Probes for measuring molecular proximity in samples

Abstract

This application relates to hybridization chain reaction (HCR). In particular, the sensitivity of hybridization chain reaction (HCR) signal amplification is combined with one or more proximity probes that can co-localize two or more fragmented initiator probes and the complete HCR initiator that triggers HCR when the target is in close proximity to each other.

Description

[Technical Field]

[0001] REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 449,543, filed March 2, 2023, and U.S. Provisional Patent Application No. 63 / 528,262, filed July 21, 2023, the disclosures of which are incorporated herein by reference in their entireties.

[0002] Statement Regarding Federally Sponsored R&D This invention is based on the findings of the National Institutes of Health This invention was made with government support under Grant No. EB006192 awarded by the National Institute of Health. The government has certain rights in this invention.

[0003] Incorporation by reference of material in the sequence listing file This application incorporates the material in the XML sequence listing provided herewith, entitled CALTE.164Aseqlist.xml, created on February 29, 2024, and 9,313 bytes in size.

[0004] background Field This application relates to hybridization chain reaction (HCR). In particular, the sensitivity of hybridization chain reaction (HCR) signal amplification is combined with one or more proximity probes that can co-localize two or more fragmented initiator probes and the complete HCR initiator that triggers HCR when the target is in close proximity to each other. Summary of the Invention

[0005] In some embodiments, a composition is provided comprising: a first fragmentary initiator probe comprising a first target binding domain, a first proximity domain, and a first fragmentary initiator configured to directly or indirectly bind to a first target; a second fragmentary initiator probe comprising a second target binding domain, a second proximity domain, and a second fragmentary initiator configured to directly or indirectly bind to a second target; and a proximity probe configured to bind to the first proximity domain and the second proximity domain. In some embodiments, a third fragmentary initiator probe may be provided comprising a third target binding domain, a third proximity domain, and a third fragmentary initiator configured to directly or indirectly bind to a third target. In some embodiments, the proximity probe is further configured to bind to the third proximity domain.

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

[0007] In some embodiments, the proximity probes are configured to detect a first target when one target is not in proximity. The proximity probe comprises one or more clamp domains configured to bind to part or all of the first fragmentary initiator and / or the second fragmentary initiator. In some embodiments, the proximity probe comprises one or more clamp domains configured to bind to part or all of the first fragmentary initiator and / or the second fragmentary initiator and / or the third fragmentary initiator when one or more targets are not in proximity.

[0008] In some embodiments, the first fragmentary initiator probe and / or the second fragmentary initiator probe comprise an antibody, a nanobody, and / or an oligonucleotide. In some embodiments, the third fragmentary initiator probe comprises an antibody, a nanobody, and / or an oligonucleotide.

[0009] In some embodiments, when a first fragmentary initiator probe is bound to a first target and a second fragmentary initiator probe is bound to a second target, and when the first and second targets are bound and / or in proximity to each other, the proximity probes can bind to the first proximity domain and the second proximity domain to co-localize the complete initiator.

[0010] In some embodiments, the proximity probes can bind to the first proximity domain, the second proximity domain, and the third proximity domain to co-localize the complete initiator when the first fragmentary initiator probe is bound to a first target and the second fragmentary initiator probe is bound to a second target, when the third fragmentary initiator probe is bound to a third target, and when the first, second, and third targets are bound to and / or in proximity to each other.

[0011] In some embodiments, the co-localized intact initiator can directly or indirectly mediate signal generation, In some embodiments, the co-localized intact initiator can trigger HCR signal amplification or otherwise signal amplification.

[0012] In some embodiments, an additional fragmentary initiator probe beyond the third fragmentary initiator probe comprises a target binding domain configured to directly or indirectly bind to a target, a proximity domain, and a fragmentary initiator; and the proximity probe is further configured to bind to the proximity domain.

[0013] According to some embodiments, a method is provided that includes providing a sample, optionally containing a first target and / or a second target; contacting the sample with a first fragmentary initiator probe comprising a first target binding domain, a first proximity domain, and a first fragmentary initiator configured to bind directly or indirectly to the first target; contacting the sample with a second fragmentary initiator probe comprising a second target binding domain, a second proximity domain, and a second fragmentary initiator configured to bind directly or indirectly to the second target; optionally contacting the sample with a third fragmentary initiator probe comprising a third target binding domain, a third proximity domain, and a third fragmentary initiator configured to bind directly or indirectly to a third target; 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 an HCR amplification agent comprising two or more HCR hairpins.

[0014] According to some embodiments, the method comprises incubating a first fragmentary initiator probe and a second fragmentary initiator probe and optionally a third fragmentary initiator probe in a sample to allow binding, optionally washing to remove unbound fragmentary initiator probes, and then rinsing the sample. In some embodiments, the method comprises incubating proximity probes in the sample to allow them to bind, optionally washing to remove unbound proximity probes, incubating an HCR amplification agent in the sample, optionally washing to remove unbound HCR hairpins, and detecting a signal. According to some embodiments, the method comprises optionally removing the signal, and optionally repeating any of the above steps to detect a signal for the same or a different target. [Brief explanation of the drawings]

