non-natural nucleic acid) may comprise a modified phosphate backbone, a modified pentose sugar, and / or a modified nucleobase. The nucleic acid analog may include, but is not limited to, 2'-O-methyl ribonucleic acid, 2'-fluoro ribonucleic acid, peptide nucleic acid, morpholino and locked nucleic acid, glycol nucleic acid, and threose nucleic acid.

[0130] In some embodiments, the imager moiety is a protein, peptide, or chemical compound as a partner to the docking strand options discussed in Section II.B above.

[0131] In some embodiments, the docking strand may be indirectly bound to the imager portion, such as through an intermediate portion. For example, if the docking strand and the imager portion are nucleic acids, an intermediate portion comprising a nucleic acid may be used, as long as it has a first region that is complementary to the docking strand and a second region that is complementary to the imager portion. In this embodiment, the docking strand does not need to be complementary to the imager portion. The intermediate portion may only perform a bridging function, or it may also perform an amplification function.

[0132] The imager strand may be provided in a liquid medium or buffer solution.

[0133] D. Primer strand In some cases, the target-specific binding partner is indirectly linked to the docking strand, such as through a primer. For example, when the docking portion and the imager portion comprise a nucleic acid, the primer strand comprising the nucleic acid can be used as a binding site for the docking strand (if the docking strand has a region complementary to the primer strand), or it can be used as a primer for nucleic acid synthesis, for example, through rolling circle amplification. The primer strand can also be used to initiate a series of binding events in hybridization chain reaction amplification. When the primer serves as a site for amplification (rolling circle amplification, hybridization chain reaction amplification, etc.), the primer is not necessarily complementary to the docking strand. Instead, it serves as a template for amplification, and the docking strand is included throughout the amplification process.

[0134] In some embodiments, the target-specific binding partner and associated primer are added to the sample as a first step, the docking strand is added as a second step, and the imager strand is added as a third step. In other embodiments, the components are not added in separate steps. Washing steps may be added between the first, second, and / or third steps.

[0135] In some embodiments, the primer strand comprises a nucleic acid. In some embodiments, the nucleic acid is a single-stranded nucleic acid, such as a single-stranded DNA, RNA, or a nucleic acid analog. The nucleic acid analog (also known as a non-natural nucleic acid) may contain a modified phosphate backbone, a modified pentose sugar, and / or a modified nucleobase. The nucleic acid analog may include, but is not limited to, 2'-O-methyl ribonucleic acid, 2'-fluoro ribonucleic acid, peptide nucleic acid, morpholino and locked nucleic acid, glycol nucleic acid, and threose nucleic acid.

[0136] In some embodiments, the primer strand comprises a single stranded nucleic acid and may be about 5-20 nucleotides in length, about 8-15, or about 10-12 nucleotides in length, in some embodiments, the primer strand is about 5, 8, 9, 10, 11, 12, 13, 14, 15, 18, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, or 70 nucleotides in length.

[0137] The primer strands may be provided in a liquid medium or a buffer solution.

[0138] E. Intermediate chain In some cases, the docking strand is attached to the imager strand through an intermediate portion (or intermediate strand). For example, if the docking portion and the imager portion comprise a nucleic acid, an intermediate strand comprising a nucleic acid may be used as long as it has a first region that is complementary to the docking strand and a second region that is complementary to the imager strand. In such an embodiment, the docking strand does not need to be complementary to the imager portion.

[0139] In some embodiments, the intermediate strand is added to a sample containing a target-specific binding partner linked, either directly or indirectly, to a docking strand as a first step, and the imager strand is added as a second step. In other embodiments, the intermediate strand and the imager strand are not added in separate steps. In some cases, the intermediate strand and the imager strand are hybridized together before being added in a single step.

[0140] In some embodiments, the intermediate strand comprises a nucleic acid. In some embodiments, the nucleic acid is a single-stranded nucleic acid, such as single-stranded DNA, RNA, or a nucleic acid analog. Nucleic acid analogs (also known as non-natural nucleic acids) may contain modified phosphate backbones, modified pentose sugars, and / or modified nucleobases. Nucleic acid analogs may include, but are not limited to, 2'-O-methyl ribonucleic acid, 2'-fluoro ribonucleic acid, peptide nucleic acid, morpholino and locked nucleic acid, glycol nucleic acid, and threose nucleic acid.

[0141] In some embodiments, the intermediate strand comprises a single stranded nucleic acid and may be about 5-30 nucleotides in length, about 8-15, or about 10-12 nucleotides in length, in some embodiments, the intermediate strand is about 5, 8, 9, 10, 11, 12, 13, 14, 15, 18, 20, 25, or 30 nucleotides in length.

[0142] The intermediate strand may be provided in a liquid medium or a buffer solution.

[0143] F. Hairpin-Formed Nucleic Acids Any of the linear nucleic acids described herein may optionally be provided in a hairpin format. This includes the imager strand, the docking strand, the primer strand, and the intermediate strand. In the hairpin format, a region of at least 1-5 nucleotides at the end of the hairpin stem region may optionally contain only G' and C'. This G / C region is known as a clamp. The G / C region prevents or reduces fraying at the end of the hairpin to prevent opening into linear DNA.

[0144] Hairpins can be used in situations where the user wishes to disrupt the interaction (direct or indirect) between the imager strand and the docking strand using a polymerase with strand displacement activity (e.g., phi29) or a polymerase with 5' to 3' exonuclease activity (e.g., DNA polymerase I). Hairpins can also be used to limit undesired binding of single-stranded nucleic acids.

[0145] G. Signs Various labels, also known as observable moieties, can be attached to the imager strand. These labels or observable moieties assist the user by allowing detection of the bound imager strand. When the application refers to detecting the bound labeled imager strand, the application refers to detecting the signal generated by the label or observable moiety attached to the imager strand.

[0146] In some embodiments, any observable moiety may be used, and in some embodiments, the moiety is optically observable. The moiety may be signal absorbing or signal emitting. Among the signal emitting molecules, fluorescent molecules such as small organic molecules, including but not limited to fluorophores, such as but not limited to fluorescein, rhodamine, cyanine dyes, Alexa dyes, DyLight dyes, Atto dyes, etc., may be used.

[0147] In some embodiments, organic polymers such as p-dots may be used. In some embodiments, the observable moiety may be a biomolecule, including but not limited to, a fluorescent protein or a fluorescent nucleic acid (including fluorescent RNA, including spinach and its derivatives). In some embodiments, the observable moiety may be an inorganic moiety, including a Q-dot. In some embodiments, the observable moiety may be a moiety that operates through scattering, either elastic or inelastic, such as nanoparticles and surface-enhanced Raman spectroscopy (SERS) reporters (e.g., 4-mercaptobenzoic acid, 2,7-mercapto-4-methylcoumarin). In some embodiments, the observable moiety may be a chemiluminescent / electrochemiluminescent emitter, such as ruthenium complexes and luciferase. The observable moiety may generate a light signal, an electromagnetic signal (across the electromagnetic spectrum), an atomic / molecular mass (e.g., detectable by mass spectrometry), a solid mass (e.g., detectable by atomic force microscopy), a current, or a voltage. EXAMPLES

[0148] Example 1: Multiple Amplification Modes There are multiple approaches to amplification, and this example demonstrates the use of three approaches including enzymatic amplification using HRP conjugates (Figures 7A-C), rolling circle amplification (Figures 7D-E), and hybridization chain reaction (HCR) (Figures 7F-G).

[0149] For HRP-based amplification, formalin-fixed / paraffin-embedded (FFPE) tonsil tissue sections were dewaxed and antigen retrieved using R-Buffer A (Fisher Scientific) in a Lab Vision PT module. Tissue sections were blocked for 1.5 h in 3% BSA and 0.2% Triton-X 100. Tissue sections were stained with mouse anti-cytokeratin primary antibody overnight at 4°C in a humidity chamber. Tissue sections were then washed with 1x PBS and stained with goat anti-mouse secondary antibody conjugated to DNA docking strand (D1) for 2 h at room temperature. Tissue sections were washed again with 1x PBS and stained against DAPI.

[0150] A fluorescent microscope was used to image the tissue sections in DAPI and Cy5 channels to serve as blanks. An imager strand (I1-650) containing a red fluorophore attached to DNA containing a domain complementary to docking strand D1 was added to the prepared tissue sections and hybridized for 25 minutes at room temperature. The sections were washed to remove unbound I1-650. Fluorescent images were then captured in DAPI and Cy5 channels using a 10x objective. See Figure 7B.

