Probe for measuring proximity of molecules in sample

Abstract

The invention relates to hybrid chain reactions (HCR). Specifically, the sensitivity of hybrid chain reaction (HCR) signal amplification is combined with two or more segmented initiator probes and one or more proximity probes that are capable of co-localizing a complete HCR initiator that will trigger an HCR reaction when the targets are in proximity to each other.

Description

INCORPORATION BY REFERENCE OF MATERIAL SUBMITTED ON COMPUTER MEDIA

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 449,543, filed March 02, 2023, and U.S. Provisional Patent Application No. 63 / 528,262, filed July 21, 2023, the disclosures of which are incorporated by reference herein in their entireties. STATEMENT REGARDING FEDERALLY ASSISTED RESEARCH AND DEVELOPMENT

[0002] This invention was made with government support under Grant No. EB006192 awarded by the National Institutes of Health. The government has certain rights in the invention. MATERIAL INCORPORATED BY REFERENCE FROM A COMPUTER MEDIA

[0003] The instant application incorporates material in an XML Sequence Listing provided herewith (named CALTE.164Aseqlist.xml, created on February 29, 2024, and having a size of 9,313 bytes). BACKGROUND Field

[0004] The present application relates to hybridization chain reaction (HCR). In particular, the sensitivity of hybridization chain reaction (HCR) signal amplification is combined with two or more segmental initiator probes and one or more proximity probes that are capable of co-localization when the targets are in proximity to each other triggers a complete HCR initiator of HCR. SUMMARY

[0005] According to some embodiments, provided compositions include a first segmental initiator probe including a first target binding domain configured to bind directly or indirectly to a first target, a first proximity domain, and a first segmental initiator; a second segmental initiator probe including a second target binding domain configured to bind directly or indirectly to a second target, a second proximity domain, and a second segmental initiator; and a proximity probe configured to bind the first proximity domain and the second proximity domain. According to some embodiments, a third segmental initiator probe can be provided including a third target binding domain configured to bind directly or indirectly to a third target, a third proximity domain, and a third segmental initiator. In some embodiments, the proximity probe is further configured to bind the third proximity domain.

[0006] According to some embodiments, the first target is a protein, nucleic acid, molecule, or combination thereof, and the second target is a protein, nucleic acid, molecule, or combination thereof. According to some embodiments, the third target is a protein, nucleic acid, molecule, or combination thereof. According to some embodiments, the first target and the second target bind to each other. According to some embodiments, the first target, the second target, and the third target bind to each other. According to some embodiments, the first target and the second target are proximal to each other. According to some embodiments, the first target, the second target, and the third target are proximal to each other. According to some embodiments, the first target and the second target are the same molecule. According to some embodiments, the first target, the second target, and the third target are the same molecule.

[0007] According to some embodiments, if one target is not proximal, the proximity probe comprises one or more clamp domains configured to bind to part or all of the first segmented initiator and / or the second segmented initiator. According to some embodiments, if one or more targets are not proximal, the proximity probe comprises one or more clamp domains configured to bind to part or all of the first segmented initiator, and / or the second segmented initiator, and / or the third segmented initiator.

[0008] According to some embodiments, the first segmented initiator probe and / or the second segmented initiator probe comprises an antibody, nanobody, and / or oligonucleotide. According to some embodiments, the third segmented initiator probe comprises an antibody, nanobody, and / or oligonucleotide.

[0009] According to some embodiments, when the first segmented initiator probe binds to the first target and the second segmented initiator probe binds to the second target, and when the first target and the second target bind to and / or are proximal to each other, then the proximity probe can bind to the first proximity domain and the second proximity domain to co-localize the complete initiator.

[0010] According to some embodiments, when the first segmented initiator probe binds to the first target and the second segmented initiator probe binds to the second target, and when the third segmented initiator probe binds to the third target, and when the first target, the second target, and the third target bind to and / or are proximal to each other, then the proximity probe can bind to the first proximity domain, the second proximity domain, and the third proximity domain to co-localize the complete initiator.

[0011] According to some embodiments, the co-localized complete initiator can directly or indirectly mediate signal generation. According to some embodiments, the co-localized complete initiator is capable of triggering HCR signal amplification or signal amplification by another method.

[0012] According to some embodiments, the additional segmented primer probe other than the third segmented primer probe comprises a target binding domain configured to bind directly or indirectly to the target, a proximity domain, and a segmented primer; and the proximity probe is further configured to bind to the proximity domain.

[0013] According to some embodiments, a method is provided comprising: providing a sample optionally comprising a first target and / or a second target; contacting the sample with a first segmented primer probe comprising a first target binding domain configured to bind directly or indirectly to the first target, a first proximity domain, and a first segmented primer; contacting the sample with a second segmented primer probe comprising a second target binding domain configured to bind directly or indirectly to the second target, a second proximity domain, and a second segmented primer; optionally, contacting the sample with a third segmented primer probe comprising a third target binding domain configured to bind directly or indirectly to a third target, a third proximity domain, and a third segmented primer; contacting the sample with a proximity probe configured to bind to the first proximity domain and the second proximity domain and optionally the third proximity domain; and contacting the sample with a HCR amplifier comprising two or more HCR hairpins.

[0014] According to some embodiments, a method is provided comprising: incubating a first segmented primer probe and a second segmented primer probe and optionally a third segmented primer probe in a sample to allow binding, optionally washing to remove unbound segmented primer probes; incubating a proximity probe in the sample to allow binding, optionally washing to remove unbound proximity probes; incubating a HCR amplifier in the sample, optionally washing to remove unbound HCR hairpins; and detecting a signal. According to some embodiments, a method comprises: optionally removing a signal, and optionally repeating any of the above steps to detect a signal for the same or a different target. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 Depiction of protein:protein interaction detection with proximity ligation assay (PLA).

[0016] Figure 2 Depiction of proximity-based HCR method for protein:protein interaction detection using a kinetic trigger mechanism.

[0017] Figure 3 Depiction of RNA:protein interaction detection with bridged strand.

[0018] Figures 4A-4C Depiction of some embodiments of HCR RNA-FISH using primer-labeled probes.

[0019] Figures 5A-5B Some embodiments of HCR RNA-FISH using segmented primer probes are depicted.

[0020] Figure 6 Some embodiments of detecting target complexes using segmented primer probes and proximity probes are depicted, where the segmented primer probes bind the sample directly.

[0021] Figure 7 Some embodiments of detecting target complexes using segmented primer probes and proximity probes are depicted, where the segmented primer probes bind the sample indirectly.

[0022] Figure 8 Some embodiments of detecting targets using segmented primer probes and proximity probes are depicted.

[0023] Figure 9 Some embodiments of detecting a first target and a second target that do not complex with each other but complex with a third target are depicted.

[0024] Figure 10 Some embodiments of detecting a first target and a second target that do not complex with each other are depicted.

[0025] Figure 11 Some embodiments of detecting targets using segmented primer probes and proximity probes are depicted, where the segmented primer probes bind the sample indirectly.

[0026] Figure 12 Some embodiments of detecting 3 targets using segmented primer probes and proximity probes are depicted.

[0027] Figure 13 Some embodiments of detecting 3 targets using segmented primer probes and two proximity probes are depicted.

[0028] Figure 14 Some embodiments of detecting protein:protein complexes are depicted.

[0029] Figure 15 Imaging of protein:protein target complexes in human cells is depicted.

[0030] Figure 16 Imaging of protein:protein target complexes in FFPE human breast tissue sections is depicted.

[0031] Figure 17 3 -plex imaging of three protein:protein target complexes in human cells is depicted.

[0032] Figure 18 Simultaneous 3-plex imaging of protein:protein target complexes, protein targets, and RNA targets in human cells is depicted.

[0033] Figures 19A-19C Quantitative HCR imaging of protein:protein target complexes in human cells is depicted.

[0034] Figure 20 Some embodiments of detecting protein:protein target complexes with segmented initiator nanobody probes and proximity probes are depicted.

[0035] Figure 21 Some embodiments of detecting protein:RNA target complexes are depicted.

[0036] Figure 22 Some embodiments of detecting RNA:RNA target complexes are depicted.

[0037] Figures 23A-23C Some embodiments of detecting target complexes using proximity probes that allow co-localization of segmented initiators when both targets are present, and clamp the entire segmented initiator when one target is not present are depicted.

[0038] Figures 24A-24C Some embodiments of detecting target complexes using proximity probes that allow co-localization of segmented initiators when both targets are present, and clamp the 5' end of the segmented initiator when one target is not present are depicted.

[0039] Figures 25A-25C Some embodiments of detecting target complexes using proximity probes that allow co-localization of segmented initiators when both targets are present, and clamp the 3' end of the segmented initiator when one target is not present are depicted.

[0040] Figures 26A-26C Some embodiments of detecting target complexes using proximity probes that allow co-localization of segmented initiators when both targets are present, and clamp the 5' and 3' ends of the segmented initiator when one target is not present are depicted.

[0041] Figures 27A-27B Some embodiments of segmented initiator probes forming a cooperative probe linkage with a target RNA or with a proximity probe are depicted.

[0042] Figures 28A-28N Some embodiments of initiator-labeled probes are depicted.

[0043] Figures 29A-29F Some embodiments of initiator-labeled probes are depicted.

[0044] Figures 30A-30D Some embodiments of probe sets comprising one or more probe units and optionally one or more helper probes are depicted.

[0045] Figures 31A-31E Some embodiments of a segmented primer probe co-localized by a target RNA are depicted.

[0046] Figures 32A-32D Some embodiments of a segmented primer probe co-localized by a target are depicted.

[0047] Figures 33A-33E Some embodiments of a segmented primer probe co-localized by a target complex are depicted.

[0048] Figures 34A-34C Some embodiments of a probe unit comprising a segmented primer probe are depicted.

[0049] Figure 35 Some embodiments of a segmented primer probe co-localized by a target RNA are depicted.

[0050] Figures 36A-36R Some embodiments of a segmented primer probe co-localized by a target directly or indirectly are depicted.

[0051] Figures 37A-37F Some embodiments of a HCR amplifier are depicted.

[0052] Figures 38A-38B Some embodiments of HCR amplification using four HCR hairpins are depicted.

[0053] Figure 39 A complete HCR primer i1 formed by two segmented primer probes co-localized by a target is depicted. Only a portion of the segmented primer probes is depicted.

[0054] Figure 40 Some embodiments of HCR amplification using two HCR hairpins are depicted.

[0055] Figures 41A-41E Some embodiments of CARD signal amplification mediated using HCR amplification to different targets and signal probes are depicted.

[0056] Figures 42A-42C Some embodiments of CARD signal amplification mediated using HCR amplification to universal targets and signal probes are depicted.

[0057] Figures 43A-43B Some embodiments of HCR amplification using HCR hairpins with reporter labels or segmented reporter labels to mediate CARD signal amplification are depicted.

[0058] Figures 44A-44CSome embodiments are depicted that use two segmental initiator probes and a proximity probe comprising one or more clamp structures to detect a target complex, such that when both segmental initiator probes bind to the proximity probe, the proximity probe allows the segmental initiators to co-localize to form a complete initiator; but if only one segmental initiator probe binds to the proximity probe, then the one or more clamp structures sequester one or more portions of the segmental initiator.

[0059] Figures 45A-45D Strong HCR signal generation in a positive sample comprising a protein:protein target complex is depicted, and no visible staining in a negative sample lacking a protein:protein target complex.

[0060] Figures 46A-46B Some embodiments of a HCR imaging method to detect a protein:protein target complex in a sample are depicted.

[0061] Figure 47 Some embodiments of a protocol for simultaneous HCR imaging of protein targets, protein:protein target complexes, and RNA targets in a sample are depicted.

[0062] Figures 48A-48D Some embodiments are depicted that use HCR signal amplification for detecting a target or target complex in a sample.

[0063] Figures 49A-49D Some embodiments of HCR immunohistochemistry and HCR protein:protein imaging using primary antibody probes with or without secondary antibody probes are depicted.

[0064] Figures 50A-50F Some embodiments of simultaneous HCR IHC / RNA-ISH using initiator-labeled antibody probes for a protein target and segmental initiator DNA probes for an RNA target are depicted. DETAILED DESCRIPTION

[0065] To study biological processes including replication, transcription, translation, and signaling, it is important to visualize not only molecules participating in these processes, such as RNA, protein, and DNA targets, but also complexes of these molecules. Making signal generation contingent on the proximity of two molecules provides sub-diffraction limit readout compared to independent imaging of the same two molecules in separate channels. Target complexes have previously been imaged using proximity ligation assays (PLA) that take advantage of enzymatic ligation and rolling circle amplification (RCA) 1-12 (see, e.g., Figure 1). To detect the complex, a 1° antibody binds to the target of interest, and a 2° antibody with an oligonucleotide tag is then bound to the 1° antibody. Two additional oligonucleotides are then bound to the oligonucleotide-tagged 2° antibody and circularized via an enzymatic ligation step. RCA is then completed by an enzymatic polymerase to generate a single-stranded DNA amplification product that is detected by a complementary fluorescent readout strand 1,12 (see, e.g., Figure 1 ). In addition to the cost and storage issues of PLA enzymes 1,12 , PLA suffers from false negatives due to formation of non-circular ligation products 13 , and false positives due to spurious amplification in the absence of a neighboring probe 12 . Furthermore, amplification signal using the PLA method does not scale linearly with target abundance 7 .

[0066] Signal amplification based on hybridization chain reaction (HCR) mechanisms 14 have been used to provide in situ signal amplification, e.g., for imaging RNA and protein targets in fixed biological samples 15-18 . In some cases, an HCR amplifier can consist of two kinetically trapped DNA hairpins (hi and h2) that are metastable in solution, storing energy to drive the conditional self-assembly of an HCR amplification polymer upon exposure to cognate initiator sequences (il; see, e.g., Figure 4A , 40 and 48A) 14 . In some cases, using HCR RNA in situ hybridization (RNA-ISH), a RNA target can be detected using one or more pairs of segmented initiator DNA probes, each pair carrying a portion of an HCR initiator il( Figure 5A and B and 48B) 17 . Pairs of probes that specifically hybridize to neighboring binding sites on a target RNA co-localize the complete HCR initiator il. In some cases, co-localization of two (or more) segmented initiators forms a complete initiator that can trigger HCR signal amplification, e.g., by binding and opening one or more metastable HCR hairpin monomers. Meanwhile, any individual probes that bind non-specifically in the sample do not co-localize the complete HCR initiator il, and do not trigger HCR, providing automatic background suppression. In some cases, using HCR immunohistochemistry (IHC), a protein target can be detected using an unlabeled primary antibody probe that in turn is detected by a segmented initiator secondary antibody probe that carries an HCR initiator il capable of triggering HCR signal amplification( Figure 48C , 50C , 50D and 50F) 18In some cases, the sample is subsequently imaged with a fluorescence microscope to map the expression pattern of the target molecule in an anatomical context. The HCR imaging protocol involves multiple stages, in which different reagents are washed into or out of the sample (e.g., see Figure 5B , 50B , 50D, 50E, and 50F) 15-19 . For example, in the detection stage, probes can be added to the fixed sample, incubated to allow the probes to bind to the target, and then un-used probes can be removed from the sample using a wash to reduce background due to non-specific probe binding; in the subsequent amplification stage, HCR amplifiers including fluorophore-labeled HCR hairpins can be added to the sample, incubated to allow HCR signal amplification to occur, and then un-used HCR hairpins can be removed from the sample using a wash to reduce background due to non-specific hairpin binding (e.g., see Figure 5B , 50B , 50D, 50E, and 50F).

[0067] To avoid the use of enzymes to detect protein: protein target complexes, proximity-based HCR methods have been developed that use a kinetic trigger mechanism to unseal an HCR initiator when two probes bind to proximal target proteins 13,20 (e.g., see Figure 2 ). To detect a target complex, two un-labeled primary antibodies bind to two target of interest, and two oligonucleotide-coupled secondary antibody probes bind to the primary antibodies. One secondary antibody probe is coupled to hairpin oligo 1, and one secondary antibody probe is coupled to hairpin oligo 2. Hairpin oligo 1 sequesters the trigger to open hairpin 2. Hairpin oligo 2 sequesters the HCR initiator. An additional activation strand is introduced that binds and opens hairpin oligo 1 to expose the trigger, which binds and opens hairpin oligo 2 to expose the HCR initiator, enabling subsequent HCR signal amplification. This method is limited to 1 -plex applications to date.

[0068] HCR signal amplification has also been used to detect ribosome interactions with mRNA 21 that uses an initiator-labeled bridge strand to link one nucleic acid probe that binds to a ribosome with a second nucleic acid probe that binds to an mRNA to detect ribosomal RNA (rRNA): messenger RNA (mRNA) interactions (e.g., see Figure 3 ). Since the bridge strand carries the complete HCR initiator, if the bridge strand binds non-specifically in the sample, amplified background will be generated. This method is limited to 1 -plex applications to date.