[0015] [Figure 1] FIG. 1 depicts the detection of protein:protein interactions using proximity ligation assay (PLA). [Figure 2] FIG. 2 depicts the detection of protein:protein interactions using a proximity-based HCR method that employs a kinetic triggering mechanism. [Figure 3] FIG. 3 depicts the detection of RNA:protein interactions using a bridge strand. [Figure 4A] FIG. 4A depicts some embodiments of HCR RNA-FISH using initiator-labeled probes. [Figure 4B] FIG. 4B depicts some embodiments of HCR RNA-FISH using initiator-labeled probes. [Figure 4C] FIG. 4C depicts some embodiments of HCR RNA-FISH using initiator-labeled probes. [Figure 5A] FIG. 5A depicts some embodiments of HCR RNA-FISH using fragmentary initiator probes. [Figure 5B] FIG. 5B depicts some embodiments of HCR RNA-FISH using fragmentary initiator probes. [Figure 6] FIG. 6 depicts some embodiments of detection of target complexes using fragmentary initiator probes and proximity probes, where the fragmentary initiator probes bind directly to the sample. [Figure 7]FIG. 7 depicts some embodiments of detection of target complexes using fragmentary initiator probes and proximity probes, where the fragmentary initiator probes bind indirectly to the sample. [Figure 8] FIG. 8 depicts some embodiments of target detection using fragmentary initiator probes and proximity probes. [Figure 9] FIG. 9 depicts some embodiments of detection of a first target and a second target that are not in a complex with each other but are in a complex with a third target. [Figure 10] FIG. 10 depicts some embodiments of detection of a first target and a second target that are not in a complex with each other. [Figure 11] FIG. 11 depicts some embodiments of target detection using fragmentary initiator probes and proximity probes, where the fragmentary initiator probes bind indirectly to the sample. [Figure 12] FIG. 12 depicts some embodiments of detection of three targets using fragmentary initiator probes and proximity probes. [Figure 13] FIG. 13 depicts some embodiments of detection of three targets using a fragmentary initiator probe and two proximity probes. [Figure 14] FIG. 14 depicts some embodiments of the detection of protein:protein complexes. [Figure 15] FIG. 15 depicts imaging of protein:protein target complexes in human cells. [Figure 16] FIG. 16 depicts imaging of protein:protein target complexes in FFPE human breast tissue sections. [Figure 17] FIG. 17 depicts 3-plex imaging of three protein:protein target complexes in human cells. [Figure 18] FIG. 18 depicts simultaneous 3-plex imaging of a protein:protein target complex, a protein target, and an RNA target in human cells. [Figure 19A] FIG. 19A depicts quantitative HCR imaging of protein:protein target complexes in human cells. [Figure 19B] FIG. 19B depicts quantitative HCR imaging of protein:protein target complexes in human cells. [Figure 19C] FIG. 19C depicts quantitative HCR imaging of protein:protein target complexes in human cells. [Figure 20] FIG. 20 depicts some embodiments of detection of protein:protein target complexes using fragmentary initiator nanobody probes and proximity probes. [Figure 21] FIG. 21 depicts some embodiments of the detection of protein:RNA target complexes. [Figure 22] FIG. 22 depicts some embodiments of detection of RNA:RNA target complexes. [Figure 23A] Figure 23A depicts some embodiments of target complex detection using proximity probes that allow colocalization of the fragmentary initiators when both targets are present and clamp the entire fragmentary initiator in the absence of one target. [Figure 23B] Figure 23B depicts some embodiments of target complex detection using proximity probes that allow colocalization of the fragmentary initiators when both targets are present and clamp the entire fragmentary initiator in the absence of one target. [Figure 23C] Figure 23C depicts some embodiments of target complex detection using proximity probes that allow colocalization of the fragmentary initiators when both targets are present and clamp the entire fragmentary initiator in the absence of one target. [Figure 24A] Figure 24A depicts some embodiments of target complex detection using proximity probes that allow colocalization of the fragmentary initiators when both targets are present and clamp the 5' end of the fragmentary initiator in the absence of one target. [Figure 24B] Figure 24B depicts some embodiments of target complex detection using proximity probes that allow colocalization of the fragmentary initiators when both targets are present and clamp the 5' end of the fragmentary initiator in the absence of one target. [Figure 24C] Figure 24C depicts some embodiments of target complex detection using proximity probes that allow colocalization of the fragmentary initiators when both targets are present and clamp the 5' end of the fragmentary initiator in the absence of one target. [Figure 25A] Figure 25A depicts some embodiments of target complex detection using proximity probes that allow colocalization of the fragmentary initiators when both targets are present and clamp the 3' end of the fragmentary initiator in the absence of one target. [Figure 25B] Figure 25B depicts some embodiments of target complex detection using proximity probes that allow colocalization of the fragmentary initiators when both targets are present and clamp the 3' end of the fragmentary initiator in the absence of one target. [Figure 25C] Figure 25C depicts some embodiments of target complex detection using proximity probes that allow colocalization of the fragmentary initiators when both targets are present and clamp the 3' end of the fragmentary initiator in the absence of one target. [Figure 26A] Figure 26A depicts some embodiments of target complex detection using proximity probes that allow colocalization of the fragmentary initiators when both targets are present, and clamp the 5' and 3' ends of the fragmentary initiators in the absence of one target. [Figure 26B] Figure 26B depicts some embodiments of target complex detection using proximity probes that allow colocalization of the fragmentary initiators when both targets are present, and clamp the 5' and 3' ends of the fragmentary initiators in the absence of one target. [Figure 26C] Figure 26C depicts some embodiments of target complex detection using proximity probes that allow colocalization of the fragmentary initiators when both targets are present, and clamp the 5' and 3' ends of the fragmentary initiators in the absence of one target. [Figure 27A] FIG. 27A depicts some embodiments of fragmentary initiator probes that form cooperative probe junctions with target RNA or with proximity probes. [Figure 27B] FIG. 27B depicts some embodiments of fragmentary initiator probes that form cooperative probe junctions with target RNA or with proximity probes. [Figure 28A] FIG. 28A depicts some embodiments of initiator-labeled probes. [Figure 28B] FIG. 28B depicts some embodiments of initiator-labeled probes. [Figure 28C] FIG. 28C depicts some embodiments of initiator-labeled probes. [Figure 28D] FIG. 28D depicts some embodiments of initiator-labeled probes. [Figure 28E] FIG. 28E depicts some embodiments of initiator-labeled probes. [Figure 28F] FIG. 28F depicts some embodiments of initiator-labeled probes. [Figure 28G] FIG. 28G depicts some embodiments of initiator-labeled probes. [Figure 28H] FIG. 28H depicts some embodiments of initiator-labeled probes. [Figure 28I] FIG. 28I depicts some embodiments of initiator-labeled probes. [Figure 28J] FIG. 28J depicts some embodiments of initiator-labeled probes. [Figure 28K] FIG. 28K depicts some embodiments of initiator-labeled probes. [Figure 28L] FIG. 28L depicts some embodiments of initiator-labeled probes. [Figure 28M] FIG. 28M depicts some embodiments of initiator-labeled probes. [Figure 28N] FIG. 28N depicts some embodiments of initiator-labeled probes. [Figure 29A] FIG. 29A depicts some embodiments of initiator-labeled probes. [Figure 29B] FIG. 29B depicts some embodiments of initiator-labeled probes. [Figure 29C]FIG. 29C depicts some embodiments of initiator-labeled probes. [Figure 29D] FIG. 29D depicts some embodiments of initiator-labeled probes. [Figure 29E] FIG. 29E depicts some embodiments of initiator-labeled probes. [Figure 29F] FIG. 29F depicts some embodiments of initiator-labeled probes. [Figure 30A] FIG. 30A depicts some embodiments of a probe set that includes one or more probe units and optionally includes one or more helper probes. [Figure 30B] FIG. 30B depicts some embodiments of a probe set that includes one or more probe units and optionally includes one or more helper probes. [Figure 30C] FIG. 30C depicts some embodiments of a probe set that includes one or more probe units and optionally includes one or more helper probes. [Figure 30D] FIG. 30D depicts some embodiments of a probe set that includes one or more probe units and optionally includes one or more helper probes. [Figure 31A] FIG. 31A depicts an embodiment of a portion of a fragmentary initiator probe that is co-localized with a target RNA. [Figure 31B] FIG. 31B depicts an embodiment of a portion of a fragmentary initiator probe that is co-localized with a target RNA. [Figure 31C] FIG. 31C depicts an embodiment of a portion of a fragmentary initiator probe that is co-localized with a target RNA. [Figure 31D] FIG. 31D depicts an embodiment of a portion of a fragmentary initiator probe that is co-localized with a target RNA. [Figure 31E] FIG. 31E depicts an embodiment of a portion of a fragmentary initiator probe that is co-localized with a target RNA. [Figure 32A] FIG. 32A depicts an embodiment of a portion of a fragmentary initiator probe that is co-localized with a target. [Figure 32B] FIG. 32B depicts an embodiment of a portion of a fragmentary initiator probe that is co-localized with a target. [Figure 32C] FIG. 32C depicts an embodiment of a portion of a fragmentary initiator probe that is co-localized with a target. [Figure 32D] FIG. 32D depicts an embodiment of a portion of a fragmentary initiator probe that is co-localized with a target. [Figure 33A] FIG. 33A depicts an embodiment of a portion of a fragmentary initiator probe that is co-localized with a target complex. [Figure 33B] FIG. 33B depicts an embodiment of a portion of a fragmentary initiator probe that is co-localized with a target complex. [Figure 33C] FIG. 33C depicts an embodiment of a portion of a fragmentary initiator probe that is co-localized with a target complex. [Figure 33D] FIG. 33D depicts an embodiment of a portion of a fragmentary initiator probe that is co-localized with a target complex. [Figure 33E] FIG. 33E depicts an embodiment of a portion of a fragmentary initiator probe that is co-localized with a target complex. [Figure 34A] FIG. 34A depicts an embodiment of a portion of a probe unit that includes a fragmentary initiator probe. [Figure 34B] FIG. 34B depicts an embodiment of a portion of a probe unit that includes a fragmentary initiator probe. [Figure 34C] FIG. 34C depicts an embodiment of a portion of a probe unit that includes a fragmentary initiator probe. [Figure 35] FIG. 35 depicts some embodiments of fragmentary initiator probes that are co-localized with a target RNA. [Figure 36A] FIG. 36A depicts some embodiments of fragmentary initiator probes co-localized with targets either directly or indirectly. [Figure 36B] FIG. 36B depicts some embodiments of fragmentary initiator probes that are co-localized with targets either directly or indirectly. [Figure 36C] FIG. 36C depicts some embodiments of fragmentary initiator probes that are co-localized with targets, either directly or indirectly. [Figure 36D] Figure 36D depicts some embodiments of fragmentary initiator probes that are co-localized with a target, either directly or indirectly. [Figure 36E] Figure 36E depicts some embodiments of fragmentary initiator probes that are co-localized with targets, either directly or indirectly. [Figure 36F] Figure 36F depicts some embodiments of fragmentary initiator probes that are co-localized with targets either directly or indirectly. [Figure 36G] Figure 36G depicts some embodiments of fragmentary initiator probes that are co-localized with a target, either directly or indirectly. [Figure 36H] Figure 36H depicts some embodiments of fragmentary initiator probes co-localized with targets either directly or indirectly. [Figure 36I] Figure 36I depicts some embodiments of fragmentary initiator probes co-localized with targets either directly or indirectly. [Figure 36J] Figure 36J depicts some embodiments of fragmentary initiator probes co-localized with targets either directly or indirectly. [Figure 36K] Figure 36K depicts some embodiments of fragmentary initiator probes co-localized with targets either directly or indirectly. [Figure 36L] Figure 36L depicts some embodiments of fragmentary initiator probes co-localized with targets either directly or indirectly. [Figure 36M] Figure 36M depicts some embodiments of fragmentary initiator probes co-localized with targets either directly or indirectly. [Figure 36N] Figure 36N depicts some embodiments of fragmentary initiator probes co-localized with targets either directly or indirectly. [Figure 36O] Figure 36O depicts some embodiments of fragmentary initiator probes co-localized with targets either directly or indirectly. [Figure 36P] Figure 36P depicts some embodiments of fragmentary initiator probes co-localized with targets either directly or indirectly. [Figure 36Q] Figure 36Q depicts some embodiments of fragmentary initiator probes co-localized with targets either directly or indirectly. [Figure 36R] Figure 36R depicts some embodiments of fragmentary initiator probes co-localized with targets either directly or indirectly. [Figure 37A] FIG. 37A depicts some embodiments of HCR amplifiers. [Figure 37B] FIG. 37B depicts some embodiments of HCR amplifiers. [Figure 37C] FIG. 37C depicts some embodiments of HCR amplifiers. [Figure 37D] FIG. 37D depicts some embodiments of HCR amplifiers. [Figure 37E] FIG. 37E depicts some embodiments of HCR amplifiers. [Figure 37F] FIG. 37F depicts some embodiments of HCR amplifiers. [Figure 38A] FIG. 38A depicts some embodiments of HCR amplification using four HCR hairpins. [Figure 38B] FIG. 38B depicts some embodiments of HCR amplification using four HCR hairpins. [Figure 39] Figure 39 depicts the complete HCR initiator i1 formed by two fragmentary initiator probes co-localized with the target. Only a portion of the fragmentary initiator probe is depicted. [Figure 40] FIG. 40 depicts some embodiments of HCR amplification using two HCR hairpins. [Figure 41A]FIG. 41A depicts some embodiments that use HCR amplification to mediate CARD signal amplification for different targets and signal probes. [Figure 41B] FIG. 41B depicts some embodiments in which HCR amplification is used to mediate CARD signal amplification for different targets and signal probes. [Figure 41C] FIG. 41C depicts some embodiments in which HCR amplification is used to mediate CARD signal amplification for different targets and signal probes. [Figure 41D] FIG. 41D depicts some embodiments in which HCR amplification is used to mediate CARD signal amplification for different targets and signal probes. [Figure 41E] FIG. 41E depicts some embodiments in which HCR amplification is used to mediate CARD signal amplification for different targets and signal probes. [Figure 42A] FIG. 42A depicts some embodiments that use HCR amplification to mediate CARD signal amplification for a generic target and signal probe. [Figure 42B] FIG. 42B depicts some embodiments that use HCR amplification to mediate CARD signal amplification for a generic target and signal probe. [Figure 42C] FIG. 42C depicts some embodiments that use HCR amplification to mediate CARD signal amplification for a generic target and signal probe. [Figure 43A] FIG. 43A depicts some embodiments in which HCR amplification using reporter-labeled or fragmentary reporter-labeled HCR hairpins is used to mediate CARD signal amplification. [Figure 43B] FIG. 43B depicts some embodiments in which HCR amplification using reporter-labeled or fragmentary reporter-labeled HCR hairpins is used to mediate CARD signal amplification. [Figure 44A]Figure 44A depicts some embodiments of detection of target complexes using a proximity probe comprising two fragmentary initiator probes and one or more clamps, where the proximity probes allow co-localization of the fragmentary initiators to form a complete initiator when both fragmentary initiator probes are bound to the proximity probe, while the one or more clamps segregate one or more component parts of the fragmentary initiator when only one fragmentary initiator probe is bound to the proximity probe. [Figure 44B] Figure 44B depicts some embodiments of detection of target complexes using a proximity probe comprising two fragmentary initiator probes and one or more clamps, where the proximity probes allow co-localization of the fragmentary initiators to form a complete initiator when both fragmentary initiator probes are bound to the proximity probe, while the one or more clamps segregate one or more component parts of the fragmentary initiator when only one fragmentary initiator probe is bound to the proximity probe. [Figure 44C] Figure 44C depicts some embodiments of detection of target complexes using a proximity probe comprising two fragmentary initiator probes and one or more clamps, where the proximity probes allow co-localization of the fragmentary initiators to form a complete initiator when both fragmentary initiator probes are bound to the proximity probe, while the one or more clamps segregate one or more component parts of the fragmentary initiator when only one fragmentary initiator probe is bound to the proximity probe. [Figure 45A] FIG. 45A depicts strong HCR signal generation in a positive sample containing a protein:protein target complex and no visible staining in a negative sample lacking the protein:protein target complex. [Figure 45B] FIG. 45B depicts strong HCR signal generation in positive samples containing protein:protein target complexes and no visible staining in negative samples lacking protein:protein target complexes. [Figure 45C]FIG. 45C depicts strong HCR signal generation in positive samples containing protein:protein target complexes and no visible staining in negative samples lacking protein:protein target complexes. [Figure 45D] FIG. 45D depicts strong HCR signal generation in positive samples containing protein:protein target complexes and no visible staining in negative samples lacking protein:protein target complexes. [Figure 46A] FIG. 46A depicts some embodiments of an HCR imaging method for detecting protein:protein target complexes in a sample. [Figure 46B] FIG. 46B depicts some embodiments of an HCR imaging method for detecting protein:protein target complexes in a sample. [Figure 47] FIG. 47 depicts some embodiments of a protocol for simultaneous HCR imaging of a protein target, a protein:protein target complex, and an RNA target in a sample. [Figure 48A] FIG. 48A depicts some embodiments that use HCR signal amplification for detection of a target or target complex in a sample. [Figure 48B] FIG. 48B depicts some embodiments that use HCR signal amplification for detection of a target or target complex in a sample. [Figure 48C] FIG. 48C depicts some embodiments that use HCR signal amplification for detection of a target or target complex in a sample. [Figure 48D] FIG. 48D depicts some embodiments that use HCR signal amplification for detection of a target or target complex in a sample. [Figure 49A] FIG. 49A depicts some embodiments of HCR immunohistochemistry and HCR protein:protein imaging using a primary antibody probe with or without a secondary antibody probe. [Figure 49B]FIG. 49B depicts some embodiments of HCR immunohistochemistry and HCR protein:protein imaging using a primary antibody probe with or without a secondary antibody probe. [Figure 49C] FIG. 49C depicts some embodiments of HCR immunohistochemistry and HCR protein:protein imaging using a primary antibody probe with or without a secondary antibody probe. [Figure 49D] FIG. 49D depicts some embodiments of HCR immunohistochemistry and HCR protein:protein imaging using a primary antibody probe with or without a secondary antibody probe. [Figure 50A] FIG. 50A depicts some embodiments of simultaneous HCR IHC / RNA-ISH using initiator-labeled antibody probes for protein targets and fragmentary initiator DNA probes for RNA targets. [Figure 50B] FIG. 50B depicts some embodiments of simultaneous HCR IHC / RNA-ISH using initiator-labeled antibody probes for protein targets and fragmentary initiator DNA probes for RNA targets. [Figure 50C] FIG. 50C depicts some embodiments of simultaneous HCR IHC / RNA-ISH using initiator-labeled antibody probes for protein targets and fragmentary initiator DNA probes for RNA targets. [Figure 50D] FIG. 50D depicts some embodiments of simultaneous HCR IHC / RNA-ISH using initiator-labeled antibody probes for protein targets and fragmentary initiator DNA probes for RNA targets. [Figure 50E] FIG. 50E depicts some embodiments of simultaneous HCR IHC / RNA-ISH using initiator-labeled antibody probes for protein targets and fragmentary initiator DNA probes for RNA targets. [Figure 50F] Figure 50F depicts some embodiments of simultaneous HCR IHC / RNA-ISH using initiator-labeled antibody probes for protein targets and fragmentary initiator DNA probes for RNA targets. DETAILED DESCRIPTION OF THE INVENTION