[0151] The imager strand I1-650 was then removed by applying 10 units of USER enzyme to the tissue sections for 15 minutes at room temperature, washing with 1x PBS, and incubating the tissue samples with 30% formamide for 5 minutes, followed by washing with 1x PBS. A fluorescent microscope was used to confirm complete removal of the fluorescent signal in the Cy5 channel. Bright field images were then taken at 10x magnification as blanks.

[0152] To amplify the cytokeratin signal for bright-field immunohistochemical detection, tissue sections were incubated with an imager strand (I1-HRP) containing horseradish peroxidase (HRP) enzyme attached to DNA containing a domain complementary to docking strand D1. The samples were washed by immersing the tissue sections in 1x PBS. Chromogenic signal amplification was then performed by applying a solution of 3,3'-diaminobenzidine (DAB) chromogen to the samples for 10 min. The samples were washed in 1x PBS. Finally, the samples were imaged using bright-field illumination. Figure 7C.

[0153] 7A-C illustrate the use of DNA exchange to allow fluorescent and brightfield (e.g., immunohistochemistry) images to be captured on the same sample. This example demonstrates the use of HRP-conjugated imager strands for chromogenic signal amplification, but the HRP-conjugated imager strands can also be applied to fluorescent signal amplification using alternative enzyme substrates (e.g., tyramide).

[0154] In a separate approach for rolling circle amplification-based amplification, cultured HeLa cells were fixed with warm 3% paraformaldehyde and 0.1% glutaraldehyde, reduced with 0.1% sodium borohydride, blocked and permeabilized with 3% bovine serum albumin and 0.2% Triton-X 100, and then stained overnight with a rabbit primary antibody against vimentin.

[0155] For the control samples, goat anti-rabbit antibody conjugated to a docking strand (Gt-a-Rb-D1) was incubated with the cells for 2 hours at room temperature as a secondary staining step. After washing of the control samples, DAPI and 100 nM fluorescently labeled imager strand were added to the samples. Fluorescence images in the DAPI and Cy5 channels of the control wells were collected. Figure 7D.

[0156] The following procedure was performed for cell samples undergoing rolling circle amplification. A goat anti-rabbit antibody conjugated to a docking strand (Gt-a-Rb-D1) was used as a template for ligation. A ligation solution was made in a PCR tube with 125 nM circular oligo (IDT DNA), 125 nM Gt-a-Rb-D1, 20 U / μL T4 ligase (NEB) in 1× ligation buffer (NEB). The ligation solution was incubated for 2 hours at room temperature. After ligation, the ligation solution was diluted 1:5 in blocking buffer and added to the cell sample and allowed to incubate for 2 hours at room temperature, then washed in 1× PBS.

[0157] In a separate vessel, a polymerase solution was made containing 0.1 mg / mL BSA, 0.2 mM dNTPs, and 1 U / μL phi29 DNA polymerase in 1× polymerase reaction buffer (New England Biolabs). The polymerase solution was applied directly to the cell samples prepared for RCA. The samples were incubated with the polymerase solution for 1.5 hours at 30° C. The cell samples were washed three times with 1× PBS. DAPI was added to stain the cell nuclei. Finally, 100 nM fluorescently labeled imager strand was added before imaging. Fluorescent images in the DAPI and Cy5 channels were collected after RCA. See FIG. 7E.

[0158] As an alternative approach for hybridization chain reaction-based amplification, formalin-fixed / paraffin-embedded (FFPE) tonsil tissue sections were dewaxed and antigen retrieved using R-Buffer A (Fisher Scientific) in a Lab Vision PT module. Tissue sections were blocked for 1.5 h in 3% BSA and 0.2% Triton-X 100. Tissue sections were stained with mouse anti-cytokeratin primary antibody overnight at 4°C in a humidity chamber. Tissue sections were then washed with 1x PBS and stained with goat anti-mouse secondary antibody conjugated to DNA docking strand (D1) for 2 h at room temperature. Tissue sections were washed again with 1x PBS and stained against DAPI.

[0159] A fluorescent microscope was used to image the tissue sections in DAPI and Cy5 channels to serve as blanks. An imager strand (I1-650) containing a red fluorophore attached to DNA containing a domain complementary to docking strand D1 was added to the prepared tissue sections and hybridized for 25 minutes at room temperature. The sections were washed to remove unbound I1-650. Fluorescent images were then captured in DAPI and Cy5 channels using a 10x objective. See Figure 7F.

[0160] The imager strand I1-650 was then removed by applying 10 units of USER enzyme to the tissue sections for 15 min at room temperature and washing with 1× PBS. Complete removal of the fluorescent signal in the Cy5 channel was confirmed using a fluorescent microscope (data not shown).

[0161] After removal of the imager strand, the primer strand containing the I1 sequence was hybridized to the D1 docking strand (20 nM) for 30 minutes at room temperature. The sections were washed with 1x PBS to wash off unbound primer strands. In parallel, hairpin strands H1 and H2-D2 were diluted to 1 uM in 5x SSX in separate microcentrifuge tubes. The tubes were heated and held at 90°C for 5 minutes, then slowly cooled to room temperature. The hairpins were then pooled and diluted to 200 nM in 5x SSC with 0.1% Tween 20 and 10% dextran sulfate. This solution was added to the tissue sections and incubated overnight in an incubation chamber at room temperature. The sections were then washed with 1x PBS. Finally, 100 nM fluorescently labeled imager strand I2 was added before imaging. Fluorescent images in the DAPI and Cy5 channels were collected after HCR amplification. Figure 7G, right.

[0162] Example 2: Amplification with pre-ligated circular oligos A nonlinear DNA strand was generated before performing the assay. The use of a nonlinear DNA strand provides the advantage that the efficiency of signal amplification can be increased, especially when the signal amplification method used is similar to rolling circle amplification (FIG. 8). In this example, a circular DNA strand was generated via a ligation step. The DNA sequence used in the ligation step is referred to as a circular oligo, which contains a domain that is equivalent to the imager strand sequence (i.e., complementary to the docking strand).

[0163] Cultured HeLa cells were fixed with warm 3% paraformaldehyde and 0.1% glutaraldehyde, reduced with 0.1% sodium borohydride, blocked and permeabilized with 3% bovine serum albumin and 0.2% Triton-X 100, and then stained overnight with rabbit primary antibody against vimentin. Goat anti-rabbit antibody conjugated to docking strand (Gt-a-Rb-D1) was then used as the secondary stain and allowed to incubate for 2 hours at room temperature.

[0164] 0.5 μM circular oligo (IDT DNA), 50 μM ATP, 2.5 mM MnCl in 1× reaction buffer (EpiCentre) 2 A ligation solution was made in a PCR tube using 5U / μL CircLigase (catalog no. CL4111K, EpiCentre). The ligation solution was placed in a thermocycler for 1 hour and incubated at 60° C. After ligation, a 1:4 dilution of the ligation solution was made in blocking buffer and added to the stained cell sample and allowed to incubate for 25 minutes to hybridize to the docking strand on the antibody-labeled target complex before washing in PBS.

[0165] As an alternative approach, the ligation step can be performed before staining the sample with the secondary antibody conjugate. After the ligation step, an excess of the ligation solution was hybridized to the antibody DNA conjugate, so that the molar ratio of the ligated circular oligo was at least 2-fold greater than the molar ratio of the docking strand bound to the antibody. After hybridization of the ligated circular oligo to Gt-a-Rb-D1, the complex was added to the sample and incubated at room temperature for 2 hours as a secondary staining step, and then washed three times in 1×PBS.

[0166] In a separate vessel, a polymerase solution was made containing 0.1 mg / mL BSA, 0.2 mM dNTPs, and 1 U / μL phi29 DNA polymerase in 1× polymerase reaction buffer (New England Biolabs). The polymerase solution was applied directly to fixed cell samples already stained with primary antibody, secondary antibody docking strand conjugate, and hybridized to the ligated circular oligos. The samples were incubated with the polymerase solution for 1.5 hours at 30° C. The cell samples were washed three times with 1× PBS. DAPI was added to stain the cell nuclei. Finally, 100 nM fluorescently labeled imager strand was added before imaging. Figure 8 shows the use of a preformed nonlinear DNA strand that can be hybridized to a docking strand as an initiation point for rolling circle polymerization.