[0069] The systems disclosed herein address the shortcomings of existing methods and, in some embodiments, enable enzyme-free, multiplexed, quantitative, high-resolution imaging of target complexes using HCR signal amplification. In some embodiments, the use of segmented primer probes enables, when two or more segmented primer probes in a primer pair bind to proximal targets in a sample, a further proximal probe to bind to proximal domains on each segmented primer probe simultaneously, thereby co-localizing a complete HCR primer. In some cases, the co-localized complete HCR primer is able to trigger HCR signal amplification by opening a metastable HCR hairpin monomer to initiate a chain reaction and polymerization of HCR monomers. In some embodiments, if a single segmented primer probe binds to a separate target in a sample, then a proximal probe will be able to bind to the separate segmented primer probe, but the binding of the proximal probe will not co-localize a complete HCR primer and thus will not generate an amplification signal at the separate target site. Synergistic probe ligation for proximity measurements in a sample

[0070] In some embodiments, the synergistic probe ligation comprises two or more segmented primer probes that are bound at their proximal domains by a proximal probe (e.g., see Figure 6 , 7 , 8, 9, 10, 11, 12, 14, 19A, 20, 21, 22, 23A, 24A, 25A, 26A, 27B, 48D, 49C, and 49D). In some embodiments, the synergistic probe ligation is used to make proximity measurements within a sample. In some embodiments, the segmented primer probe comprises a segmented primer (also referred to as an HCR segmented primer), a proximal domain, and a target binding domain configured to bind directly to a target (e.g., see Figure 6 , 20 , 21, 22, and 49C). In some embodiments, the segmented primer probe comprises a segmented primer (also referred to as an HCR segmented primer), a proximal domain, and a target binding domain configured to bind indirectly to a target (e.g., see Figure 7 , 8 , 9, 10, 11, 12, 13, 14, 19A, 21, 23A, 24A, 25A, 26A, 27B, 48D, and 49D). In some embodiments, the proximal domain is a sequence within the segmented primer probe that is configured to be bound by a proximal probe. In some embodiments, the segmented primer is a sequence within the segmented primer probe that, by itself, is not able to trigger HCR signal amplification, but when co-localized with one or more segmented primers from one or more other segmented primer probes via binding of the segmented primer probe to one or more proximal probes, forms a complete primer that is able to trigger HCR signal amplification.

[0071] In some embodiments: the first segment initiator probe comprises a first target binding domain configured to bind directly or indirectly to the first target, a first proximity domain, and a first segment initiator; the second segment initiator probe comprises a second target binding domain configured to bind directly or indirectly to the second target, a second proximity domain, and a second segment initiator; and the proximity probe is configured to bind to the first proximity domain and the second proximity domain (e.g., see Figure 6 , 7 , 9, 10, 12, 14, 19A, 20, 21, 22, 23A, 24A, 25A, 26A, 27B, 48D, 49C, and 49D). In some embodiments, binding of the proximity probe to the first proximity domain and the second proximity domain co-localizes the first segment initiator and the second segment initiator. In some embodiments, binding of the proximity probe to the first proximity domain and the second proximity domain co-localizes the first segment initiator and the second segment initiator, thereby co-localizing a complete initiator (also referred to as a complete HCR initiator) capable of triggering HCR signal amplification.

[0072] In some embodiments, the first target and the second target are sufficiently proximal to each other that, when the first segment initiator probe is bound to the first target and the second segment initiator probe is bound to the second target, the proximity probe is capable of binding the first proximity domain of the first segment initiator probe and the second proximity domain of the second segment initiator probe to form a synergistic probe linkage. In some embodiments, the first target and the second target are bound to each other in a target complex (e.g., see Figure 7 ). In some embodiments, the first target and the second target are not complexed with each other but are complexed with a third target (e.g., see Figure 9 ). In some embodiments, the first target and the second target are proximal but not bound to each other (e.g., see Figure 10 ). In some embodiments, the first target and the second target are the same molecule (e.g., see Figure 11 ).

[0073] In some embodiments, the first segment initiator probe, the second segment initiator probe, and the proximity probe form a synergistic probe linkage (e.g., see Figure 27B and 48D ). In some embodiments, triggering of HCR occurs as a result of formation of a synergistic probe linkage and co-localization of the first and second segment initiators, and thus indicates that the first target and the second target are proximal to each other in the sample.

[0074] The spatial resolution of three-dimensional fluorescence images is diffraction-limited, about 200 nm laterally, and about 500 nm axially 22,23In some embodiments, a proximity probe binds to two segment initiator probes to co-localize a complete initiator and mediate generation of an amplification signal with sub-diffraction limit spatial resolution as a condition of the proximity of two target molecules. In some embodiments, a proximity probe binds to two segment initiator probes to co-localize a complete initiator and mediate generation of an amplification signal only if the two targets are within 200 nm, or 150 nm, or 100 nm, or 90 nm, or 80 nm, or 70 nm, or 60 nm, or 50 nm, or 40 nm, or 30 nm, or 20 nm, or 10 nm, or 5 nm. In some embodiments, a proximity probe binds to two segment initiator probes to co-localize a complete initiator and mediate generation of an amplification signal only if the two targets are within 10 µm, or 5 µm, or 2 µm, or 1 µm, or 500 nm, or less than the size of a eukaryotic cell or less than the size of a prokaryote. In some embodiments, one or more proximity probes bind to two or more segment initiator probes to co-localize a complete initiator and mediate generation of an amplification signal with sub-diffraction limit spatial resolution as a condition of the proximity of two or more target molecules. In some embodiments, one or more proximity probes bind to two or more segment initiator probes to co-localize a complete initiator and mediate generation of an amplification signal only if the two or more targets are within 200 nm, or 150 nm, or 100 nm, or 90 nm, or 80 nm, or 70 nm, or 60 nm, or 50 nm, or 40 nm, or 30 nm, or 20 nm, or 10 nm, or 5 nm. In some embodiments, one or more proximity probes bind to two or more segment initiator probes to co-localize a complete initiator and mediate generation of an amplification signal only if the two or more targets are within 10 µm, or 5 µm, or 2 µm, or 1 µm, or 500 nm, or less than the size of a eukaryotic cell or less than the size of a prokaryote.

[0075] In some embodiments, a target complex is detected using a 3-stage protocol (e.g., see Figure 46Aand B): 1) In the detection phase, two unlabeled primary probes are provided to the sample to detect two targets. As shown, the primary probes can be antibody probes. Next, two secondary segmental initiator probes (pi and p2) are added, each carrying a portion of the HCR initiator il and a proximal domain, where the secondary segmental initiator probes are specific to the unlabeled primary probes. In some embodiments, the segmental initiator probes can include a target binding domain configured to specifically bind to the primary probes, such as the antibody target binding domains illustrated. 2) In the proximity phase, a proximity probe is provided to the sample that is capable of binding and co-localizing the segmental initiator portions of the segmental initiator probes by forming a synergistic probe linkage. 3) In the amplification phase, an HCR amplifier including metastable labeled HCR hairpins is added to the sample such that in the presence of co-localized intact HCR initiators, the metastable labeled HCR hairpins self-assemble into labeled HCR amplification polymers. In some embodiments, the metastable labeled HCR hairpins are fluorophore labeled. In some embodiments, in the proximity phase, if the two segmental initiator probes are proximate closely enough, then a synergistic probe linkage is formed.

[0076] In some embodiments, the use of segmental initiator probes during the detection phase, proximity probes during the proximity phase, and metastable HCR hairpins during the amplification phase, provide automatic background suppression throughout the protocol, ensuring that even if reagents bind non-specifically in the sample, they do not generate amplification background. During the detection phase, any primary or secondary probes that bind non-specifically in the sample are not co-localized with intact HCR initiators and cannot self-initiate or trigger HCR. In some embodiments, even if the primary and secondary segmental initiator probes bind specifically to their targets during the detection phase, without the presence of proximity probes provided during the proximity phase and the formation of synergistic probe linkages, the segmental initiators will not form intact HCR initiators and initiate HCR during the amplification phase. In some embodiments, even if the primary and secondary segmental initiator probes bind specifically to their targets during the detection phase, and proximity probes are provided during the proximity phase, if the segmental initiators are not proximate closely enough, the segmental initiators will not form intact HCR initiators and initiate HCR during the amplification phase. Likewise, during the proximity phase, any proximity probes that bind non-specifically in the sample lack the ability to self-initiate HCR because only when the proximity probes bind specifically to both segmental initiator probes to co-localize intact HCR initiators can they mediate HCR signal amplification during the amplification phase. During the amplification phase, any HCR hairpins that bind non-specifically in the sample are kinetically trapped and do not trigger the formation of HCR amplification polymers.

[0077] In some embodiments, each synergistic probe junction comprises: a segmental primer probe P1 comprising a target binding domain and a nucleic acid comprising a segmental primer nucleic acid sequence and a proximity domain nucleic acid sequence; a segmental primer probe P2 comprising a target binding domain and a nucleic acid comprising a segmental primer nucleic acid sequence and a proximity domain nucleic acid sequence; and a proximity probe comprising a first nucleic acid sequence complementary to the proximity domain of P1 and a second nucleic acid sequence complementary to the proximity domain of P2. In some embodiments, when the proximity probe binds, it brings the segmental primer sequences in proximity, such that they form a complete HCR primer capable of triggering HCR signal amplification. In some embodiments, a synergistic probe junction can comprise x additional segmental primer probes, where x is an integer, to detect the proximity of additional targets as the case can be.

[0078] In some embodiments, the synergistic probe junctions co-localize a complete HCR primer for generating a signal using HCR (e.g., see Figure 6 、 7 , 8, 9, 10, 11, 12, 13, 14, 19A, 20, 21, 22, 23A, 24A, 25A, 26A, 27B, 46A, 48D, 49C, and 49D). In some embodiments, the synergistic probe junctions co-localize a complete HCR primer (also referred to as a complete primer) for generating a signal using other methods than HCR. In some embodiments, the synergistic probe junctions co-localize a complete primer for generating a signal using complementary fluorescent strands, branched DNA (bDNA) methods, enzymatic methods, RCA methods, catalytic reporter deposition (CARD), and / or other signal generation methods.

[0079] In some embodiments, the target binding domain of a segmental primer probe comprises an antibody, a nanobody, a protein, a peptide, a nucleic acid, a synthetic nucleic acid analog, an aptamer, a chemically modified nucleic acid, a chemically modified protein, an RNA, a DNA, a 2’OMe-RNA, a PNA, an XNA, any other material capable of base pairing, a carbon atom, a chemical linker that cannot base pair, or any combination thereof. In some embodiments, the target binding domain is capable of binding to a desired target. In some embodiments, the target binding domain can bind directly to the target itself. In some embodiments, the target binding domain can bind indirectly to the target, such as by binding to another molecule that binds to the target, such as a primary antibody, antibody fragment, or nanobody specific for the target. In some embodiments, the target binding domain binds to the target with high affinity.

[0080] In some embodiments, the target binding domain can be selected to be specific for a target that is expected to be in close proximity to a second target. In some embodiments, the target can be a nucleic acid, a protein, a molecule, or a combination thereof. In some embodiments, the synergistic probe linkage is capable of generating a signal upon detection of a protein:protein complex, an RNA:protein complex, an RNA:RNA complex, a DNA:protein complex, a DNA:protein:protein complex, or a complex of three or more RNA, DNA, and / or protein molecules in a sample.

[0081] In some embodiments, a segmented primer probe can be used to detect the proximity of three or more targets in a sample. In some embodiments, two or more segmented primer probes can be used to detect the proximity of two or more targets in a sample. For example, 2, 3, 4, 5, 6, 7, 8, 9, 10,... n segmented primer probes can be used in combination. In some embodiments: a first segmented primer probe comprises a first target binding domain configured to bind directly or indirectly to a first target, a first proximity domain, and a first segmented primer; a second segmented primer probe comprises a second target binding domain configured to bind directly or indirectly to a second target, a second proximity domain, and a second segmented primer; a third segmented primer probe comprises a third target binding domain configured to bind directly or indirectly to a third target, a third proximity domain, and a third segmented primer; and a proximity probe is configured to bind to the first proximity domain, the second proximity domain, and the third proximity domain (see, e.g., Figure 12 ). In some embodiments, the binding of the proximity probe to the first proximity domain, the second proximity domain, and the third proximity domain co-localizes the first segmented primer, the second segmented primer, and the third segmented primer. In some embodiments, the binding of the proximity probe to the first proximity domain, the second proximity domain, and the third proximity domain co-localizes the first segmented primer, the second segmented primer, and the third segmented primer, thereby co-localizing the complete HCR primer capable of triggering HCR signal amplification.

[0082] In some embodiments, two or more different proximity probes can be used to co-localize three or more segment initiator probes. In some embodiments: a first segment initiator probe comprises a first target binding domain configured to bind directly or indirectly to a first target, a first proximity domain, and a first segment initiator; a second segment initiator probe comprises a second target binding domain configured to bind directly or indirectly to a second target, a second proximity domain, and a second segment initiator; a third segment initiator probe comprises a third target binding domain configured to bind directly or indirectly to a third target, a third proximity domain, and a third segment initiator; a first proximity probe is configured to bind to the first proximity domain and the second proximity domain; and a second proximity probe is configured to bind to the second proximity domain and the third proximity domain (e.g., see Figure 13 ). In some embodiments, the binding of the first proximity probe to the first proximity domain and the second proximity domain, and the binding of the second proximity probe to the second proximity domain and the third proximity domain, co-localize the first segment initiator, the second segment initiator, and the third segment initiator. In some embodiments, the co-localized first segment initiator, second segment initiator, and third segment initiator form a complete HCR initiator capable of triggering HCR signal amplification.

[0083] In some embodiments, the target binding domain of a segment initiator probe comprises an antibody that binds directly or indirectly to a protein in a sample (e.g., see Figure 14 、 19A , 21, 46A, 48D, 49C, and 49D). In some embodiments, the target binding domain of a segment initiator probe comprises a nanobody that binds directly or indirectly to a protein in a sample (e.g., see Figure 20 ). In some embodiments, a cooperative probe linkage is used to detect a protein:protein target complex in a sample by triggering HCR in the presence of the target complex, thereby generating a detectable signal (e.g., see Figure 14 、 46A , 48D, 49C, and 49D). In some embodiments, the sample comprises a cell (e.g., see Figure 15 、 17 , 18, 19B, 45A, and 45B). In some embodiments, the sample is a formalin-fixed paraffin-embedded (FFPE) tissue (e.g., see Figure 16 、 45C , and 45D). In some embodiments, one or more protein:protein target complexes in a sample are detected simultaneously using one or more cooperative probe linkages (e.g., see Figure 17 , and 46B) In some embodiments, two or more protein: protein complexes are detected in a sample using one or more cooperative probe ligation in a multiplexed experiment (e.g., see Figure 17 and 46B ) In some embodiments, one or more target complexes are detected in a sample using one or more cooperative probe ligation in a multiplexed experiment, including two or more segmented primer probes and one or more proximity probes, wherein one or more protein targets, and / or one or more RNA targets, and / or one or more DNA targets are detected (e.g., see Figure 18 and 47 ) In some embodiments, cooperative probe ligation generates a quantitative signal intensity (e.g., see Figure 19C ) In some embodiments, one or more RNA: protein complexes are detected in a sample using one or more cooperative probe ligation (e.g., see Figure 21 ) In some embodiments, one or more RNA: RNA complexes are detected in a sample using one or more cooperative probe ligation (e.g., see Figure 22 ), or one or more DNA: protein complexes, or one or more DNA: protein: protein complexes, or complexes of three or more of RNA, DNA, and / or protein molecules and / or other molecules. In some embodiments, the cooperative probe ligation includes two or more segmented primer probes and one or more proximity probes. In some embodiments, the cooperative probe ligation includes three or more segmented primer probes and two or more proximity probes.