[0016] Detailed Description To study biological processes including replication, transcription, translation, and signal transduction, it is important to visualize not only the molecules involved in these processes, e.g., RNA, protein, and DNA targets, but also complexes of these molecules. Making signal generation conditional on the proximity of two molecules provides a sub-diffraction-limited readout in contrast to independent imaging of the same two molecules in separate channels. Target complexes have previously been imaged using proximity ligation assays (PLA), which utilize enzymatic ligation and rolling circle amplification (RCA). 1~12 (See, e.g., Figure 1). To detect the complex, a primary antibody binds to the target of interest, and an oligonucleotide-labeled secondary antibody then binds to the primary antibody. Two additional oligonucleotides then bind to the oligonucleotide-labeled secondary antibody and are circularized via an enzymatic ligase step. RCA is then accomplished with the enzymatic polymerase to generate a single-stranded DNA amplification product, which is detected by a complementary fluorescent readout strand. 1、12 (See, for example, Figure 1.) Cost and Storage Concerns with PLA Enzymes 1、12 In addition, PLA can also produce false negatives resulting from the formation of a non-circular ligation product. 13 and false positives due to spurious amplification in the absence of adjacent probes. 12 Additionally, signal amplification using PLA methods does not scale linearly with target abundance. 7 .

[0017] Signal amplification based on the mechanism of hybridization chain reaction (HCR) 14 has been used to provide in situ signal amplification, e.g., for imaging RNA and protein targets within fixed biological specimens. 15~18In some cases, HCR amplification agents can consist of two kinetically trapped DNA hairpins (h1 and h2) that metastably coexist in solution and store energy to drive the conditional self-assembly of HCR-amplified polymers upon exposure to a cognate initiator sequence (i1) (see, e.g., Figures 4A, 40, and 48A). 14 In some cases, using HCR RNA in situ hybridization (RNA-ISH), RNA targets can be detected using one or more pairs of fragmentary initiator DNA probes, each carrying a fragment of the HCR initiator i1 (Figures 5A and B and 48B). 17 A probe pair that specifically hybridizes to adjacent binding sites on the target RNA colocalizes the complete HCR initiator i1. In some cases, the colocalization of two (or more) fragment initiators forms a complete initiator that can trigger HCR signal amplification, for example, by binding to and opening one or more metastable HCR hairpin monomers. On the other hand, any individual probe that binds nonspecifically in the sample will not colocalize the complete HCR initiator i1 and will not trigger HCR, resulting in automatic background amplification. In some cases, using HCR immunohistochemistry (IHC), protein targets can be detected using an unlabeled primary antibody probe that is then detected by a fragment initiator secondary antibody probe with HCR initiator i1 that has the ability to trigger HCR signal amplification (Figures 48C, 50C, 50D, and 50F). 18 In some cases, the specimen is then 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 introduced into the sample and washed away (see, e.g., Figures 5B, 50B, 50D, 50E, and 50F). 15~19For example, in the detection stage, a probe can be added to the fixed sample and incubated to allow the probe to bind to the target, and then washing can be used to remove unused probe from the sample to reduce background caused by non-specific probe binding; in the subsequent amplification stage, an HCR amplification agent containing a fluorophore-labeled HCR hairpin can be added to the sample and incubated to allow HCR signal amplification, and then washing can be used to remove unused HCR hairpin from the sample to reduce background caused by non-specific hairpin binding (see, for example, Figures 5B, 50B, 50D, 50E, and 50F).