[0167] Example 3: Sample re-search (0-A-0-A) This example demonstrates the ability to use exchange imaging to re-search for targets in a sample. It also shows that the enzymatic method of quenching the signal does not affect, modify, or degrade the staining of the sample.

[0168] In this example, the docking strands (D1 and D2) were conjugated to secondary antibodies goat anti-mouse and goat anti-rabbit, respectively. The imager strands I1U and I2U, complementary to D1 and D2, respectively, were labeled with a fluorescent dye (ATTO647N). The sequences of imager strands I1U and I2U contain several uracil nucleobases. An enzyme or cocktail of enzymes can cleave the bases, resulting in dehybridization of the docking-imager duplex, resulting in a decrease in the observed signal.

[0169] Cultured HeLa cells were fixed with warm 3% paraformaldehyde and 0.1% glutaraldehyde, reduced with 0.1% sodium borohydride, blocked and permeabilized with 3% bovine serum albumin and 0.2% Triton-X 100 in 1x PBS for 1.5 hours. First, cells were stained overnight at 4°C using a cocktail of primary antibodies anti-tubulin and anti-TOM20 raised in mouse and rabbit, respectively. Goat secondary antibodies against mouse and rabbit conjugated to two different docking chains (Gt-a-Ms-D1 and Gt-a-Rb-D2) were used for secondary staining at room temperature for 2 hours. DAPI was added to stain cell nuclei.

[0170] Samples were mounted on an inverted Nikon Eclipse Ti microscope (Nikon Instruments) equipped with a fluorescence module and an Andor Zyla sCMOS camera. The microscope was equipped with two fluorescence filter sets for imaging DAPI and fluorescently labeled imager strands (Cy5 channel).

[0171] First, the samples were imaged using a 20x dry objective (Nikon, 0.45NA) in both DAPI and Cy5 channels to register the cells using DAPI and measure the autofluorescence signal in the Cy5 channel (Figure 9A). A solution of 100 nM I1U was added to the sample and incubated for 15 minutes. The sample was then washed three times with 1x PBS and imaged again (Figure 9B). To quench the signal, a solution containing 5 units of USER™, 50 mM potassium acetate, 20 mM Tris-acetate, 10 mM magnesium acetate, and 100 μg / mL BSA was added to the sample and incubated at room temperature for 15 minutes. The sample was then washed three times with 1x PBS and imaged (Figure 9C). A solution of 100 nM I2U was added to the sample and incubated for 15 minutes. The sample was then washed three times with 1x PBS and imaged (Figure 9D). The signal was quenched as before and the sample was imaged again (Figure 9E). Finally, a solution of 100 nM I1U was added to the sample and incubated for 15 min. The sample was washed 3 times with 1x PBS and imaged (Figure 9F).

[0172] Figure 9 shows a series of images obtained in the Cy5 channel during this experiment. In Figures 9A, 8C, and 8E, no signal is observed. These images correspond to before introduction of the imager strand, after removal of I1U, and after removal of I2U, respectively. After addition of the imager strand I1U, a microtubule network is observed (Figures 8B, 8F). Figure 8D shows the presence of mitochondria after addition of I2U. Figure 9G shows the average signal intensity in the images of Figures 9A-F. The signal is reduced by more than 98% in both cases shown in Figures 9C and 8E. Signal recovery on the microtubule target after two exchanges is more than 95%, demonstrating the ability to reliably re-search the target after multiple exchange steps.

[0173] The images in Figures 9A-F were obtained by manually pipetting the incubation and washing solutions in and out of the imaging chamber. The exchange imaging workflow can be automated by using a flow cell and fluidics system.

[0174] Using a fluidics system synchronized with the microscope's acquisition software, the fluidic steps required for hybridization of the imager strands (injection, incubation, washing), as well as signal removal, can be performed automatically between imaging rounds.

[0175] HeLa cells were cultured, fixed, and stained as described above in a flow cell that can be mounted on a regular microscope stage. First, the sample was imaged using a 20x dry objective (Nikon, 0.45NA) in both the DAPI and Cy5 channels to register the cells using DAPI and measure the autofluorescence signal in the Cy5 channel (Figure 9, image H). A solution of 100 nM I1U was flowed into the sample chamber and incubated for 15 minutes. The sample was then washed with 1x PBS in a continuous flow for 30 seconds and imaged again (Figure 9I). Next, a solution containing 5 units of USER™, 50 mM potassium acetate, 20 mM Tris-acetate, 10 mM magnesium acetate, and 100 μg / mL BSA was injected into the flow cell and incubated for 15 minutes. The sample was then washed with 1x PBS for 30 seconds and imaged (Figure 9J). Finally, a solution of Imager I1U was again injected into the flow cell, incubated for 15 minutes, and washed with PBS for 30 seconds. The sample was imaged one last time (Figure 9K).

[0176] Example 4: Proximity detection with stable binding A pair of proximity probes (PL and PR) was conjugated to anti-mouse antibodies using the ThunderLink kit (Innova Biosciences). Cultured HeLa cells were fixed with warm 3% paraformaldehyde and 0.1% glutaraldehyde, reduced with 0.1% sodium borohydride, blocked and permeabilized with 3% bovine serum albumin and 0.2% Triton-X 100, and then stained with mouse primary antibody against alpha-tubulin. Fixed cells were then stained with a mixture of PL- and PR-conjugated secondary antibodies and washed to remove any unbound material. The majority of anti-alpha-tubulin antibodies in the sample should receive at least one molecule of the PL-conjugated secondary antibody and at least one molecule of the PR-conjugated antibody. To detect the proximity signal, DAPI and 100 nM imager strand were added to the sample, and the sample was imaged with a wide-field fluorescence microscope at 20x with an LED light source (Figure 10A (upper panel)).

[0177] In a negative control experiment (Figure 10, bottom panel), one of the proximity probes was conjugated to an off-target anti-rabbit secondary antibody. Fixed cells were stained with mouse anti-tubulin primary antibody, PL-conjugated anti-mouse secondary antibody, and PR-conjugated anti-rabbit secondary antibody. The anti-rabbit secondary antibody should not bind to the mouse-derived primary antibody, and therefore the proximity probes should not be co-localized and the complete docking site cannot bind to the imager strand.

[0178] As shown in Figure 10, microtubule structures are evident when both proximity probes are specifically bound to the target. The fluorescent signal is stable to standard fluorescent imaging.

[0179] Example 5: Control experiments for off-target cross-reactivity Cross-reactivity between off-target imagers and docking strands was assessed by measuring the signal intensity of samples before and after exposure to non-complementary imager strand sequences.

[0180] Cultured HeLa cells were fixed, reduced, blocked and permeabilized, then stained with mouse primary antibody against alpha-tubulin. Fixation buffer consisted of 3% paraformaldehyde and 0.1% glutaraldehyde. Fixed cells were reduced using a solution containing 0.1% sodium borohydride. Prior to overnight primary antibody staining, cells were blocked and permeabilized for 1.5 hours using a solution with 3% bovine serum albumin and 0.2% Triton-X 100. To test the cross-reactivity of docking strand A to imager strands B and C, an anti-mouse antibody conjugated to docking strand A (a-Ms-A) was used. d ) was added to fixed cell samples stained for alpha-tubulin. d Secondary staining with Cy5 was performed in blocking buffer for 2 hours at room temperature, followed by washing three times with 1×PBS. DAPI dye was added to all wells, and blank images were taken in both DAPI and Cy5 channels on a Nikon fluorescent microscope with a 20× objective.

[0181] After recording a blank image, an imager strand solution was prepared. In this example, the imager strand is partially double-stranded, the imager strand is composed of a universal domain and a non-universal domain, the universal domain hybridizes to a complementary sequence attached to a fluorophore, and the non-universal domain is single-stranded and is capable of interacting with a docking strand in a sample. In this example, the fluorophore is indirectly included in the imager strand through a DNA-based crosslink. In this example, the non-universal domain in the imager strand is complementary to a specific docking strand (e.g., docking strand A, B, or C).

[0182] Imager chain A (A i ), B(B i ), or C(C iA 100 nM solution of 100 nM ...

[0183] The signal intensity of each sample before and after addition of the imager strand was compared using custom software. The DAPI channel was used to align the images and create a mask to segment individual cells from the background. The mean fluorescence intensity within the cell area was calculated for the Cy5 channel and divided by the mean intensity of the background to obtain the signal-to-noise ratio. The signal-to-noise ratio obtained with the blank image was compared to the signal-to-noise ratio obtained after addition of the imager strand to obtain the signal-to-noise ratio obtained with docked strand A and imager strand A. i , B i , and C i Cross-reactivity between the

[0184] As shown in FIG. 11, the imager chain Ai on the target is a-Ms-A d Figure 11 also clearly shows specific microtubule structures stained with B. i does not cross-react with docking strand A, but C i also show cross-reactivity.