[0084] In some embodiments, the proximity probe comprises one or more clamp domains configured to bind one or more portions of the segment initiator when only one segment initiator probe is bound to the proximity probe, thereby inhibiting the generation of amplification background (in the form of undesired HCR signal amplification) in the absence of binding of both segment initiator probes to the proximity probe; and further configured such that co-localization of binding of both segment initiator probes to the proximity probe enables triggering of the complete HCR initiator for HCR signal amplification. In some embodiments, the proximity probe comprises one or more clamp domains configured to bind the entire first segment initiator and / or the second segment initiator in the absence of one of the targets in the target complex (e.g., see FIGS. 23 and 44A-44C). In some embodiments, the proximity probe comprises one or more clamp domains configured to bind the 5’ end of the first segment initiator and / or the 5’ end of the second segment initiator in the absence of one of the targets in the target complex (e.g., see FIG. 24). In some embodiments, the proximity probe comprises one or more clamp domains configured to bind the 3’ end of the first segment initiator and / or the 3’ end of the second segment initiator in the absence of one of the targets in the target complex (e.g., see FIG. 25). In some embodiments, the proximity probe comprises one or more clamp domains configured to bind the 5’ and 3’ ends of the first segment initiator and / or the 5’ and 3’ ends of the second segment initiator in the absence of one of the targets in the target complex (e.g., see FIG. 26). In some embodiments, the clamp domains can be complementary to the segment initiator domains. In some embodiments, the proximity probe does not comprise clamp domains configured to bind part or all of the first segment initiator and / or the second segment initiator in the absence of one of the targets in the target complex (e.g., see FIGS. 27A-27C). Figure 6 、 7 , 8, 9, 10, 11, 12, 13, 14, 19A, 20, 21, 22).

[0085] In some embodiments, HCR hairpin monomers (also referred to as HCR hairpins and HCR monomers) are capable of polymerizing when proximity probes bind to all proximity domains in a proximity segment initiator probe and co-localize the complete HCR initiator. In some embodiments, HCR hairpin monomers can include a fluorophore, a chromophore, a lumiphore, a phosphor, a FRET pair, or other label so that the polymer formed can be detected. In some embodiments, the signal is read using a fluorescence microscope, a fluorescence scanner, a camera, a cell phone camera, a mass spectrometer, a mass spectrometry microscope, a radioactivity scanner, or other instrument suitable for detecting the signal. In some embodiments, the signal generation includes a fluorophore, a chromophore, a lumiphore, a phosphor, a FRET pair, a member of a FRET pair, a quencher, a fluorophore / quencher pair, a rare earth element or compound, a radioactive molecule, a nucleotide, an amino acid, an oligonucleotide, a DNA, a RNA, a 2'OMe-RNA, a chemically modified nucleic acid, a synthetic nucleic acid analog, a chemically modified protein, a synthetic protein analog, a peptide, a binding substrate, a carbon atom, a chemical linker, a magnetic molecule, carbon black (CB), a carbon nanotube, a magnetized carbon nanotube, a gold nanoparticle (AuNP), a gold nanoshell, a gold nanorod, a silver shell gold nanoparticle, a latex, a magnetic nanoparticle, a silica nanoparticle, a fluorophore, a fluorophore-loaded nanoparticle, a dye-loaded nanoparticle, an enzyme, any combination thereof, or any other molecule that facilitates the measurement of a signal. In some embodiments, a hapten, a ligand, an oligonucleotide, digoxigenin (DIG), fluorescein isothiocyanate (FITC), a fluorophore, biotin, dinitrophenol, aniline, an enzyme, or other molecule or complex that can be recognized by a binding partner is utilized to facilitate the generation of a signal. Signal generation

[0086] In some embodiments, the initiator or co-localized complete initiator is used to directly or indirectly mediate signal generation. In some embodiments, the initiator or co-localized complete initiator mediates signal amplification 24-26 In some embodiments, the initiator or co-localized complete initiator mediates signal amplification via HCR 15-19 , branched DNA (bDNA) 27-33 , rolling circle amplification (RCA) 34-38 , catalyzed reporter deposition (CARD) 26,31,39-59 , polymerase chain reaction (PCR) 30,60 , proximity ligation assay (PLA) 2-6,8-12,61,62 , and / or any other signal amplification method that increases the strength of a signal. In some embodiments, the initiator or co-localized complete initiator is configured to bind to a readout probe that includes one or more reporters that directly or indirectly result in the generation of a signal. Hybridization chain reaction (HCR) signal amplification

[0087] In some embodiments, the HCR amplifier comprises two or more HCR hairpins (e.g., see Figures 5A-5B , 37A-37F, and 38A-38B). In some embodiments, each HCR hairpin (also referred to as an HCR hairpin monomer or HCR monomer) comprises an input domain having a single-stranded toehold and a stem segment, and an output domain having a single-stranded loop and the complement of a stem segment (e.g., see Figures 5A-5B , 39, 40, 37A-37F, and 38A-38B).

[0088] In some embodiments, a target is detected using a signal probe set comprising one or more initiator-labeled probes, each probe comprising a target-binding domain and an amplification domain comprising one or more HCR initiators (e.g., see Figures 28A-28N and 29A-92F). In some embodiments, a target within a sample is detected using a signal probe set comprising one or more probe units (e.g., see Figures 5A-5B and 30A-30D), wherein the probe unit comprises two or more segmented initiator probes (e.g., see Figures 31A-31E , 32A-32D, 33A-33E, and 34A-34C), wherein each segmented initiator probe comprises a target-binding domain and an amplification domain comprising a segmented initiator (e.g., see Figure 35 and 34A , 32A-32D, 33A-33E, and 34A-34C). In some embodiments, binding of each probe within the probe unit to a proximal cognate binding site on the target co-localizes the segmented initiators to form a complete HCR initiator capable of triggering HCR signal amplification (e.g., see Figures 5A-5B , 31A-31E, 32A-32D, 33A-33E, 30A-30D, and 36). In some embodiments, each segmented initiator probe within the probe unit further comprises a proximal domain (e.g., see Figure 6 , 7 , 8, 9, 10, 11, 12, 13, 46A). In some embodiments, binding of each segmented initiator probe within the probe unit to a proximal cognate binding site on the target enables binding of one or more proximal probes to a proximal domain within the probe unit, thereby co-localizing the segmented initiators to form a complete HCR initiator capable of triggering HCR signal amplification (e.g., see Figure 6 , 7 , 8, 9, 10, 11, 12, 13, 46A).

[0089] In some embodiments, one or more HCR initiators on an initiator-labeled probe each initiate a chain reaction of polymerization steps, where an initiator hybridizes to an input domain of a first HCR hairpin, opens the first hairpin to expose its output domain, which in turn hybridizes to an input domain of a second HCR hairpin, opens the second hairpin to expose its output domain, and so on, resulting in a chain reaction in which the hairpins polymerize to produce a HCR amplification polymer attached to the target (e.g., see Figure 39 , 40 , 38A-38B, 41A, 41C-41E, and 42A). In some embodiments, in the absence of an intact HCR initiator, the HCR hairpins are kinetically trapped and do not polymerize, thereby suppressing background. However, if a segmented initiator probe within a probe unit binds to its proximal cognate binding site on the target to co-localize an intact HCR initiator (e.g., see Figure 5A , 31A -31E, 32A-32D, 37C-37F), or if proximal domains within a segmented initiator probe within a probe unit bind to proximal probes to co-localize an intact HCR initiator (e.g., see Figure 6 , 7 , 8, 9, 10, 11, 12, 13, 46A), then the intact HCR initiator initiates a chain reaction of polymerization steps, where the intact initiator hybridizes to an input domain of a first HCR hairpin, opens the first hairpin to expose its output domain, which in turn hybridizes to an input domain of a second HCR hairpin, opens the second hairpin to expose its output domain, and so on, resulting in a chain reaction in which the hairpins polymerize to produce a linked HCR amplification polymer (e.g., see Figure 5A , 37C -37F, 38A-38B, 46A).

[0090] In some embodiments, the HCR hairpins further comprise zero, one, or more reporters that directly or indirectly result in the generation of an amplification signal (e.g., see Figures 37A-37F ). In some embodiments, zero, one, or more reporters on the HCR hairpins are used to mediate an additional signal amplification layer via catalytic reporter deposition (CARD) (e.g., see Figures 41A-41E , 42A-42C). In some embodiments, the reporters on the HCR hairpins comprise segmented reporters, such that an ancillary reporter-labeled readout probe does not strongly bind to a segmented reporter on an individual hairpin, but after HCR polymerization, such that adjacent hairpins in a HCR amplification polymer co-localize intact reporters, such that the co-localized intact reporters strongly bind to the ancillary reporter-labeled readout probe (e.g., Figure 37D and 43B). In some embodiments, the readout probe comprises one or more secondary reporters and further comprises a reporter binding domain configured to bind to a reporter on the HCR amplified polymer or configured to bind to a co-localized intact reporter within the HCR amplified polymer (e.g., see Figures 43A-43B In some embodiments, the amplification signal is generated by one or more reporters or secondary reporters associated with the HCR amplified polymer linked to the target within the sample. In some embodiments, the signal is removed. In some embodiments, the HCR signal is generated, detected, and removed one or more times.

[0091] In some embodiments, the HCR signal amplification increases the signal intensity by 2, 5, 10, 15, 20, 25, 30, 40, 50, 75, 100, 500, 1000, 2000, 5000, 10,000, or 20,000 fold, or a value within a range defined by any two of the preceding values.

[0092] HCR initiator. In some embodiments, the co-localized intact initiator comprises two or more segmental initiators brought into proximity by a proximity probe. In some embodiments, the intact initiator is fully complementary to the input domain of the HCR hairpin, such that it hybridizes to the input domain of the hairpin to uncap the hairpin and initiate the HCR polymerization cascade. In some embodiments, the intact initiator is partially complementary to the input domain of the HCR hairpin, but sufficiently complementary, such that it hybridizes to the input domain of the hairpin to uncap the hairpin and initiate the HCR polymerization cascade. In some embodiments, the intact initiator is shorter or longer than the input domain of the HCR hairpin, and / or has imperfect complementarity to the input domain of the HCR hairpin, but is able to hybridize to the input domain of the HCR hairpin to uncap the hairpin and initiate the HCR polymerization cascade. In some embodiments, the intact initiator can have 60%, 70%, 80%, 90%, or 100% (or any intermediate value between any of these values) complementarity to the input domain of the HCR hairpin and hybridizes to the input domain of the hairpin to uncap the hairpin and initiate the HCR polymerization cascade. In some embodiments, the segmental initiators are shielded by base pairing to reduce non-specific binding of the probe within the sample. In some embodiments, the segmental initiators can be shielded by a hairpin structure. In some embodiments, the segmental initiators can be shielded by one or more secondary oligonucleotides. In some embodiments, the segmental initiators can be shielded by self-complementarity within the probe and / or complementarity to one or more secondary strands.

[0093] Automatic background suppression of HCR segmented initiator probes. In some embodiments, segmented initiator probes automatically suppress background because the HCR initiators are split between probe pairs. In some embodiments, if a segmented initiator probe specifically binds to a target and to a proximity probe, the proximity probe co-localizes two segmented initiators in a probe pair to form a complete HCR initiator. In some embodiments, a non-specifically bound single segmented initiator probe does not trigger HCR because each probe only carries a portion of the HCR initiator and only when the complete HCR initiator is co-localized does it trigger HCR signal amplification.

[0094] Automatic background suppression of HCR hairpins. In some embodiments, HCR hairpins automatically suppress background because they are kinetically trapped, thus in the absence of HCR initiators they do not polymerize. In some embodiments, if two probes in a segmented initiator probe pair specifically bind to their proximal cognate binding sites on a target (see, e.g., Figure 5A ) and / or a proximity probe (see, e.g., Figure 46A ), the resulting co-localized complete HCR initiator triggers growth of linked HCR amplification polymers. In some embodiments, a non-specifically bound single HCR hairpin does not trigger HCR because they are kinetically trapped.

[0095] Automatic background suppression of HCR segmented initiator probes and HCR hairpins. In some embodiments, the combination of HCR segmented initiator probes for target detection and HCR amplification hairpins for signal amplification provides automatic background suppression throughout the protocol, ensuring that reagents do not generate amplified background even if non-specifically bound.

[0096] Complete HCR initiator formed by co-localization of 2 or more segmented initiator probes. Each set of segmented initiator probes that generates a complete HCR initiator is referred to as a probe unit (see, e.g., FIG. 34). In some embodiments, a complete HCR initiator is generated from a pair of segmented initiator probes, each carrying a portion of the complete HCR initiator, such that together they constitute the complete HCR initiator (the fl portion of probe PI and the f2 portion of probe P2, such that fl + f2 = 1); in this case, the probe unit is two segmented initiator probes (see, e.g., Figure 34A ). In some embodiments, the fl and f2 portions are small enough compared to the complete HCR initiator (e.g., fl = 0.5 and f2 = 0.5; or fl = 0.45 and f2 = 0.55; or fl = 0.4 and f2 = 0.6; or fl = 0.3 and f2 = 0.7) such that if the complete HCR initiator is not co-localized by a target, then HCR signal amplification is suppressed.

[0097] In some embodiments, the HCR initiator (il or i2) is split among three segmental initiator probes (fl portion of probe Pl, f2 portion of probe P2, f3 portion of probe P3, such that fl + f2 + f3 = 1); in this case, the probe unit consists of three segmental initiator probes. In some embodiments, the HCR initiator (il or i2) is split among N segmental initiator probes (fl portion of probe Pl, f2 portion of probe P2,..., fN portion of probe PN, such that fl + f2 +... + fN = 1; see, e.g., FIG. 2B) where N = 2, 3, 4, or greater; in this case, the probe unit consists of N segmental initiator probes. In some embodiments, for any of these values of N, if the complete HCR initiator is not co-localized with the target, then HCR signal amplification is inhibited. Figure 15 B), where N = 2, 3, 4, or greater; in this case, the probe unit consists of N segmental initiator probes. In some embodiments, for any of these values of N, if the complete HCR initiator is not co-localized with the target, then HCR signal amplification is inhibited.

[0098] In some embodiments, a complete HCR initiator is generated by co-localization of a pair (or group) of probes, each carrying a portion of the HCR initiator, such that the sum of the fl portion of probe PI and the f2 portion of probe P2 (fl + f2) is close enough to 1 (e.g., fl = 0.47, f2 = 0.47, fl + f2 = 0.94; or fl = 0.44, f2 = 0.42, fl + f2 = 0.86) that the HCR signal amplification is triggered by the co-localized complete initiator, which is generated from the binding of the pair of probes to their cognate binding sites on the adjacent target and the binding of their adjacent domains to the adjacent probe. In some embodiments, the segmented initiator probes within a probe unit generate a complete HCR initiator that is equivalent to 100% of the HCR initiator. In some embodiments, the segmented initiator probes within a probe unit generate a sufficient proportion of the HCR initiator to provide efficient HCR signal amplification relative to the rate of signal amplification in the absence of segmented initiator probes or in the presence of a single segmented initiator probe that is not co-localized. In some embodiments, the proportion of complete HCR initiator generated by co-localized probes within a probe unit is 99%, 95%, 90%, 80%, or 60%, including any range above any of the foregoing values or any range defined between any two of the foregoing values of complete HCR initiator. In some embodiments, a probe unit includes 2, 3, 4, 5, or more segmented initiator probes. In some embodiments, the segmented initiators in a probe unit are sufficient to function as HCR initiators when the probes within the probe unit are co-localized by binding to their cognate binding sites on the adjacent target and the binding of their adjacent domains to the adjacent probe. In some embodiments, although the HCR initiator can have a particular length of sequence (e.g., 15 nucleotides), the segmented initiators within a probe unit do not necessarily have the exact same length. For example, in some embodiments, their combined length can be 14 or 13 nucleotides if they still function as HCR initiators when co-localized.

[0099] In some embodiments, any two or more segmented initiators can be used so long as they collectively provide the function of the HCR initiator.

[0100] In some embodiments, a complete HCR initiator is generated by co-localization of a pair of probes, each carrying a portion of the HCR initiator, the HCR initiator further comprising one, several, or a number of sequence modifications, such that the sum of the fl portion of probe P1 and the f2 portion of probe P2 (fl + f2) is sufficiently close to 1 (e.g., fl = 0.45, f2 = 0.47, fl + f2 = 0.92) such that the co-localized complete initiator, which results from the binding of the pair of probes to their cognate binding sites on the adjacent target and adjacent probe, triggers HCR signal amplification. In some embodiments, the segmented initiator probes within a probe unit generate a complete HCR initiator with 100% sequence identity to the HCR initiator. In some embodiments, the segmented initiator probes within a probe unit generate a complete HCR initiator with sufficient sequence identity to the HCR initiator to allow efficient HCR signal amplification relative to the rate of signal amplification in the absence of the segmented initiator probes or in the presence of a single segmented initiator probe that is not co-localized. In some embodiments, the complete HCR initiator generated by co-localization of probes within a probe unit has 99%, 95%, 90%, 80%, or 60% sequence identity to the HCR initiator, including any range above any of the foregoing values or any range defined between any two of the foregoing values.

[0101] HCR amplifier with 2 hairpins. In some embodiments, the HCR amplifier comprises two hairpin nucleic acids (hl and h2; e.g., see Figure 5A-5B and 37A-37F). In some embodiments, each hairpin comprises an input domain with a single-stranded toehold and a stem segment, and an output domain with a single-stranded loop and a stem segment that is complementary. In the absence of the HCR initiator (il or i2), the hairpins hl and h2 coexist sub-stably, i.e., they are kinetically trapped and do not polymerize.