[0018] To avoid the use of enzymes to detect protein:protein target complexes, proximity-based HCR approaches have been developed that use a kinetic triggering mechanism to desequester the HCR initiator when the two probes are bound to the target proteins in close proximity. 13、20 (See, e.g., Figure 2.) To detect the target complex, two unlabeled primary antibodies bind to the two targets of interest, and two oligo-conjugated secondary antibody probes bind to the primary antibodies. One secondary antibody probe is conjugated to hairpin oligo 1, and one secondary antibody probe is conjugated to hairpin oligo 2. Hairpin oligo 1 sequesters a trigger capable of opening hairpin 2. Hairpin oligo 2 sequesters an HCR initiator. An additional activator strand is introduced, which binds to hairpin oligo 1, opening it to expose the trigger, and the trigger binds to hairpin oligo 2, opening it to expose the HCR initiator, allowing subsequent HCR signal amplification. This method has so far been limited to 1-plex applications.

[0019] HCR signal amplification has also been used to detect ribosome interactions with mRNA. 21This method uses an initiator-labeled bridge strand to link one nucleic acid probe bound to a ribosome with a second nucleic acid probe bound to mRNA, thereby detecting ribosomal RNA (rRNA):messenger RNA (mRNA) interactions (see, for example, Figure 3). Because the bridge strand has an intact HCR initiator, amplified background is generated if the bridge strand binds nonspecifically in the sample. This method has so far been limited to one-plex applications.

[0020] The systems disclosed herein address shortcomings in existing methods and, in some embodiments, enable enzyme-free, multiplexed, quantitative, high-resolution imaging of target complexes using HCR signal amplification. In some embodiments, fragmentary initiator probes are utilized such that when two or more fragmentary initiator probes in a probe pair bind to adjacent targets in a sample, additional proximity probes can simultaneously bind to the proximity domains on each fragmentary initiator probe to co-localize the complete HCR initiator. In some cases, the co-localized complete HCR initiator has the ability to trigger HCR signal amplification by opening a metastable HCR hairpin monomer, causing a chain reaction and polymerization of the HCR monomer. In some embodiments, when individual fragmentary initiator probes bind to isolated targets in a sample, the proximity probes can bind to the isolated fragmentary initiator probes, but the proximity probe binding does not result in complete HCR. The R initiators cannot colocalize and therefore do not generate an amplified signal at the site of the isolated target.

[0021] Cooperative probe junctions for proximity measurements in samples In some embodiments, a cooperative probe junction comprises two or more fragmentary initiator probes joined by proximity probes at their proximity domains (see, e.g., Figures 6, 7, 8, 9, 10, 11, 12, 14, 19A, 20, 21, 22, 23A, 24A, 25A, 26A, 27B, 48D, 49C, and 49D). In some embodiments, a cooperative probe junction is utilized to perform proximity measurements within a sample. In some embodiments, a fragmentary initiator probe comprises a fragmentary initiator (also known as an HCR fragmentary initiator), a proximity domain, and a target binding domain configured to directly bind to a target (see, e.g., Figures 6, 20, 21, 22, and 49C). In some embodiments, the fragmentary initiator probe comprises a fragmentary initiator (also known as an HCR fragmentary initiator), a proximity domain, and a target binding domain configured to indirectly bind to the target (see, e.g., Figures 7, 8, 9, 10, 11, 12, 13, 14, 19A, 21, 23A, 24A, 25A, 26A, 27B, 48D, and 49D). In some embodiments, the proximity domain is a sequence within the fragmentary initiator probe configured to be bound by a proximity probe. In some embodiments, the fragmentary initiator is a sequence within the fragmentary initiator probe that cannot trigger HCR signal amplification by itself, but can form a complete initiator capable of triggering HCR signal amplification when co-localized with one or more fragmentary initiators from one or more other fragmentary initiator probes via binding of the fragmentary initiator probe to one or more proximity probes.

[0022] In some embodiments, the first fragmentary initiator probe comprises a first target-binding domain configured to directly or indirectly bind to a first target, a first proximity domain, and a first fragmentary initiator; the second fragmentary initiator probe comprises a second target-binding domain configured to directly or indirectly bind to a second target, a second proximity domain, and a second fragmentary initiator; and the proximity probe is configured to bind to the first proximity domain and the second proximity domain (see, e.g., Figures 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 causes the first fragmentary initiator and the second fragmentary initiator to colocalize. In some embodiments, binding of the proximity probe to the first proximity domain and the second proximity domain causes the first fragmentary initiator and the second fragmentary initiator to co-localize to form a complete initiator (also known as a complete HCR initiator) capable of triggering HCR signal amplification.

[0023] In some embodiments, the first target and the second target are in sufficiently close proximity to each other that upon binding of the first fragmentary initiator probe to the first target and the second fragmentary initiator probe to the second target, the proximity probes can bind to both the first proximity domain of the first fragmentary initiator probe and the second proximity domain of the second fragmentary initiator probe to form a cooperative probe junction. In some embodiments, the first target and the second target are bound to each other in a target complex (see, e.g., Figure 7). In some embodiments, the first target and the second target are not in a complex with each other but are in a complex with a third target (see, e.g., Figure 9). In some embodiments, the first target and the second target are in proximity to each other but are not bound (see, e.g., Figure 10). In some embodiments, the first target and the second target are the same molecule (see, e.g., Figure 11).

[0024] In some embodiments, the first fragmentary initiator probe, the second fragmentary initiator probe , and the proximity probes form a cooperative probe junction (see, for example, Figures 27B and 48D). In some embodiments, triggering HCR occurs as a result of the formation of a cooperative probe junction and the co-localization of the first and second fragment initiators, and thus indicates that the first and second targets are in proximity to each other in the sample.

[0025] The spatial resolution of three-dimensional fluorescence images is diffraction limited to approximately 200 nm in the lateral direction and 500 nm in the axial direction. 22、23In some embodiments, upon proximity of two target molecules, the proximity probe binds to two fragmentary initiator probes, colocalizing the complete initiators and mediating the generation of an amplified signal with sub-diffraction-limited spatial resolution. In some embodiments, the proximity probe binds to two fragmentary initiator probes, colocalizing the complete initiators and mediating the generation of an amplified signal if and 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 fragmentary initiator probes, co-localizing the complete initiators and mediating the generation of an amplified signal if and only if the two targets are within 10 μm, or 5 μm, or 2 μm, or 1 μm, or 500 nm, or dimensions smaller than a eukaryotic cell, or dimensions smaller than a prokaryotic organism. In some embodiments, conditional on the proximity of two or more target molecules, one or more proximity probes bind to two or more fragmentary initiator probes, co-localizing the complete initiators and mediating the generation of an amplified signal with sub-diffraction-limited spatial resolution. In some embodiments, one or more proximity probes bind to two or more fragmentary initiator probes, allowing complete initiators to co-localize and mediate the generation of an amplified signal, if and only if 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 fragmentary initiator probes, allowing complete initiators to co-localize and mediate the generation of an amplified signal, if and only if two or more targets are within 10 μm, or 5 μm, or 2 μm, or 1 μm, or 500 nm, or dimensions smaller than a eukaryotic cell, or dimensions smaller than a prokaryotic organism.

[0026] In some embodiments, target complexes are detected using a three-stage protocol (see, e.g., Figures 46A and B): 1) In the detection stage, two unlabeled primary probes are provided to the sample to detect two targets. As shown, the primary probes may be antibody probes. Next, two secondary fragmentary initiator probes (p1 and p2) are added, each of which has a fragment of HCR initiator i1 and a proximity domain, and the secondary fragmentary initiator probes are specific for the unlabeled primary probes. In some embodiments, the fragmentary initiator probes may include a target binding domain configured to specifically bind to the primary probes, such as the antibody target binding domain shown. 2) In the proximity stage, a proximity probe is provided to the sample, which can bind to the fragmentary initiator component of the fragmentary initiator probe and colocalize them by forming a cooperative probe junction. 3) In the amplification stage, an HCR amplification agent containing a metastable labeled HCR hairpin is added to the sample, and in the presence of a co-localized complete HCR initiator, the metastable labeled HCR hairpin self-assembles into a linked labeled HCR amplification polymer. In some embodiments, the metastable labeled HCR hairpin is fluorophore-labeled. In some embodiments, in the proximity stage, when two fragmented initiator probes are in sufficiently close proximity, a cooperative probe junction is formed.

[0027] In some embodiments, the use of fragment initiator probes during the detection stage, proximity probes during the proximity stage, and metastable HCR hairpins during the amplification stage provides automatic background suppression throughout the protocol, ensuring that reagents do not generate amplified background even if they bind nonspecifically in the sample. Any primary or secondary probes that bind nonspecifically in the sample during the detection stage will not colocalize complete HCR initiators and will not initiate or trigger HCR by themselves. In some embodiments, even if the primary and secondary fragment initiator probes specifically bind to their targets in the detection stage, the fragment initiators will not form complete HCR initiators to initiate HCR in the amplification stage in the absence of proximity probes and cooperative probe junction formation provided during the proximity stage. In some embodiments, even if the primary and secondary fragment initiator probes specifically bind to their targets in the detection stage and proximity probes are provided during the proximity stage, the fragment initiators will not form complete HCR initiators to initiate HCR in the amplification stage if the fragment initiators are not in sufficiently close proximity. Similarly, any proximity-probes that bind nonspecifically in the sample during the proximity stage lack the ability to initiate HCR by themselves, because proximity-probes can only mediate HCR signal amplification during the amplification stage if they specifically bind to both fragmentary initiator probes, colocalizing the complete HCR initiator. Any HCR hairpins that bind nonspecifically in the sample during the amplification stage are kinetically trapped and do not trigger the formation of HCR-amplified polymers.