[0185] Example 6: USER and subUSER This example demonstrates the ability to eliminate signal from a target using enzymatic activity. In this example, the docking strand (D1) was conjugated to a secondary antibody goat anti-mouse. The imager strand I1U, complementary to D1, was labeled with a fluorescent dye (ATTO647N). The imager strand I1U contains several uracil nucleobases that can be cleaved by specific enzymatic activity, destabilizing the double-stranded imager / docking strand and resulting in the elimination of the fluorescent signal.

[0186] Cultured HeLa cells were fixed with warm 3% paraformaldehyde and 0.1% glutaraldehyde, reduced with 0.1% sodium borohydride, and blocked and permeabilized with 3% bovine serum albumin and 0.2% Triton-X 100 in 1x PBS for 1.5 h. First, cells were stained overnight at 4°C with primary antibody anti-tubulin raised in mouse. Secondary staining was performed for 2 h at room temperature using goat secondary antibody against mouse conjugated to D1. DAPI was added to stain cell nuclei.

[0187] Samples were mounted on an inverted Nikon Eclipse Ti microscope (Nikon Instruments) equipped with a fluorescence module and an Andor Zyla sCMOS camera. The microscope was equipped with two fluorescence filter sets for imaging DAPI and fluorescently labeled imager strands (Cy5 channel).

[0188] A solution of 100 nM I1U was added to two separate samples and incubated for 15 min. The samples were then washed 3 times with 1×PBS and imaged using a 20× dry objective (Nikon, 0.45NA) in both DAPI and Cy5 channels (FIG. 12, images A and C). To quench the signal, a solution containing 2 units of USER™, 50 mM potassium acetate, 20 mM Tris-acetate, 10 mM magnesium acetate, and 100 μg / mL BSA was added to the samples. To the other sample, 10 units of uracil-DNA glycosylase (UDG), 1 mM DTT, 1 mM EDTA in 20 mM Tris-HCl were added. Both samples were incubated for 15 min at room temperature. The samples were then washed 3 times with 1×PBS and imaged (FIG. 12, images B for USER and D for UDG).

[0189] As shown in FIG. 12, the signal can be almost completely abolished using either USER or UDG.

[0190] Example 7: Cleavage of imager strand by USER As shown in Figure 14, the extent to which the signal may be lost may depend on the structure and / or sequence design of the imager strand. In this example, formalin-fixed paraffin-embedded (FFPE) tissue sections were dewaxed, antigen-retrieved, blocked, and stained with anti-CD3 antibody conjugated to docking strand D1. The number of D1 docking strands bound to the sample was amplified by RCA and labeled with an imager strand that is complementary to D1 and contains a domain linked to a fluorophore. The imager strands contained either some uracil nucleobases (Figures 14A and C) or some uracil nucleobases and abasic sites (Figures 14B and D). Images were collected using a fluorescent microscope to detect the presence of targets bound by the labeled imager strands (Figures 14A-B).

[0191] To eliminate the signal, a solution containing USER was added and incubated with the tissue sample for 15 minutes, followed by a washing step to remove the cleaved imager strand. Fluorescent images were collected to confirm the removal of the fluorescent signal. As shown in Figure 14D, the presence of abasic sites in addition to dideoxyuridine in the imager strand sequence design results in improved removal efficiency compared to Figure 14C.

[0192] Example 8: Combined multicolor and sequential detection This example demonstrates the ability to use exchange imaging to detect multiple targets using a set of imager strands functionalized with different spectrally resolved fluorophores.

[0193] Formalin-fixed paraffin-embedded (FFPE) tonsil tissue sections were dewaxed, antigen-retrieved, blocked, and stained with DAPI and anti-CD4, anti-CD8, anti-Ki67, and anti-cytokeratin antibodies conjugated to docking strands D1, D2, D3, and D4, respectively. The number of docking strands (D1, D2, D3, D4) bound to the samples via antibodies was amplified by RCA. Imager strands I1, I2, I3, and I4, which contain domains complementary to D1, D2, D3, and D4, respectively, also contained several uracil bases for enzymatic removal. I1 and I3 are labeled with Atto565 dye, while I2 and I4 are labeled with Atto647N. Samples were imaged using an inverted Nikon Eclipse Ti microscope (Nikon Instruments) equipped with a fluorescence module and an Andor Zyla sCMOS camera. The microscope was equipped with fluorescent filter sets for imaging DAPI and fluorescently labeled imager strands (TRITC and Cy5 channels).

[0194] In the first step, samples were labeled using I1 and I2 imager strands by incubating a cocktail of the two strands on the samples for 25 minutes. Unbound strands were washed away and images were collected in TRITC and Cy5 channels to detect the presence of targets, CD4 and CD8, labeled with imager strands I1 and I2, respectively (Figures 15B and C, respectively). DAPI images were also collected to detect cell nuclei (Figure 15A).

[0195] The signal was then quenched using a solution containing USER, which was incubated with the tissue sample for 15 minutes, followed by a washing step to remove the cleaved imager strand. Fluorescent images were collected to confirm removal of the fluorescent signal.

[0196] Finally, the samples were relabeled using a cocktail solution of imager strands I3 and I4, followed by a washing step to remove unbound imager strands. Fluorescent images were acquired in the TRITC and Cy5 channels to detect the presence of Ki67 and cytokeratin (Figures 15E and F, respectively). A DAPI image was also collected (Figure 15D) and used to align and overlay the images of the four targets.

[0197] Example 9: Numbered Items The following numbered items provide further description of the embodiments herein and how they relate to each other.

[0198] Item 1. (1) contacting a sample to be tested for the presence of one or more targets with one or more target-specific binding partners, each target-specific binding partner being bound to a nucleic acid strand, and target-specific binding partners of different specificities being bound to different nucleic acid strands; (2) optionally removing unbound target-specific binding partners; and (3) The nucleic acid strand is a docking strand or a primer strand, and the nucleic acid is (a) if a docking strand, increasing the number of docking strands associated with each target-specific binding partner; or (b) if a primer strand, associating two or more docking strands with the primer strand; (4) contacting the sample with a labeled imager strand capable of binding directly or indirectly to the docking strand; (5) optionally removing unbound labeled imager strands; (6) imaging the sample to detect bound labeled imager strands; and (7) optionally removing the bound labeled imager strand from the docking strand; and (8) optionally repeating steps (1)-(6), or any subset thereof.

[0199] Item 2. (1) contacting a sample to be tested for the presence of one or more targets with one or more target-specific binding partners, each target-specific binding partner being bound to a nucleic acid strand, and target-specific binding partners of different specificities being bound to different nucleic acid strands; (2) optionally removing unbound target-specific binding partners; and (3) contacting the sample with a labeled imager strand capable of binding directly or indirectly to a docking strand, where the nucleic acid strand of (1) is either a primer strand or a docking strand, and where if the nucleic acid strand is a primer strand, it is attached to the docking strand; (4) optionally removing unbound labeled imager strands; (5) imaging the sample to detect bound labeled imager strands and determine whether amplification (step (7)) is required; (6) optionally removing the bound labeled imager strand from the docking strand; and (7) optionally increasing the number of docking strands associated with each target-specific binding partner; and (8) optionally repeating steps (1)-(7), or any subset thereof.

[0200] Item 3. A method for testing a sample for the presence of one or more targets, comprising: (1) contacting a sample to be tested for the presence of one or more targets with one or more target-specific binding partners, each target-specific binding partner being bound to a nucleic acid strand, and target-specific binding partners of different specificities being bound to different nucleic acid strands; (2) optionally removing unbound target-specific binding partners; and (3) contacting the sample with a nonlinear amplifier strand having complementarity to the nucleic acid strand, the nucleic acid strand being either a docking strand or a primer strand; (4) optionally removing unbound nonlinear amplifier strands; (5) amplifying the docking strand by rolling circle amplification in either one or two steps and contacting the sample with a labeled imager strand that has complementarity to the docking strand or the amplified strand; (6) imaging the sample to detect bound labeled imager strands; and (7) removing the bound labeled imager strand; and (8) optionally repeating steps (1)-(8), or any subset thereof.

[0201] Item 4. The method of item 3, wherein the polymerase is used for rolling circle amplification.