[0102] Initiation by initiator il. In some embodiments, the complete initiator il (1050) formed by co-localization of two or more segmented initiators comprises a domain that is complementary to the toehold (1851) of hairpin hl and a domain that is complementary to the stem segment (1755) of hl (e.g., see Figure 39). In some embodiments, if the hi hairpin (1510) encounters the intact initiator, il (1050), the intact initiator, il, hybridizes to the input domain (1852) of the hairpin hi via toehold-mediated strand displacement, opening the hairpin hi (1510) to expose the output domain (1854) of the hairpin hi and forming the complex il-hi. In some embodiments, the output domain (1854) of the hairpin hi comprises a domain complementary to the toehold (1951) of the hairpin h2 and a domain complementary to the stem (1855) of h2. In some embodiments, if the h2 hairpin (1610) encounters the il-hi complex, the exposed output domain (1854) of hi hybridizes to the input domain (1952) of the hairpin h2 via toehold-mediated strand displacement, opening the hairpin h2 to expose the output domain (1854) of the hairpin h2 and forming the complex il-hi-h2. In some embodiments, the output domain (1854) of the hairpin h2 comprises a domain complementary to the toehold (1851) of the hairpin hi and a domain complementary to the stem (1755) of hi. In some embodiments, if the hi hairpin (1510) encounters the il-hi-h2 complex, the exposed output domain (1854) of h2 hybridizes to the input domain (1852) of the hairpin hi via toehold-mediated strand displacement, opening the hairpin hi (1510) to expose the output domain (1854) of the hairpin hi and forming the complex il-hi-h2-hi. In some embodiments, this polymerization process can be repeated, with alternating hi and h2 polymerization steps, to generate a polymer of the form il-hi-h2-hi-h2-hi-h2-…, which can be denoted il-(hi-h2) N . For example, the polymer can incorporate a few molecules of hi and h2, or a few dozen molecules of hi and h2, or a few hundred molecules of hi and h2, or a few thousand molecules of hi and h2, or a few ten thousand molecules of hi and h2, or more. In some embodiments, it is possible for the polymer to end in hi or h2, so il-(hi-h2) N -hi and il-(hi-h2) N -hi-h2 are both possible, the latter being equivalent to il-(hi-h2) N+1 .

[0103] Initiated by initiator i2. In some embodiments, the complete initiator i2 formed by co-localization of two or more segment initiators includes a domain complementary to the toehold of hairpin h2 and a domain complementary to the stem segment of h2. In some embodiments, if the h2 hairpin encounters the complete initiator i2, the complete initiator i2 hybridizes to the input domain of hairpin h2 via toehold-mediated strand displacement, opening the hairpin h2 to expose the output domain of hairpin h2 and forming complex i2-h2. In some embodiments, if the h1 hairpin encounters the i2-h2 complex, the exposed output domain of h2 hybridizes to the input domain of hairpin h1 via toehold-mediated strand displacement, opening the hairpin h1 to expose the output domain of hairpin h1 and forming complex i2-h2-h1. In some embodiments, if the h2 hairpin encounters the i2-h2-h1 complex, the exposed output domain of h1 hybridizes to the input domain of hairpin h2 via toehold-mediated strand displacement, opening the hairpin h2 to expose the output domain of hairpin h2 and forming complex i2-h2-h1-h2. In some embodiments, this polymerization process can be repeated, with alternating h2 and h1 polymerization steps, to generate a polymer of the form i2-h2-h1-h2-h1-h2-h1…, which can be denoted i2-(h2-h1) N For example, the polymer can include a few h1 and h2 molecules, or tens of h1 and h2 molecules, or hundreds of h1 and h2 molecules, or thousands of h1 and h2 molecules, or tens of thousands of h1 and h2 molecules, or more. In some embodiments, it is possible for the polymer to end in h1 or h2, so i2-(h2-h1) N -h2 and i2-(h2-h1) N -h2-h1 are possible, the latter being equivalent to i2-(h2-h1) N+1 .

[0104] HCR amplifier with 4 hairpins. In some embodiments, an HCR amplifier can include more than 2 hairpins. For example, an HCR amplifier can include 4 hairpins h1, h2, h3, h4 (see, e.g., Figure 38A and 38B). In some embodiments, similar to 2-hairpin HCR, each hairpin includes an input domain comprising a single-stranded toehold and stem segment, and an output domain comprising a single-stranded loop and stem segment of the complement. In some embodiments, in the absence of the full HCR initiator (il, i2, i3, or i4), the hairpins hi, h2, h3, h4 coexist metastably, i.e., they are kinetically trapped and do not polymerize. In some embodiments, the output domain of hairpin hi includes a domain complementary to the toehold of hairpin h2 and a domain complementary to the stem segment of h2; the output domain of hairpin h2 includes a domain complementary to the toehold of hairpin h3 and a domain complementary to the stem segment of h3; the output domain of hairpin h3 includes a domain complementary to the toehold of hairpin h4 and a domain complementary to the stem segment of h4; the output domain of hairpin h4 includes a domain complementary to the toehold of hairpin hi and a domain complementary to the stem segment of hi. In some embodiments, the full initiator il formed by co-localization of two or more segmental initiators includes a domain complementary to the toehold of hairpin hi and a domain complementary to the stem segment of hi; the full initiator i2 formed by co-localization of two or more segmental initiators includes a domain complementary to the toehold of hairpin h2 and a domain complementary to the stem segment of h2; the full initiator i3 formed by co-localization of two or more segmental initiators includes a domain complementary to the toehold of hairpin h3 and a domain complementary to the stem segment of h3; the full initiator i4 formed by co-localization of two or more segmental initiators includes a domain complementary to the toehold of hairpin h4 and a domain complementary to the stem segment of h4. In some embodiments, similar to the case of 2-hairpin HCR, if hairpin hi encounters the full initiator il, the full initiator il opens hairpin hi to form complex il-hi with the exposed output domain of hi, which in turn opens hairpin h2 to form complex il-hi-h2 with the exposed output domain of h2, which in turn opens hairpin h3 to form complex il-hi-h2-h3 with the exposed output domain of h3, which in turn opens hairpin h4 to form complex il-hi-h2-h3-h4 with the exposed output domain of h4, which in turn opens hairpin hi to form complex il-hi-h2-h3-h4-hi with the exposed output domain of hi, and so on, resulting in polymerization via alternating hi, h2, h3, and h4 polymerization steps to generate a polymer of the form il-hi-h2-h3-h4-hi-h2-h3-h4-hi-h2-h3-h4…, which for a polymer incorporating N alternating copies of hi, h2, h3, and h4 can be written as il-(hi-h2-h3-h4)N In some embodiments, polymers ending in hi, h2, h3, or h4 are possible, thus i1-(hi-h2-h3-h4) N - hi, i1-(hi-h2-h3-h4) N - hi-h2, i1-(hi-h2-h3-h4) N - hi-h2-h3, and i1-(hi-h2-h3-h4) N - hi-h2-h3-h4 are all possible, the latter being equivalent to i1-(hi-h2-h3-h4) N+1 In some embodiments, HCR polymerization is triggered by any cognate complete initiator (i1, i2, i3, or i4). For example, initiation by complete initiator i3 can generate a polymer of the form i3-(h3-h4-h1-h2) N In some embodiments, HCR amplifiers with 4 hairpins facilitate generation of a signal that is not present in the un-polymerized state and is present in the polymerized state (e.g., Figure 19B FRET pairs are shown that only co-localize to generate a FRET signal when hairpins are co-localized within the amplifying polymer, to provide a basis for a no-wash method, as un-used hairpins that are not washed out of the sample do not participate in FRET, and thus avoid generating background).

[0105] HCR amplifiers with 2 or more hairpins. More generally, in some embodiments, an HCR amplifier can include M HCR hairpins (hi, h2,..., hM), where M is an integer of 2 or greater. In the absence of a complete HCR initiator (i1, i2,..., iM) formed by co-localization of two or more segmental initiators, the hairpins hi, h2,..., hM co-exist sub-stably, i.e., they are kinetically trapped and do not polymerize. In the presence of a cognate complete HCR initiator formed by co-localization of two or more segmental initiators, polymerization occurs via alternating polymerization steps analogous to 2-hairpin or 4-hairpin HCR. For example, for a polymer incorporating N alternating copies of hi, h2,..., hM, complete initiator i1 will result in growth of a polymer of the form i1-(hi-h2-...-hM) N In some embodiments, polymers ending in hi, h2, h3, or h4 are possible, thus i1-(hi-h2-h3-h4) N - hi, i1-(hi-h2-h3-h4) N - hi-h2,..., and i1-(hi-h2-...-hM) N - hi-h2-...-hM are all possible, the latter being equivalent to i1-(hi-h2-...-hM) N+1HCR polymerization is possible triggered by any cognate complete initiator (il, i2,..., iM). For example, initiation by complete initiator i3can generate a polymer of the form i3-(h3-...-hM-h1-h2). N

[0106] Reporter-labeled HCR hairpins. Each HCR hairpin includes zero, one or more reporters for a given HCR amplifier. The reporters on different hairpins within an amplifier can be the same or different. For example, an amplifier including hairpins hi and h2may have: 1) the same reporter on hi and h2, 2) different reporters on hi and h2, 3) a reporter on hi but none on h2, 4) a reporter on h2but none on hi, 5) no reporter on either hi or h2, 6) zero, one or more reporters on hi, where the zero, one or more reporters are the same or different than the zero, one or more reporters on h2. Similarly, for a HCR amplifier including hairpins hi, h2, h3, h4, each hairpin can include zero, one or more reporters (e.g., 3, 5 or 10 reporters), where the zero, one or more reporters can be the same as the zero, one or more reporters on each of the other hairpins. In some embodiments, the one or more reporters of a given hairpin can be unique within the mixture of hairpins and / or hairpin reporters. In some embodiments, there are 1, 10, 100, 1000, 10,000, 100,000 or more unique reporters within the mixture (including any range defined between any two of the prior numbers).

[0107] In some embodiments, the one or more reporters on a reporter-labeled HCR hairpin directly or indirectly contribute to the generation, alteration or elimination of a signal. For example, a reporter can be a fluorophore, a chromophore, a lumiphore, a phosphor, a FRET pair, a member of a FRET pair, a quencher, a fluorophore / quencher pair, a rare earth element or compound, a radioactive molecule, a magnetic molecule, an enzyme or any other molecule that directly or indirectly facilitates the measurement of a signal.

[0108] In some embodiments, a reporter of a modified tethered HCR amplification polymer can bind to a reporter-binding domain of a readout probe to directly or indirectly mediate the positioning of an ancillary reporter in the vicinity of the reporter, which in turn directly or indirectly mediates the generation of an amplification signal. For example:

[0109] In some embodiments, a reporter can include digoxigenin (DIG), which recruits an anti-DIG antibody as a readout probe, where the anti-DIG is directly labeled with one or more ancillary reporters, or with one or more reporters that directly or indirectly mediate the positioning of an ancillary reporter in the vicinity of the reporter. ​

[0110] In some embodiments, a reporter can include a nucleic acid domain that has full or partial sequence complementarity to a reporter binding domain within a readout probe carrying one or more helper reporters as a substrate. Figure 37C ).

[0111] In some embodiments, a reporter can include a nucleic acid domain that has full or partial sequence complementarity to a reporter binding domain within a readout probe carrying one or more substrates for mediating positioning of a helper reporter in the vicinity of the reporter.

[0112] In some embodiments, a reporter can include a nucleic acid domain that is a substrate for a readout probe that directly or indirectly mediates positioning of a helper reporter in the vicinity of the reporter.

[0113] In some embodiments, a reporter can include a substrate for recruiting a readout probe that indirectly mediates positioning of a helper reporter in the vicinity of the reporter.

[0114] In some embodiments, a reporter can include a substrate for recruiting a readout probe that includes an enzyme that mediates catalytic reporter deposition (CARD) in the vicinity of the reporter (e.g., see Figure 43A ).

[0115] In some embodiments, a reporter can include biotin that recruits streptavidin (or other biotin-binding molecule) as a readout probe, where the streptavidin is directly labeled with one or more helper reporters or is labeled with one or more substrates for directly or indirectly mediating positioning of a helper reporter in the vicinity of the reporter.

[0116] In some embodiments, a reporter can include a hapten that recruits an anti-hapten antibody readout probe or an anti-hapten nanobody readout probe that directly or indirectly mediates positioning of the reporter in the vicinity of the reporter via CARD signal amplification. For example, an anti-hapten antibody or nanobody readout probe can include an enzyme that mediates CARD (e.g., see Figures 41A-41E ).

[0117] In some embodiments, a reporter can include a hapten that recruits an anti-hapten antibody that directly or indirectly mediates positioning of a helper reporter in the vicinity of the reporter. For example, an anti-hapten readout probe can include an enzyme that mediates CARD (e.g., see Figures 42A-42C ).

[0118] In some embodiments, a reporter can include an enzyme that mediates CARD signal amplification to deposit a CARD-reporter in the vicinity of a hairpin.

[0119] In some embodiments, the reporter can include zero, one or more haptens (e.g., see Figures 37A-37B that directly or indirectly mediate localization of the helper reporter in the vicinity of the hapten.

[0120] In some embodiments, the reporter can include a hapten that recruits an anti-hapten (e.g., an antibody, nanobody, streptavidin, or other molecule) labeled with a helper reporter.

[0121] In some embodiments provided herein, HCR signal amplification is used to mediate catalyzed reporter deposition (CARD), resulting in even higher signal gain. In some embodiments, the even higher single gain is about 5, 10, 15, 20, 25, 30, 40, 50, 75, 100, 500, 1000, 2000, 5000, 10,000, 20,000, 50,000, or 100,000 fold, or a value within a range defined by any two of the preceding values.

[0122] Haptens and anti-haptens. In some embodiments, the hairpin label as a substrate including a hapten can be, for example, digoxigenin (DIG), dinitrophenyl (DNP), a fluorophore, biotin, or any small molecule, biological molecule, or non-biological molecule that can recruit an anti-hapten. Examples of anti-haptens include antibodies, nanobodies, streptavidin, aptamers, or any other molecule or complex of molecules that selectively binds to a hapten.

[0123] Enzymes for HCR-mediated catalyzed reporter deposition (CARD). In some embodiments, the reporter-modified HCR amplification polymer mediates signal amplification via catalyzed reporter deposition (CARD) by an enzyme that catalyzes a CARD-substrate to cause deposition of a CARD-reporter in the vicinity of the HCR amplification polymer (e.g., see Figures 41A-41E , 42A-42C, 43A-43B). For example:

[0124] In some embodiments, the enzyme can be horseradish peroxidase (HRP) (or a polymer HRP including multiple HRP enzymes) that acts on a CARD-substrate to catalyze deposition of a chromogenic CARD-reporter such as AEC, DAB, TMB, or StayYellow, or catalyzes a CARD-substrate to catalyze deposition of a fluorescent CARD-reporter such as a fluorophore-labeled tyramide, or catalyzes a hapten-labeled CARD-substrate such as a biotin-labeled tyramide, where the hapten is used to mediate localization of a CARD-reporter in the vicinity of the reporter-modified HCR amplification polymer.

[0125] In some embodiments, the enzyme can be alkaline phosphatase (AP) (or a polymer AP comprising multiple AP enzymes) that acts on the CARD-substrate to catalyze deposition of the CARD-reporter, e.g., a chromogenic CARD-reporter such as, but not limited to, BCIP / NBT, BCIP / TNBT, Napthol AS-MX Phosphate + Fast Blue BB, Napthol AS-MX Phosphate + Fast Red TR, StayGreen.

[0126] In some embodiments, the enzyme can be glucose oxidase that acts on the CARD-substrate to catalyze deposition of the CARD-reporter, e.g., NBT.

[0127] In some embodiments, the enzyme can be any molecule or complex that directly or indirectly mediates localization of the CARD-reporter near the reporter-modified HCR amplification polymer.

[0128] In some embodiments, the enzyme that mediates the CARD is inactivated (also referred to as deactivated) after deposition of the CARD-reporter (e.g., using chemical or thermal denaturation). For example, the enzyme that mediates the CARD can be inactivated using any combination of the following: 1. heat (e.g., 65 or above) 2. fixative (e.g., 4% PFA) 3. acid (e.g., 0.1 M glycine-HCl with 1% Tween 20 at pH 2.2, 0.2N HC1, 10% acetic acid, 10 mM HC1) 4. other chemicals (e.g., hydrogen peroxide (H2O2), hydrogen peroxide + phenol, sodium azide, DEPC, MAB with 10 mM EDTA)

[0129] In some embodiments, H2O2 is used to inactivate HRP. In some embodiments, a combination of heat and acid is used to inactivate AP. In some embodiments, a fixative is used to inactivate AP. In some embodiments, inactivation of the enzyme that mediates the CARD allows for repeated CARD for different targets using the same enzyme bound to different substrates to allow for multiplexed target analysis using HCR-mediated CARD. In some embodiments, inactivation of the enzyme that mediates the CARD allows for repeated CARD for different targets using different enzymes bound to different CARD-substrates to allow for multiplexed target analysis using HCR-mediated CARD.