[0028] In some embodiments, each cooperative probe junction includes a fragmentary initiator probe P1, which includes a nucleic acid comprising a target-binding domain and a fragmentary initiator nucleic acid sequence and a proximity domain nucleic acid sequence; a fragmentary initiator probe P2, which includes a nucleic acid comprising a target-binding domain and a fragmentary initiator nucleic acid sequence and a proximity domain nucleic acid sequence; and a proximity probe, which includes a first nucleic acid sequence complementary to the proximity domain of P1 and a second nucleic acid sequence complementary to the proximity domain P2. In some embodiments, when the proximity probes bind, they bring the fragmentary initiator sequences into proximity, so that they form a complete HCR initiator capable of triggering HCR signal amplification. In some embodiments, the cooperative probe junction may optionally include additional fragmentary initiator probes Px (where x is an integer) to detect the proximity of additional targets.

[0029] In some embodiments, the cooperative probe junction colocalizes complete HCR initiators used to generate a signal using HCR (see, e.g., Figures 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 cooperative probe junction colocalizes complete HCR initiators (also known as complete initiators) used to generate a signal using a method other than HCR. In some embodiments, the cooperative probe junction colocalizes complete initiators used to generate a signal using complementary fluorescent strands, branched DNA (bDNA) methods, enzymatic methods, RCA methods, catalyzed reporter deposition (CARD), and / or another signal generation method.

[0030] In some embodiments, the target binding domain of the fragment initiator 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, RNA, DNA, 2'OMe-RNA, PNA, XNA, any other material capable of base pairing, carbon atoms, a chemical linker without base pairing ability, or any combination thereof. In some embodiments, the target binding domain has the ability to bind to a desired target. In some embodiments, In some embodiments, the target-binding domain may bind directly to the target itself. In some embodiments, the target-binding domain may bind indirectly to the target, for example, 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.

[0031] 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 cooperative probe junction can generate a signal in the 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.

[0032] In some embodiments, a fragmentary initiator probe can be used to detect the proximity of three or more targets in a sample. In some embodiments, two or more fragmentary initiator 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 fragmentary initiator probes can be used in combination. In some embodiments, the first fragmentary 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 fragmentary initiator; the second fragmentary 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 fragmentary initiator; the third fragmentary 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 fragmentary initiator; and the 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, binding of the proximity probe to the first proximity domain, the second proximity domain, and the third proximity domain causes the first fragmentary initiator, the second fragmentary initiator, and the third fragmentary initiator to co-localize. In some embodiments, binding of the proximity probe to the first proximity domain, the second proximity domain, and the third proximity domain causes the first fragmentary initiator, the second fragmentary initiator, and the third fragmentary initiator to co-localize into a complete HCR initiator capable of triggering HCR signal amplification.

[0033] In some embodiments, two or more different proximity probes can be used to co-localize three or more fragmentary initiator probes. In some embodiments, the first fragmentary initiator probe comprises a first target binding domain, a first proximity domain, and a first fragmentary initiator, configured to directly or indirectly bind to a first target; the second fragmentary initiator probe comprises a second target binding domain, a second proximity domain, and a second fragmentary initiator, configured to directly or indirectly bind to a second target; the third fragmentary initiator probe comprises a third target binding domain, a third proximity domain, and a third fragmentary initiator, configured to directly or indirectly bind to a third target; the first proximity probe is configured to bind to the first proximity domain and the second proximity domain; and the second proximity probe is configured to bind to the second proximity domain and the third proximity domain (see, for example, Figure 13). In some embodiments, binding of the first proximity probe to the first proximity domain and the second proximity domain, and binding of the second proximity probe to the second proximity domain and the third proximity domain, causes the first fragmentary initiator, the second fragmentary initiator, and the third fragmentary initiator to co-localize. In some embodiments, the co-localized first fragmentary initiator, the second fragmentary initiator, and the third fragmentary initiator form a complete HCR initiator capable of triggering HCR signal amplification.

[0034] In some embodiments, the target binding domain of the fragmentary initiator probe comprises an antibody that directly or indirectly binds to a protein in a sample (see, e.g., Figures 14, 19A, 21, 46A, 48D, 49C, and 49D). In some embodiments, the target binding domain of the fragmentary initiator probe comprises a nanobody that directly or indirectly binds to a protein in a sample (see, e.g., Figure 20). In some embodiments, the cooperative probe junction 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 (see, e.g., Figures 14, 46A, 48D, 49C, and 49D). In some embodiments, the sample comprises cells (see, e.g., Figures 15, 17, 18, and 19B, 45A, and 45B). In some embodiments, the sample is formalin-fixed, paraffin-embedded (FFPE) tissue (see, e.g., Figures 16, 45C, and 45D). In some embodiments, one or more cooperative probe junctions are utilized to simultaneously detect one or more protein:protein target complexes in a sample (see, e.g., Figures 17 and 46B). In some embodiments, two or more cooperative probe junctions are utilized to simultaneously detect two or more protein:protein target complexes in a sample in a multiplex experiment (see, e.g., Figures 17 and 46B). In some embodiments, a cooperative probe junction comprising two or more fragmentary initiator probes and one or more proximity probes is used to simultaneously detect one or more target complexes with one or more protein targets, and / or one or more RNA targets, and / or one or more DNA targets in a multiplex experiment (see, e.g., Figures 18 and 47). In some embodiments, the cooperative probe junction generates quantitative signal intensities (see, e.g., Figure 19C). In some embodiments, one or more cooperative probe junctions are utilized to detect one or more RNA:protein complexes in a sample (see, e.g., Figure 21).In some embodiments, one or more cooperative probe junctions are utilized to detect one or more RNA:RNA complexes (see, e.g., FIG. 22), or one or more DNA:protein complexes, or one or more DNA:protein:protein complexes, or complexes of three or more RNA, DNA, and / or protein molecules, and / or other molecules in a sample. In some embodiments, a cooperative probe junction comprises two or more fragmentary initiator probes and one or more proximity probes. In some embodiments, a cooperative probe junction comprises three or more fragmentary initiator probes and two or more proximity probes.

[0035] In some embodiments, the proximity probes comprise one or more clamp domains configured to bind to one or more constituent parts of the fragmentary initiator when only one fragmentary initiator probe is bound to the proximity probe, thereby suppressing the generation of amplified background (in the form of undesired HCR signal amplification) in the absence of binding of both fragmentary initiator probes to the proximity probe, and further configured such that binding of both fragmentary initiator probes to the proximity probe co-localizes an entire HCR initiator capable of triggering HCR signal amplification. In some embodiments, the proximity probes comprise one or more clamp domains configured to bind to the entire first fragmentary initiator and / or second fragmentary initiator in the absence of one of the targets in the target complex (see, e.g., Figures 23 and 44A-44C). In some embodiments, the proximity probe comprises one or more clamp domains configured to bind to the 5' end of the first fragmentary initiator and / or the 5' end of the second fragmentary initiator in the absence of one of the targets in the target complex (see, e.g., Figure 24). In some embodiments, the proximity probe comprises one or more clamp domains configured to bind to the 3' end of the first fragmentary initiator and / or the 3' end of the second fragmentary initiator in the absence of one of the targets in the target complex. In some embodiments, the proximity probe comprises one or more clamp domains configured to bind to the 5' and 3' ends of the first fragmentary initiator and / or the 5' and 3' ends of the second fragmentary initiator in the absence of one of the targets in the target complex (see, e.g., Figure 26). In some embodiments, the clamp domains can be complementary to the fragmentary initiator domains. In some embodiments, the proximity probe does not comprise a clamp domain configured to bind to some or all of the first fragmentary initiator and / or the second fragmentary initiator in the absence of one of the targets in the target complex (see, e.g., Figures 6, 7, 8, 9, 10, 11, 12, 13, 14, 19A, 20, 21, 22).

[0036] In some embodiments, when the proximity probe binds to all of the proximity domains in the adjacent fragmentary initiator probe and co-localizes the complete HCR initiator, the HCR hairpin monomer (also known as the HCR hairpin and HCR monomer) can polymerize. In some embodiments, the HCR hairpin monomer can contain a fluorophore, chromophore, lumophore, phosphor, FRET pair, or other label that allows the formed polymer to be detected. In some embodiments, the signal is read using a fluorescence microscope, a fluorescence scanner, a camera, a mobile phone camera, a mass spectrometer, a mass spectrometry microscope, a radiation scanner, or another device suitable for detecting the signal. In some embodiments, the signal generating molecule comprises a fluorophore, a chromophore, a luminophore, 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), carbon nanotubes, magnetized carbon nanotubes, gold nanoparticles (AuNPs), gold nanoshells, gold nanorods, silver coated gold nanoparticles, latex, magnetic nanoparticles, silica nanoparticles, a fluorophore, a fluorophore-loaded nanoparticle, a dye-loaded nanoparticle, an enzyme, any combination thereof, or any other molecule that facilitates measurement of a signal. In some embodiments, a hapten, ligand, oligonucleotide, digoxigenin (DIG), fluorescein isothiocyanate (FITC), fluorophore, biotin, dinitrophenol, aniline, enzyme, or another molecule or complex that can be recognized by a binding partner is utilized to facilitate signal generation.

[0037] Signal Generation 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 is 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 signal strength. 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 direct the generation of a signal.

[0038] Hybridization chain reaction (HCR) signal amplification In some embodiments, the HCR amplifier comprises two or more HCR hairpins (see, for example, the HCR amplifiers of Figures 5A-5B, 37A-37F, and 38A-38B). In 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 stem section, and an output domain having a single-stranded loop and a complement to the stem section (see, e.g., the HCR hairpins in Figures 5A-5B, 39, 40, 37A-37F, and 38A-38B).