[0202] Item 5. The method of items 3 or 4, wherein the nonlinear amplifier strand is combined with a target-specific binding partner attached to a nucleic acid strand prior to contacting with the sample.

[0203] Item 6. The method of any one of items 3 to 5, wherein amplifying the docking strand by rolling circle amplification occurs separately from contacting the sample with a labeled imager strand having complementarity to the docking strand or the amplified strand.

[0204] Item 7. The method of any one of items 3 to 6, wherein amplifying the docking strand by rolling circle amplification occurs in the same step as contacting the sample with a labeled imager strand that has complementarity to the docking strand, and the imager strand optionally includes a 3' modification to prevent amplification of the imager strand.

[0205] Item 8. The method according to any one of items 3 to 5 or 7, wherein the imager strand is a circular imager strand for rolling circle amplification.

[0206] Item 9. The method of any one of items 3 to 7, wherein the imager strand is a linear imager strand that circularizes in the presence of the docking strand and a ligase.

[0207] Item 10. The method of any one of items 3 to 9, wherein the imager strand or the amplifier strand comprises at least two regions that are complementary to the docking strand.

[0208] Item 11. The method according to any one of items 3 to 10, wherein the labeled imager chain is linear.

[0209] Item 12. The method according to any one of items 3 to 11, wherein the nonlinear amplifier chain is a cyclic chain.

[0210] Item 13. The method of any one of items 3 to 12, wherein the nonlinear amplifier strand becomes circular after ligation.

[0211] Item 14. A method for testing a sample for the presence of one or more targets, comprising: (1) contacting the sample with one or more target-specific binding partners, each target-specific binding partner being bound, directly or indirectly, to a nucleic acid strand, and target-specific binding partners of different specificities being bound to different nucleic acid strands; (2) optionally removing unbound target-specific binding partners; and (3) The nucleic acid strand is a docking strand or a primer strand, and the nucleic acid is (a) if docking strands, optionally increasing the number of docking strands associated with each target-specific binding partner; or (b) if a primer strand, optionally associating two or more docking strands with the primer strand; (4) contacting the sample with a labeled imager strand capable of binding directly or indirectly to the docking strand; (5) optionally removing unbound labeled imager strands; (6) imaging the sample to detect bound labeled imager strands; and (7) optionally quenching the signal from the bound labeled imager strand; (8) optionally repeating steps (1)-(7), or any subset thereof.

[0212] Item 15. The method of item 14, wherein the sample is mounted on an optically transparent support.

[0213] Item 16. The method according to any one of items 14 to 15, wherein after step (6) and optionally after performing step (7), the method further comprises increasing the number of docking strands associated with each target-specific binding partner and repeating steps (4), optionally (5), (6), and optionally (7).

[0214] Item 17. The method according to any one of items 14 to 16, wherein the method comprises, in step (3), increasing the number of docking strands associated with each target-specific binding partner or associating two or more docking strands with the primer strand.

[0215] Item 18. The method of any one of items 1 to 17, wherein increasing the number of docking strands associated with each target-specific binding partner is achieved using an enzyme, which optionally is a polymerase.

[0216] Item 19. The method of any one of items 1 to 18, wherein enzymatically cleaving, modifying, or degrading the unbound labeled imager strand is accomplished by using an enzyme, optionally an enzyme selected from the group consisting of glycosylases, restriction endonucleases, nicking endonucleases, RNases, and enzymes that cleave at unnatural nucleotides.

[0217] Item 20. A method for testing a sample mounted on an optically transparent support for the presence of one or more targets, comprising: (1) contacting the sample with one or more target-specific binding partners, each target-specific binding partner being bound to a nucleic acid strand, and target-specific binding partners of different specificities being bound to different nucleic acid strands; (2) optionally removing unbound target-specific binding partners; and (3) the nucleic acid strand is a docking strand or a primer strand, and optionally the nucleic acid is (a) if a docking strand, increasing the number of docking strands associated with each target-specific binding partner; or (b) if a primer strand, associating two or more docking strands with the primer strand; (4) contacting the sample with a labeled imager strand capable of binding directly or indirectly to the docking strand; (5) optionally removing unbound labeled imager strands; (6) optionally removing the liquid to create a liquid-free sample; (7) attaching a second optically transparent material parallel to the first support; and (8) imaging the sample to detect bound labeled imager strands.

[0218] Item 21. The method of item 20, wherein the second optically transparent material is glass.

[0219] Item 22. The method of item 21, wherein the second optically transparent material is plastic.

[0220] Item 23. The method of any one of items 20 to 22, wherein the second optically transparent material is about 5 microns to 5 mm, 50 microns to 500 microns, or 500 microns to 5 mm from the first support.

[0221] Item 24. The method of item 20, wherein imaging is performed using an upright microscope.

[0222] Item 25. A method for testing a fixed sample mounted on an optically transparent support for the presence of one or more targets, comprising: (1) contacting the sample with one or more target-specific binding partners, each target-specific binding partner being bound to a nucleic acid strand, and target-specific binding partners of different specificities being bound to different nucleic acid strands; (2) optionally removing unbound target-specific binding partners; and (3) The nucleic acid strand is a docking strand or a primer strand, and the nucleic acid is (a) if a docking strand, increasing the number of docking strands associated with each target-specific binding partner; or (b) if a primer strand, associating two or more docking strands with the primer strand; (4) contacting the sample with a labeled imager strand capable of binding directly or indirectly to the docking strand; (5) optionally removing unbound labeled imager strands; (6) optionally removing the liquid to create a liquid-free sample; (7) attaching a second optically transparent material parallel to the first support; and (8) imaging the sample to detect bound labeled imager strands.

[0223] Item 26. (1) contacting a sample to be tested for the presence of one or more targets with one or more target-specific binding partners, each target-specific binding partner being bound to a nucleic acid strand, and target-specific binding partners of different specificities being bound to different nucleic acid strands; (2) optionally removing unbound target-specific binding partners; and (3) the nucleic acid strand is a docking strand or a primer strand, and optionally the nucleic acid is (a) if a docking strand, increasing the number of docking strands associated with each target-specific binding partner; or (b) if a primer strand, associating two or more docking strands with the primer strand; (4) contacting the sample with a labeled imager strand capable of binding directly or indirectly to the docking strand; (5) optionally removing unbound labeled imager strands; (6) imaging the sample to detect the presence, location, and number of bound, labeled imager strands; (7) quenching the signal from the bound labeled imager strand; and (8) repeating steps (3)-(6) or (3)-(7), wherein each labeled imager strand optionally has a unique nucleotide sequence relative to every other labeled imager strand.

[0224] Item 27. A composition comprising a sample bound to two or more target-specific binding partners, each binding partner being bound to a nucleic acid strand and at least one docking strand stably bound directly or indirectly to a labeled imager strand, the nucleic acid strand being a docking strand or a primer strand, the nucleic acid being: (a) if a docking strand, increasing the number of docking strands associated with each target-specific binding partner; or (b) a composition which, when a primer strand, associates two or more docking strands with the primer strand.

[0225] Item 28. The composition of item 27, wherein the docking strand bound directly or indirectly to the labeled imager strand comprises at least 90% binding for 30 minutes.

[0226] Item 29. (a) a docking strand, or (b) the docking strand and any intermediate strands is 80 nucleotides or less, 70, 60, 50, 40, or 30 nucleotides or less.

[0227] Item 30. The composition of any one of items 27 to 29, wherein the imager strand is 60 nucleotides or less.

[0228] Item 31. (a) A composition using a half docking domain, comprising: (i) at least one first binding partner-oligonucleotide conjugate comprising a binding partner attached to an oligonucleotide comprising the half docking domain, an optional stability domain, and optionally a spacer domain; and (ii) at least one second binding partner-oligonucleotide conjugate comprising a binding partner attached to an oligonucleotide comprising the half docking domain, an optional stability domain, and optionally a spacer domain, wherein the stability domains of (a)(i) and (a)(ii) are complementary to each other, and (a)(i) and (a)(ii) are complementary to each other. (a)(ii) half docking domains that can be linearly combined to form a full docking domain; and (iii) at least one labeled imager strand or intermediate strand comprising a 5' domain, a 3' domain, and a linker domain located between the 5' domain and the 3' domain, wherein the 5' domain is complementary to the half docking domain of (a)(i) and the 3' domain is complementary to the half docking domain of (a)(ii), and the labeled imager strand or intermediate strand is stably attached to the full docking domain and, if an intermediate strand is used, also provides a labeled imager strand; or (b) a composition using a half primer domain, comprising: (i) at least one first binding partner-oligonucleotide conjugate comprising a binding partner attached to an oligonucleotide comprising the half primer domain, an optional stable domain, and optionally a spacer domain; (ii) at least one second binding partner-oligonucleotide conjugate comprising a binding partner attached to an oligonucleotide comprising the half primer domain, an optional stable domain, and optionally a spacer domain, wherein the stable domains of (b)(i) and (b)(ii) are complementary to each other, and the half primer domains of (b)(i) and (b)(ii) can be linearly combined to form a complete primer domain; (iii) at least one docking strand capable of stably binding to the complete primer domain; and (iv) at least one labeled imager strand capable of stably binding to the docking strand or an intermediate strand capable of stably binding to the docking strand, and a labeled imager strand capable of stably binding to the intermediate strand.