[0130] In some embodiments, the CARD allows the stained sample to be stored for 10 years or more to allow for reanalysis in compliance with regulatory requirements. In some embodiments, the CARD-stained sample is stable for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 years or more and still in compliance with regulatory requirements. In some embodiments, the CARD staining provides long-term storage for archival samples.

[0131] Target types. In some embodiments, a segmented primer probe includes a target binding domain, a segmented primer, and a proximity domain. In some embodiments, a probe unit includes two or more segmented primer probes such that when their target binding domains bind to proximal cognate targets and their proximity domains bind to one (or more) cognate proximity probes, their segmented primers co-localize to form a complete HCR primer capable of triggering HCR.

[0132] In some embodiments, a probe unit can detect a target that includes any molecule, including but not limited to an RNA molecule (e.g., mRNA, rRNA, IncRNA, siRNA, shRNA, microRNA, non-coding RNA, synthetic RNA, or modified RNA), a DNA molecule, a non-natural nucleic acid molecule, a protein molecule, a small molecule, a biological molecule, a chemically modified biological molecule, a non-biological molecule.

[0133] In some embodiments, a probe unit can detect a target that includes any molecule complex, including any combination of RNA, DNA, protein, small molecule, biological molecule, and / or non-biological molecule (e.g., RNA:RNA complex, RNA:protein complex, DNA:protein complex, RNA:DNA:protein complex, protein:protein complex), a complex of 2, 3, or more molecules.

[0134] In some embodiments, a probe unit can detect a target that includes any set of proximal molecules or complexes such that when the segmented primer probes comprising the probe unit bind to their respective targets within the set of proximal molecules or complexes and also bind to one (or more) cognate proximity probes, the segmented primers in the probe unit can co-localize to form a complete HCR primer.

[0135] In some embodiments, the target-binding domains within a probe unit are configured to bind to overlapping or non-overlapping regions of a target. In some embodiments, the proximity domains within a probe unit are configured to bind to overlapping or non-overlapping regions of a proximity probe. In some embodiments, the segmental initiator within a probe unit is designed to hybridize to overlapping or non-overlapping regions of a HCR hairpin. In some embodiments, the non-specifically bound individual segmental initiator probes do not co-localize the complete HCR initiator, thereby inhibiting the generation of false HCR background amplification. In some embodiments, all segmental initiator probes within a probe unit must bind to one or more proximity probes to trigger HCR signal amplification.

[0136] In any of the embodiments provided herein, the segmental initiator within a probe unit is designed to be complementary to (or the segmental initiator within a probe unit is) a non-overlapping region (e.g., a region spaced by 0, 1, 2, or more nucleotides) of a HCR hairpin, or the segmental initiator within a probe unit is designed to be complementary to (or the segmental initiator within a probe unit is) an overlapping region (e.g., a region overlapping by 1, 2, or more nucleotides) of a HCR hairpin, or the segmental initiator within a probe unit is designed to be substantially complementary to (e.g., complementary except for 0, 1, 2, few, or several mismatches) (or the segmental initiator within a probe unit is) a HCR hairpin, or is configured to bind to a HCR hairpin.

[0137] In any of the embodiments provided herein, the target-binding region within a probe unit is configured to bind to a non-overlapping region (e.g., a region spaced by 0, 1, 2, or more nucleotides, or a region spaced by 0, 1, 2, or more nanometers) of a target, or is configured to bind to an overlapping region (e.g., a region overlapping by 1, 2, or more nucleotides, or a region overlapping by 1, 2, or more nanometers) of a target.

[0138] In any of the embodiments provided herein, the proximity domain within a probe unit is configured to bind to a non-overlapping region (e.g., a region spaced by 0, 1, 2, or more nucleotides, or a region spaced by 0, 1, 2, or more nanometers) of one or more proximity probes, or is configured to bind to an overlapping region (e.g., a region overlapping by 1, 2, or more nucleotides, or a region overlapping by 1, 2, or more nanometers) of one or more proximity probes.

[0139] In some embodiments, the segmental initiator probe comprises a kissing domain. In some embodiments, the probe unit comprises two segmental initiator probes each comprising a kissing domain. In some embodiments, the kissing domains within a probe unit are configured to bind to each other. In some embodiments, the probe unit comprises two or more segmental initiator probes each comprising a kissing domain. In some embodiments, the kissing domains within a probe unit are configured to bind to each other.

[0140] Segmented initiator probes for multiplex detection. In some embodiments, segmented initiator probes are designed for multiplex experiments, in which 2, 3, 4, 5, 10, 20, or 100 or more segmented initiator probes are used to bind different targets in the same sample. In some embodiments, multiplex experiments employing segmented initiator probes can be used to detect multiple target complexes, or multiple targets within a sample that are proximal to one another.

[0141] Materials and compositions of initiator-labeled probes. In some embodiments, initiator-labeled probes comprise one or more target-binding domains and one or more HCR initiators (e.g., see Figures 28A-28N and 29A-29F). In some embodiments, each domain can comprise one or more materials, including DNA, RNA, 2’OMe-RNA, PNA, XNA, chemically modified nucleic acids, synthetic nucleic acid analogs, amino acids, chemical linkers, synthetic amino acid analogs, and / or any other molecule suitable for the purpose of that domain. For example:

[0142] In some embodiments, initiator-labeled probes can comprise one or more initiators made of DNA and target-binding domains made of DNA.

[0143] In some embodiments, initiator-labeled probes can comprise one or more initiators made of DNA, chemical linkers, and target-binding domains made of amino acids (e.g., antibodies or nanobodies or antibody fragments).

[0144] In some embodiments, initiator-labeled probes can comprise initiators made of synthetic nucleic acid analogs and target-binding domains made of a combination of DNA and 2’OMe-RNA.

[0145] In some embodiments, initiator-labeled probes can comprise initiators made of 2’OMe-RNA and target-binding domains made of a combination of RNA and proteins.

[0146] In some embodiments, initiator-labeled probes can comprise initiators made of DNA and target-binding domains made of PNA.

[0147] In some embodiments, initiator-labeled probes can comprise one or more initiators made of any nucleic acid or nucleic acid analog, and one or more target-binding domains made of any combination of materials suitable for binding a target molecule.

[0148] In some embodiments, the initiator-labeled probe can comprise an antibody or nanobody coupled to one or more oligonucleotides, each comprising one or more initiators.

[0149] In some embodiments, the initiator-labeled probe can comprise a single covalently linked molecule, or can comprise two or more molecules that non-covalently interact to form a complex (each covalently linked). For example:

[0150] In some embodiments, the initiator-labeled probe can comprise an initiator made of DNA, covalently linked to a target-binding domain made of DNA.

[0151] In some embodiments, the initiator-labeled probe can comprise an initiator made of nucleic acid or nucleic acid analog, covalently linked or non-covalently bound to a target-binding domain comprising one or more molecules.

[0152] In some embodiments, the initiator-labeled probe can comprise an antibody or nanobody coupled to one or more oligonucleotides, each comprising one or more segmented initiators, a proximity binding domain, wherein the antibody or nanobody is a secondary probe that binds to a primary probe that binds to a target.

[0153] Materials and composition of segmented initiator probes. In some embodiments, a segmented initiator probe comprises one or more target-binding domains, one or more segmented initiators, and optionally one or more proximity domains (see, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 31A-31E, 32A-32D, 33A-33E, 36, 46A). In some embodiments, each domain can comprise one or more materials, including DNA, RNA, 2’OMe-RNA, PNA, XNA, chemically modified nucleic acids, synthetic nucleic acid analogs, chemical linkers, amino acids, synthetic amino acid analogs, and / or any other molecule suitable for the purpose of the domain. For example: Figure 6 7 In some embodiments, a segmented initiator probe can comprise one or more segmented initiators made of DNA, an optional proximity domain made of DNA, and a target-binding domain made of DNA.

[0154] In some embodiments, a segmented initiator probe can comprise one or more segmented initiators made of DNA, an optional proximity domain made of DNA, a chemical linker, and a target-binding domain made of amino acids (e.g., an antibody or nanobody or antibody fragment).

[0155] In some embodiments, a segmented initiator probe can comprise one or more segmented initiators made of DNA, an optional proximity domain made of DNA, a chemical linker, and a target-binding domain made of amino acids (e.g., an antibody or nanobody or antibody fragment).

[0156] ​In some embodiments, a segmented primer probe can include a segmented primer made of a synthetic nucleic acid analog, an optional proximity domain made of a synthetic nucleic acid analog, and a target binding domain made of a combination of DNA and 2'OMe-RNA.

[0157] In some embodiments, a segmented primer probe can include a segmented primer made of 2'OMe-RNA, an optional proximity domain made of 2'OMe-RNA, and a target binding domain made of a combination of RNA and protein.

[0158] In some embodiments, a segmented primer probe can include a segmented primer made of DNA, an optional proximity domain made of DNA, and a target binding domain made of PNA.

[0159] In some embodiments, a segmented primer probe can include one or more segmented primers made of any nucleic acid or nucleic acid analog, one or more optional proximity domains made of a nucleic acid or nucleic acid analog, and one or more target binding domains made of a combination of any material suitable for binding a target molecule.

[0160] In some embodiments, a segmented primer probe can include an antibody or nanobody coupled to an oligonucleotide comprising a segmented primer and a proximity domain.

[0161] In some embodiments, a segmented primer probe can include a target binding domain, a segmented primer, and a proximity domain.

[0162] In some embodiments, a segmented primer probe can include a single covalently linked molecule, or can include two or more molecules that non-covalently interact to form a complex (each covalently linked). For example:

[0163] In some embodiments, a segmented primer probe can include a segmented primer and an optional proximity domain made of DNA, covalently linked to a target binding domain made of DNA.

[0164] In some embodiments, a segmented primer probe can include one or more segmented primers and one or more optional proximity domains made of DNA, covalently linked to a dCas9 (or another Cas) of a non-covalently bound guide RNA (gRNA) such that the target binding domain includes a gRNA:dCas9 complex (or a gRNA:Cas complex using another Cas).

[0165] In some embodiments, a segmented primer probe can include one or more segmented primers and one or more optional proximal domains made of DNA covalently linked to a gRNA that non-covalently binds to a dCas9 (or another Cas), such that the target binding domain includes a gRNA:dCas9 complex (or a gRNA:Cas complex using another Cas).

[0166] In some embodiments, a segmented primer probe can include a segmented primer and an optional proximal domain made of a nucleic acid or nucleic acid analog covalently linked to or non-covalently bound to a target binding domain that includes one or more molecules. Each segmented primer probe within a probe unit can have the same or different material composition as other segmented primer probes in the probe unit. Each segmented primer probe within a probe unit can have a target binding region that binds to a different detection site on the same target molecule, or a different detection site within a target complex, or a different detection site within a proximal set of molecules or complex targets.

[0167] In some embodiments, a segmented primer probe can include an antibody or nanobody coupled to one or more oligonucleotides, each including a segmented primer and an optional proximal domain, wherein the antibody or nanobody is a secondary antibody probe that binds to a primary antibody probe that binds to a target.

[0168] In some embodiments, a segmented primer probe can include a target binding domain, a proximal domain, and a segmented primer, wherein the target is a primary antibody probe that binds to a primary target.

[0169] Removing signal from the sample. In some embodiments, signal is removed from the target after detecting the HCR signal. In some embodiments, signal can be removed by any method that reduces the number of signal generating reporters and / or helper reporters, such as: photobleaching fluorescent reporters using light and / or chemical reactions, chemically cleaving reporters from HCR hairpins (e.g., TCEP), chemically cleaving hairpins to fragment HCR amplification polymers, chemically cleaving probes to de-ligate HCR amplification polymers from targets, using helper strands to de-hybridize hairpins from HCR amplification polymers, using helper strands to de-hybridize probes from targets, using chemical denaturants and / or elevated temperatures to destabilize HCR amplification polymers, using chemical denaturants and / or elevated temperatures to destabilize probes and targets, using enzymes to degrade HCR amplification polymers, using enzymes to degrade probes from targets, using DNAse to degrade DNA amplification polymers, using DNAse to degrade DNA probes, using DNAse to degrade DNA targets, using RNAse to degrade RNA targets, using two or more of the above methods or any other method for removing signal from a target at the same time or at different times.

[0170] Assay formats. In some embodiments, signals can be measured in different assay formats, including but not limited to: blotting, northern blotting, western blotting, Southern blotting, dot blotting, paper assays, flow cytometry assays, fluorescent flow cytometry assays, cell sorting assays, fluorescence activated cell sorting assays, magnetic activated cell sorting assays, microscopy assays, light microscopy assays, epifluorescence microscopy assays, confocal microscopy assays, light sheet microscopy assays, microarray assays, bead-based assays, mass spectrometry assays, fluorescence microscopy assays, mass spectrometry microscopy assays, mass spectrometry flow cytometry assays, fluorescence assays, chemiluminescence assays, bioluminescence assays, colorimetric assays, electrochemical impedance assays, electrochemical chemiluminescence assays, energy dissipation assays, assays using the human eye, assays using a cell phone camera, gel electrophoresis assays, in situ hybridization (ISH) assays, RNA-ISH assays, DNA-ISH assays, immunohistochemistry (IHC) assays, autoradiography assays, or any assay capable of detecting a signal generated by HCR amplified polymers.

[0171] Sample types. In some embodiments, initiator-labeled probes and / or segmented initiator probes can be used with HCR amplification hairpins to detect targets in a sample, including molecules, complexes, or collections of proximal molecules or complexes. Target molecules can be included in a sample, including, for example: bacteria, zebrafish embryos, chicken embryos, mouse embryos, human biopsies, human tissue sections, FFPE tissue sections, urine samples, blood samples, fecal samples, mouse tissue sections, brain sections, sea urchin embryos, nematode larvae, fruit fly embryos, model organisms, non-model organisms, multi-species biological mixtures, environmental samples including unknown organisms, biological communities (e.g., a mixture of prokaryotes and bacteria within another organism’s gut), termites, microbiomes, clinical samples, diagnostic samples, sputum samples, tumor biopsy samples, research samples, samples including material from humans, samples including material from pets (e.g., dogs, cats, rabbits, lizards, snakes, or fish), material from wild animals (e.g., cheetahs, elephants, rhinoceroses, or chimpanzees), material from extinct animals (e.g., mammoths, dodos, great auks, triceratops, or passenger pigeons), living cells (e.g., bacteria or cultured mammalian cells), or living organisms (e.g., living mice or living humans).

[0172] In some embodiments, targets can be free in solution within a sample. For example, targets can be free in solution within a test tube, a cell, an embryo, an organism, a tissue section, a biological sample, or other sample.

[0173] In some embodiments, the targets can be covalently cross-linked or non-covalently bound to one or more capture probes that are covalently or non-covalently attached to a solid support. For example, directly or indirectly bound to a capture probe covalently linked to a microarray or a bead.

[0174] In some embodiments, the targets can be immobilized, covalently cross-linked, or non-covalently directly or indirectly bound to a solid support. For example, the targets can be bound, immobilized, or covalently cross-linked to a glass slide, a blot, a membrane, a paper substrate, or any other substrate. The targets can be immobilized or covalently cross-linked to a cell, an embryo, an organism, a tissue section, a biological sample, or any other sample. The targets can be covalently linked within a sample that is fixed and permeabilized, fixed but not permeabilized, or not fixed but permeabilized.

[0175] In some embodiments, the targets can be free within a living cell, a living embryo, a living organism, a living ecosystem, or a living biological community (e.g., the microbiome within a mammalian gut). The targets can be associated externally to a cell or organism, or can be included within a cell or organism. The targets can be covalently cross-linked within a living cell, a living embryo, a living organism, a living ecosystem, or a living biological community. The targets can or can not be present in one or more cell types within a sample. The targets can or can not be present in one or more biological species within a sample. The targets can be present in a sample that includes one or more off-targets that exhibit varying degrees of similarity to the target molecules. The targets can be present within an inflated sample. The targets can be present within a compressed sample. A sample can be inflated prior to detecting the targets to increase the degree of spatial separation between molecules. A sample can be compressed prior to detecting the targets to decrease the degree of spatial separation between molecules. The targets and / or other molecules can be cross-linked with an inflated sample to preserve the relative positions of molecules within the sample as the sample is inflated. The targets and / or other molecules can be cross-linked with a gel, a matrix, or other reagent introduced to the sample to preserve the relative positions and / or orientations of molecules within the sample as the sample is inflated. A sample can be differentially inflated and / or compressed with different inflation and / or compression factors in different tissues and / or organs within the sample.