[0039] In some embodiments, the target is detected using a signal probe set comprising one or more initiator-labeled probes, each comprising a target binding domain and an amplification domain comprising one or more HCR initiators (e.g., Figures 28A-28N and 29A-92F). In some embodiments, the target is detected in a sample using a signal probe set comprising one or more probe units (see, e.g., the probe sets in Figures 5A-5B and 30A-30D), where the probe units comprise two or more fragmentary initiator probes (see, e.g., the probe units in Figures 31A-31E, 32A-32D, 33A-33E, and 34A-34C), each of which comprises a target binding domain and an amplification domain comprising a fragmentary initiator (see, e.g., the fragmentary initiator probes in Figures 35 and 34A-34C). In some embodiments, binding of each probe within a probe unit to adjacent cognate binding sites on the target causes the fragment initiators to colocalize to form a complete HCR initiator (see, e.g., Figures 5A-5B, 31A-31E, 32A-32D, 33A-33E, 30A-30D, and 36 complete HCR initiators), which are capable of triggering HCR signal amplification. In some embodiments, each fragment initiator probe within a probe unit further comprises a proximity domain (see, e.g., Figures 6, 7, 8, 9, 10, 11, 12, 13, 46A). In some embodiments, binding of each fragmentary initiator probe within a probe unit to its cognate binding site on a nearby target allows one or more proximity probes to bind to proximity domains within the probe unit, colocalizing the fragmentary initiators to form a complete HCR initiator (see, e.g., Figures 6, 7, 8, 9, 10, 11, 12, 13, 46A), which has the ability to trigger HCR signal amplification.

[0040] In some embodiments, one or more HCR initiators on the initiator-labeled probe each initiate a chain reaction of polymerization steps, in which the initiator hybridizes to the input domain of a first HCR hairpin, opening the first hairpin to expose its output domain, which then hybridizes to the input domain of a second HCR hairpin, opening the second hairpin to expose its output domain, and so on, leading to a chain reaction in which the hairpins polymerize to yield target-linked HCR amplification polymers (see, for example, the amplification polymers in Figures 39, 40, 38A-38B, 41A, 41C-41E, and 42A). In some embodiments, in the absence of complete HCR initiators, the HCR hairpins are kinetically trapped and do not polymerize, resulting in reduced background. However, when the fragmentary initiator probes within a probe unit bind to their adjacent cognate binding sites on the target and colocalize the complete HCR initiator (see, e.g., Figures 5A, 31A-31E, 32A-32D, 37C-37F), or when the proximity domains within the fragmentary initiator probes within a probe unit bind to proximity probes and colocalize the complete HCR initiator (see, e.g., Figures 6, 7, 8, 9, 10, 11, 12, 13, 46A), the complete HCR initiator initiates a chain reaction of polymerization steps, and in this chain reaction, In this case, the intact initiator hybridizes to the input domain of the first HCR hairpin, opening the first hairpin to expose its output domain, which then hybridizes to the input domain of the second HCR hairpin, opening the second hairpin to expose its output domain, and so on, leading to a chain reaction in which the hairpins polymerize to yield linked HCR amplified polymers (see, e.g., the amplified polymers in Figures 5A, 37C-37F, 38A-38B, and 46A).

[0041] In some embodiments, the HCR hairpin further comprises zero or more reporters that directly or indirectly guide the generation of an amplified signal (see, e.g., Figures 37A-37F). In some embodiments, the zero or more reporters on the HCR hairpin serve to mediate an additional layer of signal amplification via catalyzed reporter deposition (CARD) (see, e.g., Figures 41A-41E, 42A-42C). In some embodiments, the reporters on the HCR hairpin comprise fragmentary reporters such that auxiliary reporter-labeled readout probes do not bind strongly to fragmentary reporters on individual hairpins, but after HCR polymerization, adjacent hairpins in the HCR-amplified polymer colocalize the complete reporters, resulting in strong binding of the colocalized complete reporters to the auxiliary reporter-labeled readout probes (see, e.g., Figures 37D and 43B). In some embodiments, the readout probe comprises one or more auxiliary reporters and further comprises a reporter binding domain configured to bind to a reporter on the HCR-amplified polymer or to a co-localized intact reporter within the HCR-amplified polymer (see, e.g., readout probes in Figures 43A-43B). In some embodiments, the amplified signal is generated by one or more reporters or auxiliary reporters associated with the HCR-amplified polymer linked to the target in the sample. In some embodiments, the signal is removed. In some embodiments, the HCR signal is generated, detected, and removed one or more times.

[0042] In some embodiments, HCR signal amplification increases 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 range value defined by any two of the above values.

[0043] HCR initiator. In some embodiments, the co-localized complete initiator comprises two or more fragment initiators brought into proximity by proximity probes. In some embodiments, the complete initiator is perfectly complementary to the input domain of the HCR hairpin, such that it hybridizes to the input domain of the hairpin, opening the hairpin and initiating the HCR polymerization cascade. In some embodiments, the complete initiator is partially complementary to the input domain of the HCR hairpin, but is sufficiently complementary, such that it hybridizes to the input domain of the hairpin, opening the hairpin and initiating the HCR polymerization cascade. In some embodiments, the complete initiator is shorter or longer than the input domain of the HCR hairpin and / or has incomplete complementarity to the input domain of the hairpin, but is still able to hybridize to the input domain of the hairpin, opening the hairpin and initiating the HCR polymerization cascade. In some embodiments, the complete initiator may have 60%, 70%, 80%, 90%, or 100% complementarity to the input domain of the HCR hairpin (or any intermediate value between any of these values) and may hybridize to the input domain of the hairpin to open the hairpin and initiate the HCR polymerization cascade. In some embodiments, the fragmentary initiator is shielded by base pairing to reduce nonspecific binding of the probe within the sample. In some embodiments, the fragmentary initiator may be shielded by the hairpin structure. In some embodiments, the fragmentary initiator may be shielded by one or more auxiliary oligos. In some embodiments, the fragmentary initiator may be shielded by self-complementarity within the probe and / or complementarity to one or more auxiliary strands.

[0044] Automatic background suppression using HCR fragmental initiator probes. In some embodiments, fragmental initiator probes automatically suppress background because the HCR initiator is split between the pair of probes. In some embodiments, when a fragmental initiator probe specifically binds to a target and to a proximity probe in close proximity, The proximity probe allows the two fragmentary initiators within the probe pair to co-localize to form a complete HCR initiator. In some embodiments, non-specifically binding individual fragmentary initiator probes do not trigger HCR because each probe contains only a fragment of the HCR initiator, and HCR signal amplification is triggered only when the complete HCR initiator is co-localized.

[0045] Automatic background suppression using HCR hairpins. In some embodiments, HCR hairpins automatically suppress background because they are kinetically trapped and therefore do not polymerize in the absence of an HCR initiator. In some embodiments, when both probes in a fragmentary initiator probe pair specifically bind to their adjacent cognate binding sites on the target (see, e.g., FIG. 5A) and / or on a proximity probe (see, e.g., FIG. 46A), the resulting colocalized complete HCR initiator triggers the growth of linked HCR amplification polymers. In some embodiments, non-specifically binding individual HCR hairpins do not trigger HCR because they are kinetically trapped.

[0046] Automatic background suppression using HCR fragment-like initiator probes and HCR hairpins. In some embodiments, the combination of HCR fragment-like 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 they bind nonspecifically.

[0047] A complete HCR initiator is formed by the co-localization of two or more fragmentary initiator probes. Each set of fragmentary initiator probes that makes up a complete HCR initiator is referred to as a probe unit (see, for example, Figure 34). In some embodiments, a complete HCR initiator is made up of a pair of fragmentary initiator probes, each of which has a fragment of the complete HCR initiator, so that together they make up the complete HCR initiator (fragment f1 for probe P1 and fragment f2 for probe P2; f1 + f2 = 1); in this case, a probe unit is two fragmentary initiator probes (see, for example, Figure 34A). In some embodiments, fragments f1 and f2 are sufficiently small compared to the complete HCR initiator (e.g., f1=0.5 and f2=0.5; or f1=0.45 and f2=0.55; or f1=0.4 and f2=0.6; or f1=0.3 and f2=0.7), such that HCR signal amplification is suppressed when the complete HCR initiator is not co-localized with the target.

[0048] In some embodiments, the HCR initiator (i1 or i2) is divided among three fragmentary initiator probes (fragment f1 for probe P1, fragment f2 for probe P2, fragment f3 for probe P3; f1+f2+f3=1); in this case, the probe unit consists of three fragmentary initiator probes. In some embodiments, the HCR initiator (i1 or i2) is divided among N (N=2, 3, or 4 or more) fragmentary initiator probes (fragment f1 for probe P1, fragment f2 for probe P2, ..., fragment fN for probe P N; f1+f2+...fN=1; see, e.g., Figure 15B); in this case, the probe unit consists of N fragmentary initiator probes. In some embodiments, for any of these values ​​of N, HCR signal amplification is suppressed when the complete HCR initiator is not co-localized with the target.