[0229] Item 32. The composition of item 31, wherein at least one imager strand is bound to the complete docking domain for at least 30 minutes.

[0230] Item 33. The composition of item 31, wherein at least 70% of the imager chain bound to the complete docking domain remains bound for at least 30 minutes.

[0231] Item 34. The composition of item 31, wherein at least 90% of the imager chain bound to the complete docking domain is resistant to non-enzymatic buffer washes.

[0232] Item 35. A method for testing a fixed sample mounted on an optically transparent support for the presence of one or more targets, comprising: (1) contacting the sample with one or more target-specific binding partners, each target-specific binding partner being bound to a nucleic acid strand, and target-specific binding partners of different specificities being bound to different nucleic acid strands; (2) optionally removing unbound target-specific binding partners; and (3) the nucleic acid strand is a docking strand or a primer strand, and optionally the nucleic acid is (a) if a docking strand, increasing the number of docking strands associated with each target-specific binding partner; or (b) if a primer strand, associating two or more docking strands with the primer strand; (4) contacting the sample with a labeled imager strand capable of binding directly or indirectly to the docking strand, where indirect binding, if used, can occur through an intermediate strand; (5) optionally removing unbound labeled imager strands; (6) imaging the sample to detect bound labeled imager strands; and (7) quenching the signal from the bound labeled imager strand; and (8) contacting the sample with a labeled imager strand, or intermediate strand, if used, having a nucleotide sequence that is not complementary to the docking strand; (9) optionally removing unbound labeled imager strands; (10) imaging the sample to detect bound labeled imager strands; (11) optionally, quenching the signal from the bound labeled imager strand.

[0233] Item 36. A method for testing a fixed sample mounted on an optically transparent support for the presence of one or more targets, comprising: (1) contacting the sample with one or more target-specific binding partners, each target-specific binding partner being bound to a nucleic acid strand, and target-specific binding partners of different specificities being bound to different nucleic acid strands; (2) optionally removing unbound target-specific binding partners; and (3) the nucleic acid strand is a docking strand or a primer strand, and optionally the nucleic acid is (a) if a docking strand, increasing the number of docking strands associated with each target-specific binding partner; or (b) if a primer strand, associating two or more docking strands with the primer strand; (4) contacting the sample with a labeled imager strand, or any intermediate strand, if used, having a nucleotide sequence that is not complementary to the docking strand; (5) optionally removing unbound labeled imager strands; (6) imaging the sample to detect the presence, location, and / or number of bound labeled imager strands; (7) quenching the signal from the bound labeled imager strand; and (8) contacting the sample with a labeled imager strand capable of binding directly or indirectly to the docking strand, where indirect binding, if used, can occur through an intermediate strand; (9) optionally removing unbound labeled imager strands; (10) imaging the sample to detect the presence, location, and / or number of bound labeled imager strands; (11) optionally, quenching the signal from the bound labeled imager strand.

[0234] Item 37. The method of any one of Items 35 or 36, wherein the method further comprises repeating steps (4) to (11).

[0235] Item 38. The method of any one of items 35-37, wherein the method further comprises repeating the steps to detect binding of additional non-complementary imaging strands.

[0236] Item 39. The method according to any one of items 35 to 38, wherein the labeled imager strand capable of binding directly or indirectly to the docking strand used in the repeated steps optionally has a unique nucleotide sequence relative to at least one other labeled imager strand capable of binding directly or indirectly to another docking strand.

[0237] Item 40. (1) Signs; (2) a first nucleic acid domain, a second nucleic acid domain, and a third nucleic acid domain, each nucleic acid domain being 1 to 9 nucleotides in length; (3) a first binding moiety that links the first nucleic acid domain and the second nucleic acid domain; (4) a second binding moiety that links the second nucleic acid domain and the third nucleic acid domain; A composition, wherein both binding moieties are independently selected from (a) an abasic site having an intact phosphodiester backbone, (b) a linker cleavable by a nucleic acid glycosylase, (c) a non-naturally occurring nucleotide, or (d) a restriction site or a nicking site.

[0238] Item 41. The composition of item 40, wherein the additional nucleic acid domain is linked by an additional binding moiety.

[0239] Item 42. The composition of any one of items 40-41, wherein at least one binding moiety is an abasic site (apyrimidine) having an intact phosphodiester backbone.

[0240] Item 43. The composition of any one of items 40 to 42, wherein at least one binding moiety is susceptible to cleavage from endonuclease VIII.

[0241] Item 44. The composition of any one of items 40 to 43, wherein the nucleic acid domain comprises DNA.

[0242] Item 45. The composition of any one of items 40 to 44, wherein the nucleic acid domain comprises RNA.

[0243] Item 46. The composition of any one of items 40 to 45, wherein at least one binding moiety comprises at least one non-natural nucleotide.

[0244] Item 47. The composition of any one of items 40 to 46, wherein at least one binding moiety comprises 8-oxoguanine.

[0245] Item 48. A method for testing a sample for the presence of one or more targets, comprising: (1) contacting the sample with one or more target-specific binding partners, each target-specific binding partner being bound, directly or indirectly, to a nucleic acid strand, and target-specific binding partners of different specificities being bound to different nucleic acid strands; (2) optionally removing unbound target-specific binding partners; and (3) The nucleic acid strand is a docking strand or a primer strand, and the nucleic acid is (a) if a docking strand, increasing the number of docking strands associated with each target-specific binding partner; or (b) if a primer strand, associating two or more docking strands with the primer strand; (4) contacting the sample with a labeled imager strand capable of directly or indirectly binding to the docking strand, the labeled imager strand comprising a composition according to any one of items 34 to 41; (5) optionally removing unbound labeled imager strands; (6) imaging the sample to detect bound labeled imager strands; and (7) removing the bound labeled imager strand from the docking strand, wherein the labeled imager strand is removed from the docking strand by enzymatically cleaving, modifying, or degrading the labeled imager nucleic acid; and (8) optionally repeating steps (1)-(7), or any subset thereof.

[0246] Item 49. The method of item 48, wherein the labeled imager nucleic acid is removed by enzymatic cleavage of the labeled imager strand.

[0247] Item 50. A method for testing a sample for the presence of one or more targets, comprising: (1) treating the sample to expose one or more previously unavailable targets; (2) contacting the sample with one or more target-specific binding partners, each target-specific binding partner being bound to a nucleic acid strand, and target-specific binding partners of different specificities being bound to different nucleic acid strands; (3) optionally removing unbound target-specific binding partners; and (4) The nucleic acid strand is a docking strand or a primer strand, and optionally the nucleic acid is (a) if a docking strand, increasing the number of docking strands associated with each target-specific binding partner; or (b) if a primer strand, associating two or more docking strands with the primer strand; (5) contacting the sample with a labeled imager strand capable of binding directly or indirectly to the docking strand; (6) optionally removing unbound labeled imager strands; (7) imaging the sample to detect bound labeled imager strands; and (8) quenching the signal from the bound labeled imager strand; and (9) Optionally, repeating steps (1)-(8), each time (a) exposing a different set of previously unavailable targets, (b) using one or more different target-specific binding partners, and (c) using a labeled imager strand having a unique nucleotide sequence relative to at least one other labeled imager strand.