[0176] Fixing a sample. In some embodiments, the target molecules can be cross-linked to a sample for preservation in subsequent steps of an experiment. For example, a chemical reagent (e.g., formaldehyde, paraformaldehyde, EDC (1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide)) can be used to cross-link the target molecules to the sample.

[0177] Permeabilizing a sample. In some embodiments, a sample can be treated to enhance the accessibility of target molecules to HCR probes and amplifiers. For example, a chemical reagent (e.g., methanol, ethanol, a detergent) or an enzyme (e.g., proteinase K) can be used to permeabilize a sample (e.g., a cell, a tissue section, or a whole embryo). A sample can also be permeabilized via homogenization, microdissection, electroporation, sectioning, heat treatment (e.g.,Smith JJ Gunasekera TS Barardi CR Veal D Vesey G (2004) J Appl Microbiol 96(2):409-417) and / or microwave treatment (e.g., Lan HY, Mu W, NG YY, Nikolic-Paterson DH, & Atkins RC (1996) J Histochem Cytochem 44(3):281-287) to enhance target accessibility. An additional option is to use chemical transfection reagents to deliver HCR probes and amplifiers across the cell membrane.

[0178] Samples are washed to remove unbound reagents from the sample. In some embodiments, background can be reduced by eluting unused imaging reagents from the sample. For example, a wash can be used to remove probes, initiator-labeled probes, segmented initiator probes, HCR amplification hairpins, amplification reagents, labeled probes, antibodies, and / or other imaging reagents from the sample. The wash can be performed using a chemical reagent at a particular temperature such that specifically bound imaging reagents are primarily not removed (signal is preserved) and non-specifically bound imaging reagents are primarily removed (background is reduced). For example, a wash buffer can include a denaturant (e.g., formamide, urea), a salt buffer (e.g., sodium chloride sodium citrate (SSC), phosphate buffered saline (PBS)), an acid (e.g., citric acid), a surfactant (e.g., Tween 20, Triton-X, SDS), or a blocking agent (e.g., tRNA, salmon sperm DNA, BSA, ficoll, polyvinylpyrrolidone, heparin). The wash buffer can be combined with a wash temperature (e.g., 25-80 ) to optimize wash stringency. Exemplary embodiments

[0179] Figure 6 Depiction of detecting a target complex comprising two target molecules using segmented initiator probes and a proximity probe, where a segmented initiator probe directly binds its target. Segmented initiator probe 1, comprising a target binding domain against target 1, binds target 1, and segmented initiator probe 2, comprising a target binding domain against target 2, binds target 2. As both segmented initiator probes bind to their targets and are proximal to each other, a proximity probe can bind to proximity domains 1 and 2, allowing segmented initiators 1 and 2 to co-localize and form a complete initiator capable of initiating HCR.

[0180] Figure 7 ​​​​Depicted is the detection of a target complex comprising two target molecules using segmented primer probes and a proximity probe, where the segmented primer probes indirectly bind their targets. Primary probe anti-target 1 binds target 1, and primary probe anti-target 2 binds target 2. Segmented primer probe 1, comprising a target binding domain anti-anti-target 1, binds to primary probe anti-target 1, and segmented primer probe 2, comprising a target binding domain anti-anti-target 2, binds to primary probe anti-target 2. As both segmented primer probes bind to their targets and are in proximity to each other, a proximity probe can bind to proximity domains 1 and 2, allowing segmented primers 1 and 2 to co-localize and form a complete primer capable of priming HCR.

[0181] Figure 8 Depicted is the detection of a target molecule using two segmented primer probes and a proximity probe, where the segmented primer probes indirectly bind different sites on the target. Two separate sites on the target are bound by anti-target primary probes. Segmented primer probe 1 binds one anti-target primary probe through its anti-anti-target domain, and segmented primer probe 2 binds the other anti-target primary probe through its anti-anti-target domain. As both segmented primer probes bind to their targets and are in proximity to each other, a proximity probe can bind to proximity domains 1 and 2, allowing segmented primers 1 and 2 to co-localize and form a complete primer capable of priming HCR.

[0182] Figure 9 Depicted is the detection of a first target molecule and a second target molecule using two segmented primer probes and a proximity probe, where the first and second target molecules are not complexed with each other but are complexed with a third target molecule. Primary probe anti-target 1 binds target 1, and primary probe anti-target 2 binds target 2, where target 1 and 2 do not directly bind to each other but both bind to target 3. Segmented primer probe 1 binds anti-target 1 through its anti-anti-target 1 domain, and segmented primer probe 2 binds anti-target 2 through its anti-anti-target 2 domain. As both segmented primer probes bind to their targets and are in proximity to each other, a proximity probe can bind to proximity domains 1 and 2, allowing segmented primers 1 and 2 to co-localize and form a complete primer capable of priming HCR.

[0183] Figure 10Depicted is the detection of a first target and a second target using two segmented primer probes and a proximity probe, where the first and second targets are adjacent but not complexed with each other. Targets 1 and 2 of the target complex are bound by anti-target 1 and 2, respectively, where targets 1 and 2 are not directly bound to each other. Segmented primer probe 1 binds to anti-target 1 through its anti-anti-target 1 domain, and segmented primer probe 2 binds to anti-target 2 through its anti-anti-target 2 domain. As both segmented primer probes are bound to their targets and adjacent to each other, a proximity probe can bind to proximity domains 1 and 2, allowing segmented primers 1 and 2 to co-localize and form a complete primer capable of priming HCR.

[0184] Figure 11 Depicted is the detection of a target using two segmented primer probes and a proximity probe, where the segmented primer probes indirectly bind to the target via binding to different sites on a primary probe that directly binds to the target. The primary probe anti-target binds to the target. Segmented primer probe 1 and segmented primer probe 2 bind to different sites on the anti-target primary probe through their respective anti-anti-target domains. As both segmented primer probes are bound to their targets and adjacent to each other, a proximity probe can bind to proximity domains 1 and 2, allowing segmented primers 1 and 2 to co-localize and form a complete primer capable of priming HCR.

[0185] Figure 12 Depicted is the detection of three targets using a segmented primer probe and a proximity probe, where the three targets are complexed together. Targets 1, 2, and 3 of the target complex are bound by primary probe anti-target 1, 2, and 3, respectively. Segmented primer probe 1 binds to anti-target 1 through its anti-anti-target 1 domain, segmented primer probe 2 binds to anti-target 2 through its anti-anti-target 2 domain, and segmented primer probe 3 binds to anti-target 3 through its anti-anti-target 3 domain. As all segmented primer probes are bound to their targets and adjacent to each other, a proximity probe can bind to proximity domains 1, 2, and 3, allowing segmented primers 1, 2, and 3 to co-localize and form a complete primer capable of priming HCR.

[0186] Figure 13 Depicted is the detection of three targets using a segmented primer probe and two proximity probes, where the three targets are complexed together. Targets 1, 2, and 3 of the target complex are bound by primary probe anti-target 1, 2, and 3, respectively. Segmented primer probe 1 binds to anti-target 1 through its anti-anti-target 1 domain, segmented primer probe 2 binds to anti-target 2 through its anti-anti-target 2 domain, and segmented primer probe 3 binds to anti-target 3 through its anti-anti-target 3 domain. As all segmented primer probes are bound to their targets and adjacent to each other, proximity probe 1 can bind to proximity domains 1 and 2, and proximity probe 2 can bind to proximity domains 2 and 3, allowing segmented primers 1, 2, and 3 to co-localize and form a complete primer capable of priming HCR.

[0187] Figure 14 Depicted is the detection of two protein targets using segmented primer probes and a proximity probe, where the targets are in a protein: protein complex. Proteins 1 and 2 of the target complex are bound by primary antibody probe anti-protein 1 and anti-protein 2, respectively. Segmented primer probe 1 binds anti-protein 1 through its secondary antibody domain, and segmented primer probe 2 binds anti-protein 2 through its secondary antibody domain. As both segmented primer probes bind to their targets and are in proximity to each other, a proximity probe can bind to the proximity domains 1 and 2, allowing segmented primers 1 and 2 to co-localize and form a complete primer capable of priming HCR. Additional embodiments

[0188] Any of the embodiments, compositions, and / or methods provided herein can be used in conjunction with, or in alternative form to, any of the following. Thus, for example, the compositions and / or methods mentioned above can employ any of the compositions or methods mentioned below. Similarly, the compositions and / or methods mentioned above are to be understood as also providing methods that employ or are part of the methods mentioned below.

[0189] Similarly, the embodiments and / or methods provided herein are also to be understood to provide embodiments directed to the methods, e.g., compositions, components of the methods, kits, etc. In some embodiments, any of the components of one or more methods and / or steps provided herein can be provided as a kit, which includes one or more of the mentioned components (and optionally a target or target sequence or sample). Compositions

[0190] Some embodiments of compositions are summarized in Figure 46A and 46B and other figures provided herein. In some embodiments, a composition is provided that includes a first segmented primer probe that includes: a first target binding domain configured to bind directly or indirectly to a first target, a first proximity domain, a first segmented primer; a second segmented primer probe that includes: a second target binding domain configured to bind directly or indirectly to a second target, a second proximity domain, a second segmented primer, a proximity probe configured to bind the first proximity domain and the second proximity domain; and an HCR amplifier that includes two or more HCR hairpins. Optionally, the composition can include additional segmented primer probes or proximity probes.

[0191] In some embodiments, the first and second segmental initiator together form a complete HCR initiator when bound proximally, which is capable of binding and opening the first HCR hairpin. In some embodiments, the first segmental initiator probe further comprises a first target binding domain, and the second segmental initiator probe further comprises a second target binding domain, wherein the first target binding domain is configured to bind to a first target, and the second target binding domain is configured to bind to a second target. In some embodiments, these target binding domains, upon their binding to the respective targets, are in proximity to each other, such that when both segmental initiator probes are bound to both target domains, they can be bound proximally by a proximal probe at their respective proximal domains, and the first and second segmental initiators (within the segmental initiator probes) are co-localized to form a complete HCR initiator capable of binding and opening the first HCR hairpin.

[0192] Some embodiments of HCR monomer compositions are outlined in the Figure 39 and Figure 40 Some embodiments of HCR monomer compositions are outlined in the

[0193] In some embodiments, a composition is provided comprising a first HCR hairpin (1510) comprising: a) a first input domain (1852) comprising a first toehold (1851) and a first stem segment (1755); b) a first output domain (1854) comprising a first loop (1853) and a complement of the first stem segment (1756); and optionally c) one or more reporters (1850). In some embodiments, the composition further comprises a second HCR hairpin (1610) comprising: a) a second input domain (1952) comprising a second toehold (1951) and a second stem segment (1855); b) a second output domain (1954) comprising a second loop (1953) and a complement of the second stem segment (1856); and optionally c) one or more reporters (1950).

[0194] In some embodiments, the first toehold (1851) is complementary to the second loop (1953). In some embodiments, the second toehold (1951) is complementary to the first loop (1853). In some embodiments, this circularity allows HCR polymerization to occur in the presence of an initiator capable of binding the first input domain, as described herein. In some embodiments, the first toehold is not 100% complementary to the second loop, but is sufficient to allow hybridization. Methods

[0195] In some embodiments, the provided methods comprise: A) providing: a sample comprising a first target and a second target; a first segmental initiator probe comprising: a first target binding domain configured to bind directly or indirectly to the first target, a first proximity domain, a first segmental initiator; a second segmental initiator probe comprising: a second target binding domain configured to bind directly or indirectly to the second target, a second proximity domain, a second segmental initiator; a proximity probe configured to bind the first proximity domain and the second proximity domain; a HCR amplifier comprising two or more HCR hairpins; B) incubating the first segmental initiator probe and the second segmental initiator probe in the sample to allow binding; C) incubating the proximity probe in the sample to allow binding; D) incubating the HCR amplifier in the sample; E) and detecting a signal. In some embodiments, the first segmental initiator probe binds to the first target and the second segmental initiator probe binds to the second target, and when the first target and the second target bind to and / or are proximal to each other, then the proximity probe binds the first proximity domain and the second proximity domain to co-localize the complete initiator. In some embodiments, the co-localized complete initiator is used to trigger hybridization chain reaction (HCR) signal amplification.

[0196] In some embodiments, the methods further comprise providing a third segmental initiator probe comprising: a third target binding domain configured to bind directly or indirectly to a third target, a third proximity domain, and a third segmental initiator. In some embodiments, the proximity probe is configured to bind the first proximity domain, the second proximity domain, and the third proximity domain. In some embodiments, a washing step is performed after B) to remove unbound segmental initiator probes. In some embodiments, a washing step is performed after C) to remove unbound proximity probes. In some embodiments, a washing step is performed after D) to remove unbound HCR hairpins. In some embodiments, the signal is removed after E). In some embodiments, any of the above steps are repeated to detect a signal of the same or different targets.

[0197] In some embodiments, the signal is detected by fluorescence microscopy, fluorescence scanner, camera, cell phone camera, mass spectrometer, mass spectrometry microscope, or radioactivity scanner. In some embodiments, the methods further comprise providing an auxiliary probe to maximize signal generation. In some embodiments, the auxiliary probe does not comprise a segmental initiator. In some embodiments, the auxiliary probe does not comprise a proximity domain. In some embodiments, binding of one or more auxiliary probes to a target increases target accessibility to facilitate binding of one or more segmental initiator probes to the target (see, e.g., Figure 30D). In some embodiments, the first target is a protein, nucleic acid, molecule, or combination thereof, and wherein the second target is a protein, nucleic acid, molecule, or combination thereof. In some embodiments, the first target and the second target bind to each other. In some embodiments, the first target and the second target are the same molecule. In some embodiments, the proximity probe comprises one or more clamp domains configured to bind to part or all of the first segment initiator and / or the second segment initiator if one target is not proximal. In some embodiments, the first segment initiator probe and / or the second segment initiator probe comprises an antibody, nanobody, and / or oligonucleotide. In some embodiments, the co-localized complete initiator is used to directly or indirectly mediate signal generation. In some embodiments, the co-localized complete initiator triggers the self-assembly of a metastable fluorophore-labeled HCR hairpin, forming a linked fluorescent amplification polymer to generate an amplification signal at the target site. In some embodiments, the co-localized complete initiator is used to detect one or more protein: protein complexes, RNA: protein complexes, RNA: RNA complexes, DNA: protein complexes, DNA: protein: protein complexes, or complexes of three or more of RNA, DNA, and / or proteins or other molecules in a sample. In some embodiments, the non-specifically bound segment initiator probes do not co-localize the complete initiator and do not initiate HCR. In some embodiments, the three synergistic probe connections comprise the oligonucleotide sequences set forth in Table 1, wherein each synergistic probe connection comprises: a segment initiator probe P1 comprising a segment initiator and a proximity domain, a segment initiator probe P2 comprising a segment initiator and a proximity domain, and a proximity probe. In some embodiments, the signal generation comprises a fluorophore, a chromophore, a lumiphore, a phosphor, a FRET pair, a member of a FRET pair, a quencher, a fluorophore / quencher pair, a rare earth element or compound, a radioactive molecule, a nucleotide, an amino acid, an oligonucleotide, DNA, RNA, 2’Ome-RNA, a chemically modified nucleic acid, a synthetic nucleic acid analog, a chemically modified protein, a synthetic protein analog, a peptide, a binding substrate, a carbon atom, a chemical linker, a magnetic molecule, carbon black (CB), a carbon nanotube, a magnetized carbon nanotube, a gold nanoparticle (AuNP), a gold nanoshell, a gold nanorod, a silver shell gold nanoparticle, a latex, a magnetic nanoparticle, a silica nanoparticle, a fluorophore, a fluorophore-loaded nanoparticle, a dye-loaded nanoparticle, an enzyme, any combination thereof. In some embodiments, the method further comprises providing a hapten, a ligand, an oligonucleotide, digoxigenin (DIG), fluorescein isothiocyanate (FITC), a fluorophore, biotin, dinitrophenol, aniline, or an enzyme to facilitate signal generation.

[0198] In some embodiments, the target in the sample is subjected to blocking for about one hour prior to incubating the segmented primer probe or anti-target molecule with the target in the sample. In some embodiments, one or more anti-target molecules are incubated with the target in the sample, and then two or more segmented primer probes are incubated with the target. In some embodiments, the one or more anti-target molecules comprise unlabeled primary antibody probes. In some embodiments, the two or more segmented primer probes are incubated with the sample after incubation with the one or more anti-target molecules. In some embodiments, the two or more segmented primer probes comprise segmented primer secondary antibody probes. In some embodiments, the two or more segmented primer probes are incubated with the sample overnight or for about 17 hours. In some embodiments, one or more proximity probes are added to the sample after overnight incubation of the one or more proximity probes with the two or more segmented primer probes. In some embodiments, the one or more proximity probes are incubated with the sample for about four hours. In some embodiments, HCR amplifiers are added to the sample after incubation of the HCR amplifiers with the proximity probes. In some embodiments, the HCR amplifiers are incubated with the sample overnight or for about 20 hours. In some embodiments, a washing step is performed prior to detection.