[0049] In some embodiments, the complete HCR initiator is generated by co-localization of a pair (or set) of probes, each of which carries a fragment of the HCR initiator, such that the sum (f1+f2) of fragment f1 for probe P1 and fragment f2 for probe P2 is sufficiently close to 1 (e.g., f1=0.47, f2=0.47, f1+f2=0.94; or f f1=0.44, f2=0.42, f1+f2=0.86), such that HCR signal amplification is triggered by the co-localized complete initiator resulting from the binding of the probe pair to their cognate binding sites on the adjacent target and the binding of their proximity domains to the proximity probe. In some embodiments, the fragmentary initiator probe within the probe unit generates a complete HCR initiator corresponding to 100% of the HCR initiator. In some embodiments, the fragmentary initiator probe within the probe unit generates sufficient HCR initiator fragments to provide efficient HCR signal amplification compared to the rate of signal amplification when the fragmentary initiator probe is absent or when individual fragmentary initiator probes are present but not co-localized. In some embodiments, the complete HCR initiator fragment generated by the co-localized probe within the probe unit is 99%, 95%, 90%, 80%, or 60% of the complete HCR initiator (including any range greater than any one of the preceding values, or defined between any two of the preceding values). In some embodiments, a probe unit comprises two, three, four, or five or more fragmentary initiator probes. In some embodiments, the fragmentary initiators in a probe unit are sufficient to function as HCR initiators when the probes in the probe unit are co-localized by binding to their cognate binding sites on adjacent targets and binding their proximity domains to proximity probes. In some embodiments, the HCR initiators may have a sequence of a specific length (e.g., 15 nucleotides), but the fragmentary initiators in a probe unit do not need to be exactly the same length. For example, in some embodiments, their combined length can be 14 or 13 nucleotides, provided that they still function as HCR initiators when co-localized.

[0050] In some embodiments, any two or more fragmentary initiators can be used as long as together they provide the functionality of an HCR initiator.

[0051] In some embodiments, the complete HCR initiator is generated by colocalization of a pair of probes, each of which has a fragment of the HCR initiator further comprising one, two, three, or several sequence modifications, and the sum (f1 + f2) of fragment f1 for probe P1 and fragment f2 for probe P2 is sufficiently close to 1 (e.g., f1 = 0.45, f2 = 0.47, f1 + f2 = 0.92), such that HCR signal amplification is triggered by the colocalized complete initiator resulting from binding of the pair of probes to their cognate binding sites on the adjacent target and proximity probe. In some embodiments, the fragmentary initiator probes within the probe unit generate a complete HCR initiator that has 100% sequence identity with the HCR initiator. In some embodiments, the fragmentary initiator probes within the probe unit generate sufficient sequence identity to the HCR initiator to enable efficient HCR signal amplification compared to the rate of signal amplification when the fragmentary initiator probes are absent or when individual fragmentary initiator probes are present but not colocalized. In some embodiments, the complete HCR initiator generated by the co-localized probes within the probe unit has 99%, 95%, 90%, 80%, or 60% sequence identity with the HCR initiator (including any range defined above any one of the preceding values ​​or between any two of the preceding values).

[0052] HCR amplification agent having two hairpins. In some embodiments, the HCR amplification agent comprises two hairpin nucleic acids (h1 and h2) (see, e.g., Figures 5A-5B and 37A-37F). In some embodiments, each hairpin comprises an input domain with a single-stranded toehold and stem section, and an output domain with a single-stranded loop and a complement to the stem section. In the absence of an HCR initiator (i1 or i2), hairpins h1 and h2 coexist metastably, i.e., they are kinetically trapped and do not polymerize.

[0053] Initiation with initiator i1. In some embodiments, the complete initiator i1 (1050) formed from the colocalization of two or more fragment initiators comprises a domain complementary to the toehold (1851) of hairpin h1 and a domain complementary to the stem section (1755) of h1 (see, for example, Figure 39). In some embodiments, when the h1 hairpin (1510) encounters the complete initiator i1 (1050), the complete initiator i1 hybridizes to the input domain (1852) of hairpin h1 via toehold-mediated strand displacement, opening hairpin h1 (1510) to expose the output domain (1854) of hairpin h1 and forming the complex i1-h1. In some embodiments, the output domain (1854) of hairpin h1 comprises a domain complementary to the toehold (1951) of hairpin h2 and a domain complementary to the stem section (1855) of h2. In some embodiments, when h2 hairpin (1610) encounters the i1-h1 complex, the exposed output domain (1854) of h1 hybridizes to the input domain (1952) of hairpin h2 via toehold-mediated strand displacement, opening hairpin h2 to expose the output domain (1854) of hairpin h2 and forming the complex i1-h1-h2. In some embodiments, the output domain (1854) of hairpin h2 comprises a domain complementary to the toehold (1851) of hairpin h1 and a domain complementary to the stem section (1755) of h1. In some embodiments, when h1 hairpin (1510) encounters an i1-h1-h2 complex, the exposed output domain (1854) of h2 hybridizes to the input domain (1852) of hairpin h1 via toehold-mediated strand displacement, opening hairpin h1 (1510) to expose the output domain (1854) of hairpin h1 and forming the complex i1-h1-h2-h1. In some embodiments, this polymerization process can be repeated with alternating h1 and h2 polymerization steps to produce a polymer of the form i1-h1-h2-h1-h2-h1-h2-..., which is i1-(h1-h2) for a polymer incorporating N alternating copies of hairpins h1 and h2.N For example, a polymer may incorporate 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, the polymer can be terminated with either h1 or h2, such that i1-(h1-h2) N -h1 and i1-(h1-h2) N -h1-h2 are both possible, the latter is i1-(h1-h2) N+1 is equivalent to

[0054] Initiation with initiator i2. In some embodiments, the complete initiator i2 formed from the colocalization of two or more fragmentary initiators comprises a domain complementary to the toehold of hairpin h2 and a domain complementary to the stem section of h2. In some embodiments, when 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 hairpin h2 to expose the output domain of hairpin h2 and forming the complex i2-h2. In some embodiments, when 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 hairpin h1 to expose the output domain of hairpin h1 and forming the complex i2-h2-h1. In some embodiments, when the h2 hairpin encounters an i2-h2-h1 complex, the exposed output domain of h1 hybridizes to the input domain of hairpin h2 via toehold-mediated strand displacement, opening hairpin h2 to expose the output domain of hairpin h2 and forming the complex i2-h2-h1-h2. In some embodiments, this polymerization process can be repeated with alternating h2 and h1 polymerization steps to produce polymers of the form i2-h2-h1-h2-h1-h2-h1..., which is called i2-(h1...) for polymers incorporating N alternating copies of h2 and h1. h2-h1) N For example, a polymer may incorporate 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, the polymer can be terminated with either h1 or h2, such that i2-(h2-h1) N -h2 and i2-(h2-h1) N -h2-h1 are both possible, the latter is i2-(h2-h1) N+1 is equivalent to

[0055] HCR amplifier with four hairpins. In some embodiments, the HCR amplifier can contain more than two hairpins. For example, the HCR amplifier can contain four hairpins: h1, h2, h3, and h4 (see, for example, Figures 38A and 38B). In some embodiments, as with a two-hairpin HCR, each hairpin contains an input domain containing a single-stranded toehold and stem section, and an output domain containing a complement to the single-stranded loop and stem section. In some embodiments, in the absence of a complete HCR initiator (i1, i2, i3, or i4), hairpins h1, h2, h3, and h4 coexist metastably, i.e., they are kinetically trapped and do not polymerize. In some embodiments, the output domain of hairpin h1 comprises a domain complementary to the toehold of hairpin h2 and a domain complementary to the stem section of h2; the output domain of hairpin h2 comprises a domain complementary to the toehold of hairpin h3 and a domain complementary to the stem section of h3; the output domain of hairpin h3 comprises a domain complementary to the toehold of hairpin h4 and a domain complementary to the stem section of h4; and the output domain of hairpin h4 comprises a domain complementary to the toehold of hairpin h1 and a domain complementary to the stem section of h1. In some embodiments, complete initiator i1 formed from the colocalization of two or more fragment initiators comprises a domain complementary to the toehold of hairpin h1 and a domain complementary to the stem section of h1; complete initiator i2 formed from the colocalization of two or more fragment initiators comprises a domain complementary to the toehold of hairpin h2 and a domain complementary to the stem section of h2; complete initiator i3 formed from the colocalization of two or more fragment initiators comprises a domain complementary to the toehold of hairpin h3 and a domain complementary to the stem section of h3; and complete initiator i4 formed from the colocalization of two or more fragment initiators comprises a domain complementary to the toehold of hairpin h4 and a domain complementary to the stem section of h4.In some embodiments, similar to the case of two-hairpin HCR, when hairpin h1 encounters complete initiator i1, complete initiator i1 opens hairpin h1 to form complex i1-h1 exposing the h1 output domain, the h1 output domain then opens hairpin h2 to form complex i1-h1-h2 exposing the h2 output domain, the h2 output domain then opens hairpin h3 to form complex i1-h1-h2-h3 exposing the h3 output domain, and the h3 output domain then opens hairpin h4 to form complex i1-h1-h2-h3 exposing the h4 output domain. The h1 output domain exposes the complex i1-h1-h2-h3-h4, and the h4 output domain then opens the hairpin h1 to form the complex i1-h1-h2-h3-h4-h1 with the h1 output domain exposed, and so on, leading to polymerization through alternating h1, h2, h3, and h4 polymerization steps to produce a polymer of the form i1-h1-h2-h3-h4-h1-h2-h3-h4-h1-h2-h3-h4..., which is i1-(h1-h2-h3-h4) for a polymer incorporating N alternating copies of h1, h2, h3, and h4. N In some embodiments, the polymer can be terminated with h1, h2, h3, or h4, so that i1-(h1-h2-h3-h4) N -h1, i1-(h1-h2-h3-h4) N -h1-h2, i1-(h1-h2-h3-h4) N -h1-h2-h3, and i1-(h1-h2-h3-h4) N -h1-h2-h3-h4 are all possible, the latter being i1-(h1-h2-h3-h4) N+1 is equivalent to In some embodiments, HCR polymerization can be triggered by any of the cognate intact initiators (i1, i2, i3, or i4). For example, initiation by intact initiator i3 results in a polymerization of the form i3-(h3-h4-h1-h2). NIn some embodiments, an HCR amplification agent with four hairpins is advantageous for generating a signal that is present in the polymeric state but not in the unpolymerized state (e.g., Figure 19B illustrates a FRET pair that colocalizes and generates a FRET signal only when the hairpins are colocalized within the amplification polymer; this provides the basis for a wash-free method, as unused hairpins that are not washed from the sample do not participate in FRET and thus avoid background generation).