[0248] Item 51. A method for testing a fixed sample mounted on a first optically transparent support for the presence of one or more targets, comprising: (1) contacting the sample with one or more target-specific binding partners, each target-specific binding partner being bound to a nucleic acid strand, and target-specific binding partners of different specificities being bound to different nucleic acid strands; (2) optionally removing unbound target-specific binding partners; and (3) the nucleic acid strand is a docking strand or a primer strand, and optionally the nucleic acid is (a) if a docking strand, increasing the number of docking strands associated with each target-specific binding partner; or (b) if a primer strand, associating two or more docking strands with the primer strand; (4) contacting the sample in an imaging chamber (e.g., a flow cell) with a labeled imager strand capable of binding directly or indirectly to a docking strand; (5) optionally removing unbound labeled imager strands; (6) imaging the sample to detect the presence, location, and / or number of bound labeled imager strands; (7) quenching the signal from the bound labeled imager strand; and (8) repeating steps (4)-(6) or (4)-(7), at least once, wherein the labeled imager strand optionally has a unique nucleotide sequence relative to at least one other labeled imager strand.

[0249] Item 52. The method of item 51, wherein the imaging chamber is removed after the imaging step and before repeating steps (4)-(6) or (4)-(7).

[0250] Item 53. The method of any one of items 51-52, wherein the imaging chamber does not allow fluid flow and the imaging chamber is removed after the imaging step and before repeating steps (4)-(6) or (4)-(7).

[0251] Item 54. The method of any one of items 51-53, wherein the flow cell is formed of a second optically transparent material.

[0252] Item 55. The method of item 54, wherein the second optically transparent material comprises glass.

[0253] Item 56. The method of item 54, wherein the second optically transparent material comprises plastic.

[0254] Item 57. The method of any one of items 51-52, wherein the imaging chamber (such as a flow cell) comprises a gasket having at least one fluid inlet and / or outlet port.

[0255] Item 58. The method of any one of items 54-56, wherein the flow cell comprises a second optically transparent material parallel to the first optically transparent support.

[0256] Item 59. The method of any one of items 1-26, 35-39, or 48-58, wherein all fluid exchange steps are performed using a fluid system including electronic, and / or pneumatic, and / or hydraulic, and / or electro-fluidic actuators and systems.

[0257] Item 60. The method of item 59, wherein the fluidic system is controlled by software.

[0258] Item 61. The method of item 59, wherein the fluidic system is automatically controlled by software that synchronizes steps (1, and / or 2, and / or 3, and / or 4, and / or 5, and / or 7) with the imaging step (6), with reference to the numbering provided in item 1.

[0259] Item 62. The method of item 59, wherein the fluidic system is controlled by software that synchronizes steps (1, and / or 2, and / or 3, and / or 4, and / or 5, and / or 7) with the imaging step (6) by communicating with the imaging software with reference to the numbering provided in item 1.

[0260] Item 63. The method of any one of items 59-62, wherein all fluidic steps are performed while the sample is on the imaging device.

[0261] Item 64. The method of any one of items 59 to 63, wherein steps 4, 5, and 7 are performed while the sample is on the imaging device, with reference to the numbering provided in item 1.

[0262] Item 65. The method of any one of items 59-64, wherein the sample is immobilized in a disposable imaging chamber (such as a flow cell).

[0263] Item 66. The method of any one of items 59-65, wherein the sample is fixed in a reusable imaging chamber (such as a flow cell).

[0264] Item 67. (1) one or more reagent(s) including, but not limited to, a target-specific binding partner attached to a nucleic acid strand (where target-specific binding partners of different specificities are attached to different nucleic acid strands), a labeled imager strand, a buffer, an amplification reagent, and / or a reagent for removing bound imager strand (where the nucleic acid strand is a docking strand or a primer strand, and where the nucleic acid strand is a docking strand, either increasing the number of docking strands associated with each target-specific binding partner, or where the nucleic acid strand is a primer strand, causing two or more docking strands to associate with the primer strand); (2) a fluidics system for performing all fluid exchange steps; software for controlling the fluidic system and timing and / or synchronizing the fluidic steps with the imaging steps; (3) a flow cell having at least one optically transparent side attached to a sample of interest to enable imaging of the sample.

[0265] Item 68. The kit of item 67, wherein the flow cell is a disposable flow cell.

[0266] Item 69. The method of any one of items 1-26, 35-39, or 48-66, wherein imaging the sample to detect bound labeled imager strands detects the presence of bound labeled imager strands.

[0267] Item 70. The method of any one of items 1-26, 35-39, or 48-66, wherein imaging the sample to detect bound labeled imager strands detects the presence, location, and / or number of bound labeled imager strands.

[0268] Item 71. The method of item 70, wherein the method includes imaging the sample to detect and / or measure a background signal, and subtracting the background signal from the image of the sample to detect the bound labeled imager strand.

[0269] Item 72. The method of item 71, wherein the background signal comprises autofluorescence.

[0270] Item 73. The method of items 71 or 72, wherein the background comprises residual fluorescence associated with incomplete loss of signal from bound labeled imager strands.

[0271] Item 74. The method of any one of items 71 to 73, wherein the background signal is measured prior to imaging of the sample to detect bound labeled imager strands.

[0272] Item 75. The method of any one of items 71 to 74, wherein the background signal is measured after imaging of the sample to detect bound labeled imager strands.

[0273] Item 76. The method of any one of items 1 to 26, 35 to 39, or 48 to 75, wherein the sample is a fixed sample.

[0274] Item 77. The method of any one of items 1 to 26, 35 to 39, or 48 to 76, wherein the sample is a cell, a cell lysate, a tissue, a tissue lysate, a body fluid, and / or a whole organism.

[0275] Item 78. The method of any one of items 1-26, 35-39, or 48-77, wherein the method is useful for identifying biomarkers.

[0276] Item 79. The method of item 78, wherein at least 96 samples are imaged and data analysis is performed on those samples.

[0277] Item 80. The method of any one of items 78-79, wherein at least 15 targets are tested using a corresponding target-specific binding partner for each target.

[0278] Item 81. The method of any one of items 1 to 26, 35 to 39, or 48 to 80, wherein imaging is performed using optical microscopy, widefield, confocal (line and point scanning, spinning disk), total internal reflection illumination (TIR), stimulated emission depletion (STED), light sheet illumination, fluorescence microscopy including structured illumination (SIM), and expansion microscopy.

[0279] Item 82. The method of any one of items 1-26, 35-39, or 48-81, wherein the docking strand bound directly or indirectly to the labeled imager strand comprises at least 90% binding for 30 minutes.

[0280] Item 83. The method of any one of items 1-26, 35-39, or 48-82, wherein the target-specific binding partner is directly attached to the docking strand.

[0281] Item 84. The method of any one of items 1 to 26, 35 to 39, or 48 to 83, wherein the target-specific binding partner is indirectly linked to the docking strand through the primer strand.

[0282] Item 85. The method of any one of items 1-26, 35-39, or 48-84, wherein the docking strand is directly attached to the imager strand.

[0283] Item 86. The method of any one of items 1-26, 35-39, or 48-85, wherein the docking strand has complementarity to the imager strand.

[0284] Item 87. The method of any one of items 1-26, 35-39, or 48-86, wherein the docking strand is indirectly attached to the imager strand through an intermediate strand.

[0285] Item 88. The method of any one of items 1 to 26, 35 to 39, or 48 to 87, or the kit of items 67 to 68, wherein the imager strand and / or the intermediate strand contains at least one U that is cleavable by USER.

[0286] Item 89. The method of any one of items 1 to 26, 35 to 39, or 48 to 88, or the kit of items 66 to 67, and 88, wherein the imager strand and / or the intermediate strand comprises at least one abasic site.

[0287] Item 90. The method of any one of items 1 to 25, 34 to 38, or 47 to 89, or the kit of items 66 to 67, and 89, wherein the imager strand and / or the intermediate strand comprises a hairpin.

[0288] Item 91. The method of any one of items 1 to 25, 34 to 38, or 47 to 90, or the kit of items 66 to 67, or 88 to 90, wherein the imager strand and / or the intermediate strand comprises a hairpin having a clamp.

[0289] Item 92. The method of any one of items 1-25, 34-38, or 47-91, wherein at least two targets are imaged using at least two labels in the same imaging step.

[0290] Item 93. The method of any one of items 1-25, 34-38, or 47-92, wherein at least two targets are imaged using the same label in different imaging steps.

[0291] Item 94. The method of any one of items 1-25, 34-38, or 47-93, wherein at least two targets are imaged using at least two labels, the signal is quenched, and then at least one more label is imaged using at least one of the same labels, and the imaging steps can be performed in any order.