[0199] In some embodiments, the target in the sample is subjected to blocking for about one hour prior to incubating the one or more anti-target molecules with the target in the sample. In some embodiments, some or all of three targets in the same sample are detected simultaneously in a multiplexed detection experiment (e.g., Figure 47) : 1) a target that is a single protein molecule, 2) a target that is two proteins that form a complex or are proximal, 3) a target that is a single RNA molecule. In some embodiments, in the protein detection phase, one or more unlabeled primary anti-target probes are incubated in the sample, optionally followed by one or more wash steps, and then one or more initiator-labeled secondary probes and two or more segmented initiator secondary probes are incubated in the sample, optionally followed by one or more wash steps. In some embodiments, the total incubation time of the primary probes plus the total incubation time of the secondary probes is about 4, or 8, or 16 or 24 hours. In some embodiments, the primary probes comprise primary antibodies or nanobodies, and the secondary probes comprise secondary antibodies or nanobodies. In some embodiments, in the proximity phase, one or more proximity probes are incubated in the sample, optionally followed by one or more wash steps. In some embodiments, the one or more proximity probes are incubated with the sample for about 0.5, or 1, or 2, or 4, or 8, or 16 or 24 hours. In some embodiments, in the RNA detection phase, two or more segmented initiator DNA probes are incubated with the sample, optionally followed by one or more wash steps. In some embodiments, the DNA probes are incubated with the sample for about 1, or 2, or 4, or 8, or 16, or 20 or 24 hours. In some embodiments, in the amplification phase, an HCR amplifier is added to the sample, optionally followed by one or more wash steps. In some embodiments, the HCR amplifier is incubated with the sample for about 0.5, or 1, or 2, or 10, or 16 or 24 hours. In some embodiments, after the signal for one or more targets is detected, the signal is removed from the sample and one or more of the above steps are repeated to detect the signal for one or more new targets. Examples Example 1 - Imaging of protein:protein target complexes

[0200] Figure 15 Imaging of protein:protein target complexes in fixed adherent A-431 human cells using segmented initiator probe pairs, proximity probes, and HCR amplifiers was depicted. For each protein:protein complex, background signal was assessed by a technical control in which the antibody corresponding to one protein of the complex was omitted, mimicking a situation in which the two proteins were not complexed with each other. For each of the three protein:protein target complexes, a high signal-to-noise ratio was obtained: 170 ± 10 (SC35: SON), 50 ± 10 (beta-catenin: E-cadherin), and 140 ± 10 (alpha-tubulin: beta-tubulin) (mean standard error, N = 3 representative regions within cells).

[0201] Figure 16 Imaging of the β-catenin:E-cadherin target complex in high autofluorescence FFPE human breast tissue sections (5 µm thick) using segmented initiator probe pairs, proximity probes, and an HCR amplifier was depicted. Background was assessed by fluorescence in sample regions containing low / absent protein:protein complexes. High signal-to-noise ratios (70 ± 20) were obtained (mean standard error, N = 3 representative regions). Example 2 - Proteins: Multiple Imaging of Protein-Target Complexes

[0202] Figure 17 This study depicts triple imaging of protein:protein complexes in immobilized A-431 human cells using different segmented initiator probe pairs, orthogonal HCR amplifiers, and neighboring probes for each target complex. Three orthogonal HCR amplifiers carry spectrally different fluorophores to enable multiple imaging. Imaging revealed fluorescence signals from the nuclear SC35:SON complex, the membrane β-catenin:E-cadherin complex, and the cytoskeleton α-tubulin:β-tubulin complex. Example 3 - Proteins: Simultaneous Multiple Imaging of Protein Complexes, Protein Targets, and RNA Targets

[0203] Figure 18 Simultaneous hematocrit (HCR) imaging of proteins, protein complexes, protein targets, and RNA targets was described in fixed-attached A-431 human cells. Reagents and protocols were described in... Figure 47 In this study, protein targets were detected using unlabeled primary antibody probes, which were then bound to initiator-labeled secondary antibody probes. RNA targets were detected using probe units comprising two fragmented initiator DNA probe pairs. Protein: Protein target complexes were detected using two unlabeled primary antibody probes, which were then bound to fragmented initiator secondary antibody probes, which were in turn bound to neighboring probes to co-localize intact HCR initiators. Each of the three target types (protein: protein, protein, RNA) triggered an orthogonal HCR amplifier carrying a spectrally differentially fluorophore. The amplified HCR signals revealed membrane β-catenin: E-cadherin complex, cytoskeleton β-tubulin, and nuclear U6 RNA. Example 4 - Proteins: Quantitative HCR (qHCR) Imaging of Protein Complexes

[0204] via Figure 19A The 2-channel redundancy detection experiment demonstrated quantitative imaging of protein:protein complexes. Figure 19B Redundancy detection was depicted in the membrane β-catenin:E-cadherin protein:protein complex of fixed-attached A-431 human cells. Figure 19C Showing Figure 19BA 2-channel scatter plot of voxel intensities of subcellular 2x2x0.8 pm voxels in a rectangular region of 100x100 pm reveals a tight linear distribution (Pearson correlation coefficient, r) corresponding to accurate and precise relative quantification. Accuracy corresponds to a line with zero intercept, and precision corresponds to the degree of dispersion around the line. Example 5 - High signal-to-noise ratio imaging of protein:protein complexes

[0205] Figure 45 demonstrates the performance of HCR protein:protein imaging by comparing the fluorescence intensities between two pairs of biological sample types using the same imaging settings. The positive sample includes the protein:protein complex of interest; the negative sample does not include the protein:protein complex of interest. For each pair of sample types, the signal plus background is estimated using the positive sample type, and the background is estimated using the negative sample, thereby calculating the signal-to-noise ratio.

[0206] A-431 adherent human cells were used as positive samples, and HeLa adherent human cells were used as negative samples for imaging of the beta-catenin:E-cadherin target complex. While A-431 cells form beta-catenin:E-cadherin complexes at the cell membrane at intercellular junctions 63 , HeLa cells express N-cadherin instead of E-cadherin 64,65 , and thus lack beta-catenin:E-cadherin complexes. A-431 cells Figure 45A exhibit strong signal at intercellular junctions, and HeLa cells exhibit no visible staining Figure 45B , with a signal-to-noise ratio of 26 ± 4 (average ± SEM of representative regions from N=3 replicate wells on a slide).

[0207] The beta-catenin:E-cadherin target complex was detected in highly autofluorescent FFPE human breast tissue sections. Beta-catenin:E-cadherin complexes are robustly formed in normal breast epithelial cells, but the expression and interaction between beta-catenin and E-cadherin is disrupted as breast epithelial cells become cancerous during the course of invasive lobular carcinoma lesions 66,67 . Paired normal and invasive lobular carcinoma FFPE breast tissue sections from the same patient were evaluated for the beta-catenin:E-cadherin target complex, producing strong HCR signal in the normal breast tissue Figure 45C ; positive sample) and no visible staining in the cancerous tissue Figure 45D ; negative sample) with a signal-to-noise ratio of 30 ± 3 (average ± SEM of representative regions from N=3 replicate sections). Example 6 - Sequences of synergistic probe ligation

[0208] Table 1 shows the sequence of three synergistic probe ligation. Each synergistic probe ligation includes: a) a PI oligonucleotide comprising a segmental primer (underlined sequence) and a proximity domain (bold sequence); b) a P2 oligonucleotide comprising a segmental primer (underlined sequence) and a proximity domain (bold sequence); c) a proximity probe comprising a binding site (bold sequence) for the P1 and P2 proximity domains. Note that the segmental primer probe P1 includes the PI oligonucleotide, and the segmental primer probe P2 includes the P2 oligonucleotide (see, e.g., Figure 46A schematic).

[0209] Figures 45A-45B and Figures 45C-45D Two 1 -plex imaging studies each employed segmental primer antibody probe pairs (P1 and P2) and a proximity probe that formed a synergistic probe ligation J1 (oligonucleotide sequences in Table 1).

[0210] Figure 17 A 3-plex imaging study of employed segmental primer probe pairs and proximity probes that formed different synergistic probe ligations for each target complex: for beta-catenin: E-cadherin was ligation J1, for alpha-tubulin: beta-tubulin was ligation J2, and for SC35: SON was ligation J3. Table 1. Oligonucleotide sequences of synergistic probe ligations Additional arrangements

[0211] Arrangement 1 : A composition comprising: a first segmental primer probe comprising: a first target binding domain configured to bind directly or indirectly to a first target, a first proximity domain, a first segmental primer; a second segmental primer probe comprising: a second target binding domain configured to bind directly or indirectly to a second target, a second proximity domain, a second segmental primer; a proximity probe configured to bind the first proximity domain and the second proximity domain; a hybridization chain reaction (HCR) amplifier comprising two or more HCR hairpin monomers, the at least one HCR hairpin monomer comprising a reporter; and wherein when the first segmental primer probe is bound to the first target, and the second segmental primer probe is bound to the second target; and the first target and the second target are bound to and / or proximal to each other, the proximity probe is capable of binding the first proximity domain and the second proximity domain to co-localize a complete primer comprising the first segmental primer and the second segmental primer, and wherein the co-localized complete primer is configured to prime HCR signal amplification, whereupon the HCR hairpin monomers self-assemble into a linked HCR amplification polymer, thereby generating a signal.

[0212] Arrangement 2: The composition of arrangement 1, wherein at least one HCR hairpin monomer comprises an input domain, and wherein the first and second segmented primers collectively form a complete primer configured to hybridize to the input domain.

[0213] Arrangement 3: The composition of any one of arrangements 1-2, further comprising: a third segmented primer probe comprising: a third target binding domain configured to bind directly or indirectly to a third target, a third proximity domain, a third segmented primer; and a proximity probe further configured to bind to the third proximity domain.

[0214] Arrangement 4: The composition of any one of arrangements 1-3, wherein the first target is a protein, a nucleic acid, or a combination thereof, and wherein the second target is a protein, a nucleic acid, or a combination thereof.

[0215] Arrangement 5: The composition of arrangement 4, wherein the first target and the second target bind to each other.

[0216] Arrangement 6: The composition of arrangement 4, wherein the first target and the second target are the same molecule.

[0217] Arrangement 7: The composition of any one of arrangements 1-6, wherein if one target is not proximal, the proximity probe comprises one or more clamp domains configured to bind to part or all of the first and / or second segmented primers.

[0218] Arrangement 8: The composition of any one of arrangements 1-7, wherein the first and / or second segmented primer probes comprise an antibody, a nanobody, and / or an oligonucleotide.

[0219] Arrangement 9: The composition of any one of arrangements 1-8, wherein the co-localized complete primers are used to directly or indirectly mediate signal generation.

[0220] Arrangement 10: The composition of any one of arrangements 1-9, wherein the co-localized complete primers trigger the self-assembly of the metastable fluorophore-labeled HCR hairpins into a connected fluorescent amplification polymer from to generate an amplification signal at the target site.

[0221] Arrangement 11: The composition of any one of arrangements 1-10, wherein the first and second targets are selected from a protein, an RNA, a DNA, or other nucleic acid.

[0222] Arrangement 12: The composition of any one of arrangements 1-11, wherein the first and second targets form a complex.

[0223] Arrangement 13: The composition of arrangement 12, wherein the composition comprises an additional segmental initiator for each of the N target complexes to be detected in a sample.

[0224] Arrangement 14: The composition of arrangement 13, wherein each of the N target complexes is detected in the same sample using a different segmental initiator probe, proximity probe, and HCR amplifier for each target complex.

[0225] Arrangement 15: The composition of any one of arrangements 1-14, wherein the sequence of the first segmental initiator is selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 4, and SEQ ID NO: 7; the sequence of the second segmental initiator is selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 5, and SEQ ID NO: 8; and the sequence of the proximity probe is selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 6, and SEQ ID NO: 9.

[0226] Arrangement 16: The composition of any one of arrangements 1-15, wherein the at least one reporter comprises a fluorophore, a chromophore, a lumiphore, a phosphor, a FRET pair, a member of a FRET pair, a quencher, a fluorophore / quencher pair, a rare earth element or compound, a radioactive molecule, a nucleotide, an amino acid, an oligonucleotide, DNA, RNA, 2’Ome-RNA, a chemically modified nucleic acid, a synthetic nucleic acid analog, a chemically modified protein, a synthetic protein analog, a peptide, a binding substrate, a carbon atom, a chemical linker, a magnetic molecule, carbon black (CB), a carbon nanotube, a magnetized carbon nanotube, a gold nanoparticle (AuNP), a gold nanoshell, a gold nanorod, a silver shell gold nanoparticle, a latex, a magnetic nanoparticle, a silica nanoparticle, a fluorophore-loaded nanoparticle, a dye-loaded nanoparticle, an enzyme, or any combination thereof.

[0227] Arrangement 17: The composition of any one of arrangements 1-16, wherein the at least one reporter directly or indirectly mediates signal generation.

[0228] Arrangement 18: The composition of any one of arrangements 1-17, wherein the at least one reporter comprises a hapten, a ligand, an oligonucleotide, digoxigenin (DIG), fluorescein isothiocyanate (FITC), a fluorophore, biotin, dinitrophenol, aniline, or an enzyme for facilitating signal generation.

[0229] Arrangement 19: The composition of any one of arrangements 1-18, wherein the HCR amplification polymer mediates catalytic reporter deposition (CARD).

[0230] Arrangement 20: A method comprising: providing: a sample comprising a first target and a second target; a first segmental primer probe comprising: a first target binding domain configured to bind directly or indirectly to the first target, a first proximity domain, a first segmental primer; a second segmental primer probe comprising: a second target binding domain configured to bind directly or indirectly to the second target, a second proximity domain, a second segmental primer; a proximity probe configured to bind the first proximity domain and the second proximity domain; a hybridization chain reaction (HCR) amplifier comprising two or more HCR hairpin monomers, wherein at least one HCR hairpin monomer comprises a reporter; adding the first segmental primer probe and the second segmental primer probe to the sample; adding the proximity probe to the sample; adding the HCR amplifier to the sample; detecting a signal indicative of the presence of the HCR polymer; wherein when the first segmental primer probe binds to the first target and the second segmental primer probe binds to the second target, and when the first target and the second target are bound or proximal to each other, then the proximity probe binds the first proximity domain and the second proximity domain to co-localize the complete primer; and wherein the co-localized complete primer initiates polymerization of the HCR monomers, thereby generating the signal.

[0231] Arrangement 21 : The method of arrangement 20, wherein the method further comprises providing a third segmental primer probe comprising: a third target binding domain configured to bind directly or indirectly to a third target, a third proximity domain, and a third segmental primer.

[0232] Arrangement 22: The method of arrangement 21, wherein the proximity probe is configured to bind the first proximity domain, the second proximity domain, and the third proximity domain.

[0233] Arrangement 23: The method of any one of arrangements 20-22, wherein a washing step is performed after b and before c to remove unbound segmental primer probes.

[0234] Arrangement 24: The method of any one of arrangements 20-23, wherein a washing step is performed after c and before d to remove unbound proximity probes.

[0235] Arrangement 25: The method of any one of arrangements 20-24, wherein a washing step is performed after d to remove unbound HCR hairpins.

[0236] Arrangement 26: The method of any one of arrangements 20-25, wherein the signal is removed after e.

[0237] Arrangement 27: The method of arrangement 26, wherein any of the above steps are repeated to detect a signal of the same or a different target.

[0238] Arrangement 28: The method of any one of arrangements 20-27, wherein the signal is detected by a fluorescence microscope, a fluorescence scanner, a camera, a cell phone camera, a mass spectrometer, a mass spectrometry microscope, or a radioactivity scanner.

[0239] Arrangement 29: The method of any one of arrangements 20-28, further comprising providing an auxiliary probe to maximize signal generation.

[0240] Arrangement 30: The method of any one of arrangements 20-29, wherein the first target is a protein, a nucleic acid, or a combination thereof, and the second target is a protein, a nucleic acid, or a combination thereof.

[0241] Arrangement 31 : The method of arrangement 30, wherein the first target and the second target bind to each other.

[0242] Arrangement 32: The method of arrangement 30, wherein the first target and the second target are the same molecule.

[0243] Arrangement 33: The method of any one of arrangements 20-32, wherein if one target is not proximal, the proximal probe comprises one or more clamp domains configured to bind to part or all of the first segment initiator and / or the second segment initiator.