[0292] Item 95. The method of any one of items 1-25, 34-38, or 47-93, wherein eliminating the signal from the bound labeled imager strand comprises removing the nucleic acid strand from the target specific binding partner.

[0293] Item 96. The method of item 95, wherein removing the nucleic acid strand comprises enzymatically cleaving, modifying, or degrading the nucleic acid strand bound to the target specific binding partner.

[0294] Item 97. The method of item 97, wherein the nucleic acid strand is a docking strand.

[0295] Item 98. The method of item 97, wherein the nucleic acid strand is a primer strand.

[0296] Item 99. The method of any one of items 1-25, 34-38, or 47-94, wherein when the nucleic acid strand bound to the target specific binding partner is a primer strand, eliminating the signal from the bound labeled imager strand comprises removing the docking strand from the primer strand.

[0297] Item 100. The method of item 99, wherein removing the docking strand comprises enzymatically cleaving, modifying, or degrading the docking strand.

[0298] Item 101. The method of any one of items 1-25, 34-38, or 47-94, wherein eliminating the signal from the bound labeled imager strand comprises removing the imager strand.

[0299] Item 102. The method of any one of items 1-25, 34-38, or 47-94, wherein removing the imager strand comprises enzymatically cleaving, modifying, or degrading the imager strand.

[0300] Item 103. The method of any one of items 1-25, 34-38, or 47-94, wherein eliminating the signal from the bound labeled imager strand comprises removing the label from the imager strand.

[0301] Equivalent The above written specification is sufficient to enable one skilled in the art to practice the present invention. The above description and examples detail certain embodiments and describe the best mode contemplated by the inventors. However, no matter how detailed the above appears in text, it will be understood that the embodiments can be practiced in many ways and should be construed in accordance with the appended claims and any equivalents thereof.

[0302] As used herein, the term about refers to numerical values, including, for example, integers, fractions, and percentages, whether or not explicitly indicated. The term about generally refers to a range of numerical values ​​(e.g., + / - 5 to 10% of the recited range) that one of ordinary skill in the art would consider equivalent to the recited value (e.g., having the same function or result). When a term such as at least and about precedes a list of numerical values ​​or ranges, the term modifies all of the values ​​or ranges provided in the list. In some cases, the term about may include numerical values ​​that are rounded to the nearest significant figure.

Claims

1. 1. A method for testing a sample for the presence of one or more targets, comprising: (1) contacting a sample to be tested for the presence of one or more targets with one or more target-specific binding partners, each target-specific binding partner being bound to a nucleic acid strand, and target-specific binding partners of different specificities being bound to different nucleic acid strands; (2) optionally removing unbound target-specific binding partners; and (3) contacting the sample with a nonlinear amplifier strand having complementarity to a nucleic acid strand, the nucleic acid strand being either a docking strand or a primer strand; (4) optionally removing unbound nonlinear amplifier strands; and (5) amplifying the docking strand by rolling circle amplification in either one or two steps and contacting the sample with a labeled imager strand having complementarity to the docking strand or the amplified strand; (6) imaging the sample to detect bound labeled imager strands; and (7) removing the bound labeled imager strand; and (8) optionally repeating steps (1)-(8), or any subset thereof.

2. The method of claim 1 , wherein the polymerase is used for rolling circle amplification.

3. The method of claim 1 , wherein the nonlinear amplifier strand is combined with the target-specific binding partner attached to a nucleic acid strand prior to contacting the sample.

4. 2. The method of claim 1, wherein amplifying the docking strand by rolling circle amplification occurs separately from contacting the sample with a labeled imager strand having complementarity to the docking strand or the amplified strand.

5. 2. The method of claim 1, wherein amplifying the docking strand by rolling circle amplification occurs in the same step as contacting the sample with a labeled imager strand having complementarity to the docking strand, the imager strand optionally comprising a 3' modification to prevent amplification of the imager strand.

6. 2. The method of claim 1, wherein the imager strand is a circular imager strand for rolling circle amplification.

7. The method of claim 1 , wherein the imager strand or amplifier strand comprises at least two regions that are complementary to the docking strand.

8. The method of claim 1 , wherein the labeled imager strand is linear.

9. The method of claim 1 , wherein the nonlinear amplifier chain is a cyclic chain.

10. The method of claim 1 , wherein the nonlinear amplifier strand becomes circular after ligation.

11. 1. A method for testing a sample for the presence of one or more targets, comprising: (1) contacting the sample with one or more target-specific binding partners, each target-specific binding partner being bound, directly or indirectly, to a nucleic acid strand, and target-specific binding partners of different specificities being bound to different nucleic acid strands; (2) optionally removing unbound target-specific binding partners; and (3) The nucleic acid strand is a docking strand or a primer strand, and the nucleic acid is (a) if docking strands, optionally increasing the number of docking strands associated with each target-specific binding partner; or (b) if a primer strand, optionally associating two or more docking strands with said primer strand; (4) contacting the sample with a labeled imager strand capable of binding directly or indirectly to a docking strand; (5) optionally removing unbound labeled imager strands; and (6) imaging the sample to detect bound labeled imager strands; and (7) optionally quenching the signal from the bound labeled imager strand; and (8) optionally repeating steps (1)-(7), or any subset thereof.

12. 12. The method of claim 11, wherein after step (6), and optionally after performing step (7), the method further comprises increasing the number of docking strands associated with each target-specific binding partner and repeating steps (4), optionally (5), (6), and optionally (7).

13. 12. The method of claim 11, wherein the method comprises, in step (3), increasing the number of docking strands associated with each target-specific binding partner or associating two or more docking strands with the primer strand.

14. 12. The method of claim 11, wherein said increase in the number of docking strands associated with each target-specific binding partner is achieved using an enzyme, which optionally is a polymerase.

15. The method of claim 11, wherein enzymatically cleaving, modifying, or degrading the unbound labeled imager strand is accomplished by using an enzyme, optionally selected from glycosylases, restriction endonucleases, nicking endonucleases, RNases, and enzymes that cleave at unnatural nucleotides.

16. A composition comprising a sample bound to two or more target specific binding partners, each binding partner bound to a nucleic acid strand and at least one docking strand stably bound directly or indirectly to a labeled imager strand, the nucleic acid strand being a docking strand or a primer strand, the nucleic acid comprising: (a) if a docking strand, increasing the number of docking strands associated with each target-specific binding partner; or (b) when it is a primer strand, said composition having two or more docking strands associated with said primer strand.

17. 17. The composition of claim 16, wherein the docking strand bound directly or indirectly to the labeled imager strand comprises at least 90% binding for 30 minutes.

18. The above (a) a docking strand, or (b) the docking strand and any intermediate strands is no more than 80 nucleotides, no more than 70, 60, 50, 40, or no more than 30 nucleotides.

19. The composition of claim 16 , wherein the imager strand is 60 nucleotides or less.

20. (1) A sign; (2) a first nucleic acid domain, a second nucleic acid domain, and a third nucleic acid domain, each nucleic acid domain being 1-9 nucleotides in length; (3) a first binding moiety that links the first nucleic acid domain and the second nucleic acid domain; and (4) a second binding moiety linking the second nucleic acid domain and the third nucleic acid domain, The composition, wherein both binding moieties are independently selected from: (a) an abasic site having an intact phosphodiester backbone, (b) a linker cleavable by a nucleic acid glycosylase, (c) a non-naturally occurring nucleotide, or (d) a restriction site or a nicking site.

21. The composition of claim 20 , wherein the additional nucleic acid domain is linked by an additional binding moiety.

22. 21. The composition of claim 20, wherein at least one binding moiety is an abasic site (apyrimidine) having an intact phosphodiester backbone.

23. 21. The composition of claim 20, wherein at least one binding moiety is susceptible to cleavage by endonuclease VIII.

24. The composition of claim 20 , wherein the nucleic acid domain comprises DNA.

25. The composition of claim 20 , wherein the nucleic acid domain comprises RNA.

26. The composition of claim 20 , wherein at least one binding moiety comprises at least one non-naturally occurring nucleotide.

27. The method of claim 1 , wherein the imager strand and / or the intermediate strand comprises at least one U that is cleavable by a USER.

28. The method of claim 1 , wherein the imager strand and / or the intermediate strand comprises at least one abasic site.

29. The method of claim 1 , wherein at least two targets are imaged using at least two labels in the same imaging step.

30. 2. The method of claim 1, wherein at least two targets are imaged using at least two labels, the signal is quenched, and then at least one further label is imaged using at least one of the same labels, and the imaging steps can be performed in any order.

Images