[0244] Arrangement 34: The method of any one of arrangements 20-33, wherein the first segment initiator probe and / or the second segment initiator probe comprises a target binding region comprising an antibody, a nanobody, and / or an oligonucleotide.

[0245] Arrangement 35: The method of any one of arrangements 20-34, wherein the co-localized complete initiator is used to directly or indirectly mediate signal generation.

[0246] Arrangement 36: The method of any one of arrangements 20-34, wherein the co-localized complete initiator triggers a sub-stable fluorophore-labeled HCR hairpin to self-assemble into a linked fluorescent amplification polymer to generate an amplification signal at the proximal target site.

[0247] Arrangement 37: The method of any one of arrangements 20-36, wherein the co-localized complete initiator is used to detect one or more protein: protein complexes, RNA: protein complexes, RNA: RNA complexes, DNA: protein complexes, DNA: protein: protein complexes, or complexes of three or more of RNA, DNA, and / or proteins or other molecules in a sample.

[0248] Arrangement 38: The method of any one of arrangements 20-37, wherein the non-specifically bound segment initiator probe does not co-localize the complete initiator and does not initiate HCR.

[0249] Arrangement 39: The method of any one of arrangements 20-38, wherein N target complexes are each detected in the same sample using a different set of segmental primer probes, proximity probes, and HCR amplifiers for each target complex.

[0250] Arrangement 40: The method of any one of arrangements 20-39, wherein the sequence of the first segmental primer is selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 4, and SEQ ID NO: 7; the sequence of the second segmental primer is selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 5, and SEQ ID NO: 8; and the sequence of the proximity probe is selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 6, and SEQ ID NO: 9.

[0251] Arrangement 41: The method of any one of arrangements 20-40, wherein the at least one reporter comprises a fluorophore, a chromophore, a lumiphore, a phosphor, a FRET pair, a member of a FRET pair, a quencher, a fluorophore / quencher pair, a rare earth element or compound, a radioactive molecule, a nucleotide, an amino acid, an oligonucleotide, DNA, RNA, 2’Ome-RNA, a chemically modified nucleic acid, a synthetic nucleic acid analog, a chemically modified protein, a synthetic protein analog, a peptide, a binding substrate, a carbon atom, a chemical linker, a magnetic molecule, carbon black (CB), a carbon nanotube, a magnetized carbon nanotube, a gold nanoparticle (AuNP), a gold nanoshell, a gold nanorod, a silver shell gold nanoparticle, a latex, a magnetic nanoparticle, a silica nanoparticle, a fluorophore, a fluorophore-loaded nanoparticle, a dye-loaded nanoparticle, an enzyme, or any combination thereof.

[0252] Arrangement 42: The method of any one of arrangements 20-41, wherein the at least one reporter directly or indirectly mediates signal generation.

[0253] Arrangement 43: The method of any one of arrangements 20-42, wherein the at least one reporter comprises a hapten, a ligand, an oligonucleotide, digoxigenin (DIG), fluorescein isothiocyanate (FITC), a fluorophore, biotin, dinitrophenol, aniline, or an enzyme for facilitating signal generation.

[0254] Arrangement 44: The method of any one of arrangements 20-43, wherein the HCR amplification polymer mediates catalytic reporter deposition (CARD).

[0255] While the foregoing application has been described in terms of certain preferred embodiments, other embodiments will be apparent to those of ordinary skill in the art. Additionally, other combinations, omissions, substitutions and modifications will be contemplated by those of ordinary skill in the art, in light of the disclosure. Therefore, the application is not intended to be limited to the preferred embodiments described herein, but rather the scope of the application is to be defined by the claims and their equivalents.

[0256] Any of the embodiments, compositions, and / or methods provided herein can be used in combination with, or in alternative form to, any of the following. Thus, for example, the above-mentioned compositions and / or methods can employ any of the following compositions or methods. Similarly, it is to be understood that the above-mentioned compositions and / or methods also provide methods that employ or are part of the following methods.

[0257] Similarly, the embodiments and / or methods provided herein are also to be understood to provide embodiments directed to the practice of the methods, e.g., compositions, components of the methods, kits, etc. In some embodiments, any of the components of one or more methods and / or steps provided herein can be provided as a kit comprising one or more of the recited components (and optionally comprising a target or target sequence or sample).

[0258] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. All literature and similar materials cited in this application, including but not limited to, patents, patent applications, articles, books, treatises, and internet web pages are expressly incorporated by reference for any purpose. When definitions of terms in incorporated references appear to conflict with those provided in the present teachings, the definition provided in the present teachings controls. It is understood that where the application is discussed in terms of "about" and "at or about" and numbers exist between the recited values, that all intervening numbers are contemplated, so as to have the "about" and "at or about" provide support for either the lesser or the greater value or any number between the stated range. In the application, the use of the singular includes the plural, unless specifically stated otherwise. Furthermore, "comprise," "comprises," "comprising," "contain," "contains," "containing," "include," "includes," "including" and "have" are not intended to be limiting. 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 application, as claimed. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the application pertains. See, e.g., Singleton et al., Dictionary of Microbiology and Molecular Biology, 2nded., J. Wiley & Sons (New York, N.Y. 1994); Sambrook et al., Molecular Cloning, A Laboratory Manual, 3rded., Cold Springs Harbor Press (Cold Springs Harbor, N.Y. 2001). It is to be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the application, as claimed. In this application, the use of the singular includes the plural, unless specifically stated otherwise. In this application, the use of "or" means "and / or" unless stated otherwise. Furthermore, the use of the term "including" as well as "comprising" and variations thereof, is intended to be broad and inclusive, not restrictive. Also, the use of the term "about" in relation to a given value is intended to encompass minor variations (plus or minus ten percent) from the given value, unless specifically stated otherwise. Likewise, the term "substantially" is used herein to represent the somewhat imprecise approximation as understood by those of ordinary skill in the art. Also, the use of the term "portion" can include a half portion or an entire half.

[0259] In some embodiments, any one or more optional elements in any one or more figures herein can be combined with any one or more other optional elements in any one or more other figures herein. In some embodiments, any one or more compositions or steps provided in any one figure provided herein can be combined with any other composition or step provided herein. As used herein, a general reference to a set of figures (e.g., FIGS. 36) means all of the different figures included within that number (e.g., Figures 36A-36R ), combined together, one or more of them in combination, or each figure separately, unless otherwise indicated. References (1) Soderberg, O.; Gullberg, M.; Jarvius, M.; Ridderstrale, K.; Leuchowius, K. J.; Jarvius, J.; Wester, K.; Hydbring, P.; Bahram, F.; Larsson, L. G.; Landegren, U. Direct Observation of Individual Endogenous Protein Complexes in Situ by Proximity Ligation. Nat. Methods 2006, 3

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Claims

1. A composition comprising: a. A first-segment initiator probe, comprising: i. A first target binding domain configured to bind directly or indirectly to the first target. ii. First neighboring structural domain, iii. First-segment initiator, b. A second segmented initiator probe, comprising: i. A second target binding domain configured to bind directly or indirectly to the second target. ii. Second neighboring structural domain, iii. Second-stage initiator, c. Configured as a proximity probe that combines the first and second proximity domains; d. A hybridization chain reaction (HCR) amplifier comprising two or more HCR hairpin monomers, wherein at least one HCR hairpin monomer includes a reporter; and Wherein, when the first segmented initiator probe binds to the first target and the second segmented initiator probe binds to the second target; and when the first target and the second target are bound to each other and / or adjacent to each other, the adjacent probe can bind to the first adjacent structural domain and the second adjacent structural domain to co-localize the complete initiator including the first segmented initiator and the second segmented initiator, and wherein the co-localized complete initiator is configured to initiate HCR signal amplification, and then the HCR hairpin monomer self-assembles into a connected HCR amplification polymer, thereby generating a signal.

2. The composition of claim 1, wherein at least one HCR hairpin monomer comprises an input domain, and wherein the first segmented initiator and the second segmented initiator together form a complete initiator configured to hybridize with the input domain.

3. The composition according to claim 1, further comprising: a) The third-segment initiator probe includes: i) A third target binding domain configured to bind directly or indirectly to a third target. ii) Third neighboring domain, iii) The third-stage initiator; and b) Further configured as a neighbor probe that incorporates a third neighboring structural domain.

4. The composition according to claim 1, wherein the first target is a protein, nucleic acid, or a combination thereof, and wherein the second target is a protein, nucleic acid, or a combination thereof.

5. The composition according to claim 4, wherein the first target and the second target are bound to each other.

6. The composition according to claim 4, wherein the first target and the second target are the same molecule.

7. The composition of claim 1, wherein if a target is not adjacent, the adjacent probe comprises one or more clamp-shaped domains configured to bind partially or completely to a first segmented initiator and / or a second segmented initiator.

8. The composition of claim 1, wherein the first segmented initiator probe and / or the second segmented initiator probe comprise antibodies, nanobodies, and / or oligonucleotides.

9. The composition of claim 1, wherein the co-localized intact initiator is used to directly or indirectly mediate signal generation.

10. The composition of claim 1, wherein the co-localized intact initiator triggers the self-assembly of metastable fluorophore-tagged HCR hairpins into linked fluorescent amplification polymers to generate an amplification signal at the target site.

11. The composition of claim 1, wherein the first target and the second target are selected from proteins, RNA, DNA or other molecules.

12. The composition of claim 1, wherein the first target and the second target form a complex.

13. The composition of claim 12, wherein the composition comprises an additional segmentation initiator for each of the N target complexes to be detected in the sample.

14. The composition of claim 13, wherein different segmented initiator probe pairs, neighboring probes, and HCR amplifiers are used for each target complex to detect each of the N target complexes in the same sample.

15. The composition of claim 1, wherein the sequence of the first segmented initiator is selected from SEQ ID NO: 1, SEQ ID NO: 4 and SEQ ID NO: 7; the sequence of the second segmented initiator is selected from SEQ ID NO: 2, SEQ ID NO: 5 and SEQ ID NO: 8; and the sequence of the adjacent probe is selected from SEQ ID NO: 3, SEQ ID NO: 6 and SEQ ID NO:

9.

16. The composition of claim 1, wherein at least one reporter comprises: Fluorescein, chromophore, luminescent group, phosphor, FRET pair, FRET pair member, quencher, fluorophore / quencher pair, rare earth element or compound, radioactive molecule, nucleotide, amino acid, oligonucleotide, DNA, RNA, 2'Ome-RNA, chemically modified nucleic acid, synthetic nucleic acid analog, chemically modified protein, synthetic protein analog, peptide, binding substrate, carbon atom, chemical linker, magnetic molecule, carbon black (CB), carbon nanotube, magnetized carbon nanotube, gold nanoparticle (AuNP), gold nanoshell, gold nanorod, silver-shelled gold nanoparticle, latex, magnetic nanoparticle, silica nanoparticle, fluorophore-loaded nanoparticle, dye-loaded nanoparticle, enzyme or any combination thereof.

17. The composition of claim 1, wherein at least one reporter directly or indirectly mediates signal generation.

18. The composition of claim 1, wherein at least one reporter comprises a hapten, ligand, oligonucleotide, digoxigenin (DIG), fluorescein isothiocyanate (FITC), fluorophore, biotin, dinitrophenol, aniline, or enzyme for promoting signal generation.

19. The composition of claim 1, wherein the HCR amplification polymer mediates catalytic reporter deposition (CARD).

20. A method comprising: a). Provide: i) Samples including the first target and the second target; ii) A first segmented initiator probe, comprising: 1) A first target binding domain configured to directly or indirectly bind to the first target. 2) First neighboring structural domain, 3) First-stage initiator; iii) A second segmented initiator probe, comprising: 1) A second target binding domain configured to directly or indirectly bind to the second target. 2) Second neighboring structural domain, 3) Second-stage initiator; iv). Configured to combine a first neighboring domain and a second neighboring domain as a proximity probe; v). A hybridization chain reaction (HCR) amplifier comprising two or more HCR hairpin monomers, wherein at least one HCR hairpin monomer includes a reporter; b) Add the first and second segmented initiator probes to the sample; c) Add adjacent probes to the sample; d) Add the HCR amplifier to the sample; e). Detect signals indicating the presence of HCR polymers; Wherein, when the first segmented initiator probe binds to the first target and the second segmented initiator probe binds to the second target, and when the first target and the second target are bound to each other or adjacent to each other, then the adjacent probe binds to the first adjacent structural domain and the second adjacent structural domain to co-localize the intact initiator; and The co-located complete initiator initiates the polymerization of HCR monomers, thereby generating a signal.

21. The method of claim 20, wherein the method further comprises providing a third segmented initiator probe, the third segmented initiator probe comprising: 1) A third target binding domain configured to bind directly or indirectly to a third target. 2) The third neighboring structural domain, and 3) Third-segment initiator.

22. The method of claim 21, wherein the proximity probe is configured to combine a first proximity domain, a second proximity domain, and a third proximity domain.

23. The method of claim 20, wherein a washing step is performed after b and before c to remove unbound segmented initiator probes.

24. The method of claim 20, wherein a washing step is performed after c and before d to remove unbound adjacent probes.

25. The method of claim 20, wherein a washing step is performed after d to remove unbonded HCR hair clips.

26. The method of claim 20, wherein the signal is removed after e.

27. The method of claim 26, wherein any of the above steps are repeated to detect signals of the same or different targets.

28. The method of claim 20, wherein the signal is detected by a fluorescence microscope, a fluorescence scanner, a camera, a mobile phone camera, a mass spectrometer, a mass spectrometry microscope, or a radiometric scanner.

29. The method of claim 20, further comprising providing an auxiliary probe to maximize signal generation.

30. The method of claim 20, wherein the first target is a protein, nucleic acid, or a combination thereof, and wherein the second target is a protein, nucleic acid, or a combination thereof.

31. The method of claim 30, wherein the first target and the second target are combined with each other.

32. The method of claim 30, wherein the first target and the second target are the same molecule.

33. The method of claim 20, wherein if a target is not adjacent, then the adjacent probe comprises one or more clamp-shaped domains configured to bind partially or entirely to a first segmented initiator and / or a second segmented initiator.

34. The method of claim 20, wherein the first segmented initiator probe and / or the second segmented initiator probe includes a target binding region, said target binding region including an antibody, nanobody, and / or oligonucleotide.

35. The method of claim 20, wherein the co-localized intact initiator is used to directly or indirectly mediate signal generation.

36. The method of claim 20, wherein the co-localized intact initiator triggers the self-assembly of metastable fluorophore-tagged HCR hairpins into linked fluorescent amplification polymers to generate an amplification signal at a proximal target site.

37. The method of claim 20, wherein the co-localized intact initiator is used to detect one or more proteins in a sample: protein complex, RNA: protein complex, RNA: RNA complex, DNA: protein complex, DNA: protein: protein complex, or a complex of three or more RNA, DNA and / or proteins or other molecules.

38. The method of claim 20, wherein the non-specifically bound segmented initiator probe does not co-localize the intact initiator and does not initiate HCR.

39. The method of claim 20, wherein different segmented initiator probe pairs, neighboring probes, and HCR amplifiers are used for each target complex to detect each of the N target complexes in the same sample.

40. The method of claim 20, wherein the sequence of the first segmented initiator is selected from SEQ ID NO: 1, SEQ ID NO: 4 and SEQ ID NO: 7; the sequence of the second segmented initiator is selected from SEQ ID NO: 2, SEQ ID NO: 5 and SEQ ID NO: 8; and the sequence of the adjacent probe is selected from SEQ ID NO: 3, SEQ ID NO: 6 and SEQ ID NO:

9.

41. The method of claim 20, wherein at least one reporting item comprises: Fluorescein, chromophore, luminescent group, phosphor, FRET pair, FRET pair member, quencher, fluorophore / quencher pair, rare earth element or compound, radioactive molecule, nucleotide, amino acid, oligonucleotide, DNA, RNA, 2'Ome-RNA, chemically modified nucleic acid, synthetic nucleic acid analog, chemically modified protein, synthetic protein analog, peptide, binding substrate, carbon atom, chemical linker, magnetic molecule, carbon black (CB), carbon nanotube, magnetized carbon nanotube, gold nanoparticle (AuNP), gold nanoshell, gold nanorod, silver-shelled gold nanoparticle, latex, magnetic nanoparticle, silica nanoparticle, fluorophore, fluorophore-loaded nanoparticle, dye-loaded nanoparticle, enzyme or any combination thereof.

42. The method of claim 20, wherein at least one reporter directly or indirectly mediates signal generation.

43. The method of claim 20, wherein at least one reporter comprises a hapten, ligand, oligonucleotide, digoxigenin (DIG), fluorescein isothiocyanate (FITC), fluorophore, biotin, dinitrophenol, aniline, or enzyme for promoting signal generation.

44. The method of claim 20, wherein the HCR amplification polymer-mediated catalytic reporter deposition (CARD).

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