[0056] HCR amplifiers having two or more hairpins. More generally, in some embodiments, an HCR amplifier may comprise M HCR hairpins (h1, h2, ..., hM), where M is an integer greater than or equal to 2. In the absence of a complete HCR initiator (i1, i2, ..., iM) formed from the colocalization of two or more fragmentary initiators, hairpins h1, h2, ..., hM coexist metastably, i.e., they are kinetically trapped and do not polymerize. In the presence of a cognate complete HCR initiator formed from the colocalization of two or more fragmentary initiators, polymerization occurs via alternating polymerization steps similar to two-hairpin or four-hairpin HCR. For example, complete initiator i1 may be of the form i1-(h1-h2-...-hM) for a polymer incorporating N alternating copies of h1, h2, ..., hM. N The polymer can terminate at h1, h2, ..., hM, so that i1-(h1-h2-...-hM) N -h1, i1-(h1-h2-...-hM) N -h1-h2, ..., and i1-(h1-h2-...-hM) N -h1-h2-...-hM are all possible, the latter being i1-(h1-h2-...-hM) N+1 The HCR polymerization can be triggered by any of the cognate intact initiators (i1, i2, ..., iM). For example, initiation by intact initiator i3 occurs in the form i3-(h3-...-hM-h1-h2). N The polymer may be produced.

[0057] Reporter-labeled HCR hairpins. For a given HCR amplification agent, each HCR hairpin contains zero or more reporters. The reporters on different hairpins within the amplification agent may be the same or different. For example, an amplification agent containing hairpins h1 and h2 may have: 1) the same reporter on h1 and h2, 2) different reporters on h1 and h2, 3) a reporter on h1 but not on h2, 4) a reporter on h2 but not on h1, 5) no reporter on either h1 or h2, or 6) zero or more reporters on h1, zero or more of which may be the same as or different from zero or more reporters on h2. Similarly, for an HCR amplification agent containing hairpins h1, h2, h3, and h4, each hairpin may contain zero or more reporters (e.g., 3, 5, or 10 reporters), zero or more of which may be the same as zero or more reporters in each of the other hairpins. In some embodiments, one or more of the reporters for a given hairpin can be unique within the mixture of hairpins and / or hairpin reporters, hi some embodiments, there are 1, 10, 100, 1000, 10,000, or 100,000 or more unique reporters (including any range defined between any two of the preceding numbers) within the mixture.

[0058] In some embodiments, one or more reporters on the reporter-labeled HCR hairpin directly or indirectly contribute to the generation, modification, or elimination of a signal. For example, the reporter may be a fluorophore, a chromophore, a luminophore, 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 compound that directly or indirectly facilitates the measurement of a signal. It can be a molecule.

[0059] In some embodiments, the reporter decorating the linked HCR amplification polymer may bind to the reporter binding domain of the readout probe and directly or indirectly mediate the localization of an auxiliary reporter in the vicinity of the reporter, which may then directly or indirectly mediate the generation of an amplified signal. For example:

[0060] In some embodiments, the reporter can comprise an anti-digoxigenin (DIG) that recruits an anti-DIG antibody as a readout probe, and the anti-DIG is directly labeled with one or more auxiliary reporters or with one or more reporters that serve to directly or indirectly mediate the localization of the auxiliary reporter in the vicinity of the reporter.

[0061] In some embodiments, the reporter can include a nucleic acid domain that serves as a substrate with complete or partial sequence complementarity to a reporter binding domain in a readout probe with one or more auxiliary reporters (see, e.g., Figure 37C).

[0062] In some embodiments, the reporter may comprise a nucleic acid domain that serves as a substrate having full or partial sequence complementarity to a reporter binding domain in the readout probe with one or more substrates that serve to mediate localization of an auxiliary reporter in the vicinity of the reporter.

[0063] In some embodiments, a reporter can include a nucleic acid domain that serves as a substrate for a readout probe that directly or indirectly mediates the localization of an auxiliary reporter in the vicinity of the reporter.

[0064] In some embodiments, the reporter may comprise a substrate that serves to recruit a readout probe that indirectly mediates the localization of an auxiliary reporter in the vicinity of the reporter.

[0065] In some embodiments, the reporter can include a substrate that serves to recruit a readout probe that includes an enzyme that mediates catalyzed reporter deposition (CARD) in the vicinity of the reporter (see, for example, Figure 43A).

[0066] In some embodiments, the reporter can include biotin that recruits streptavidin (or another biotin-binding molecule) as a readout probe, and the streptavidin is directly labeled with one or more auxiliary reporters or with one or more substrates that serve to directly or indirectly mediate the localization of the auxiliary reporter in the vicinity of the reporter.

[0067] In some embodiments, the reporter can comprise a hapten that recruits an anti-hapten antibody or nanobody readout probe that directly or indirectly mediates localization of the reporter in its vicinity via CARD signal amplification. For example, the anti-hapten antibody or nanobody readout probe can comprise an enzyme that mediates CARD (see, e.g., Figures 41A-41E).

[0068] In some embodiments, the reporter can include a hapten that recruits an anti-hapten that directly or indirectly mediates localization of an auxiliary reporter in the vicinity of the reporter. For example, an anti-hapten readout probe can include an enzyme that mediates CARD (see, e.g., Figures 42A-42C).

[0069] In some embodiments, the reporter can include an enzyme that mediates CARD signal amplification and deposits a CARD reporter molecule in the vicinity of the hairpin.

[0070] In some embodiments, a reporter can include zero or more haptens that directly or indirectly mediate the localization of an auxiliary reporter in the vicinity of the hapten (see, eg, Figures 37A-37B).

[0071] In some embodiments, the reporter can include a hapten that recruits an anti-hapten (eg, an antibody, nanobody, streptavidin, or another molecule) labeled with an auxiliary reporter.

[0072] In some embodiments provided herein, HCR signal amplification is used to mediate catalyzed reporter deposition (CARD) to achieve higher signal gains. In some embodiments, the higher signal gains are about 5, 10, 15, 20, 25, 30, 40, 50, 75, 100, 500, 1000, 2000, 5000, 10,000, 20,000, 50,000, or 100,000 times, or a range defined by any two of the above values.

[0073] Haptens and anti-haptens. In some embodiments, the hairpin label, which is a substrate containing 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 molecule complex that selectively binds to a hapten.

[0074] Enzymes for HCR-mediated Catalytic Reporter Deposition (CARD). In some embodiments, reporter-decorated HCR amplification polymers mediate signal amplification via catalytic reporter deposition (CARD) by enzymes that catalyze a CARD substrate, leading to the deposition of a CARD reporter in the vicinity of the HCR amplification polymer (see, e.g., Figures 41A-41E, 42A-42C, 43A-43B). For example:

[0075] In some embodiments, the enzyme can be horseradish peroxidase (HRP) (or a polymeric HRP comprising multiple HRP enzymes) that acts on a CARD substrate to catalyze the deposition of a chromogenic CARD reporter, such as AEC, DAB, TMB, or StayYellow, or that catalyzes the deposition of a fluorescent CARD reporter, such as a fluorophore-labeled tyramide, or that catalyzes the deposition of a hapten-labeled CARD substrate, such as a biotin-labeled tyramide, where the hapten serves to mediate the localization of the CARD reporter in the vicinity of the reporter-decorated HCR amplified polymer.

[0076] In some embodiments, the enzyme can be an alkaline phosphatase (AP) (or a polymeric AP comprising multiple AP enzymes) that acts on a CARD substrate to catalyze the deposition of a CARD reporter, such as a chromogenic CARD reporter, for example, but not limited to, BCIP / NBT, BCIP / TNBT, Napthol AS-MX phosphate + FastBlue BB, Napthol AS-MX phosphate + FastRed TR, StayGreen.

[0077] In some embodiments, the enzyme can be glucose oxidase, which acts on a CARD substrate to catalyze the deposition of a CARD reporter, such as NBT.

[0078] In some embodiments, the enzyme is in the vicinity of the reporter-decorated HCR amplified polymer. It can be any molecule or complex that directly or indirectly mediates the localization of the CARD reporter.

[0079] In some embodiments, the CARD-mediating enzyme is inactivated (or inactivated) after CARD reporter deposition (e.g., using chemical or heat denaturation). For example, the CARD-mediating enzyme can be inactivated using any combination of the following: 1. Heat (e.g., 65°C or higher) 2. Fixation (e.g., 4% PFA) 3. Acid (e.g., 0.1 M glycine-HCl + 1% Tween 20, pH 2.2, 0.2 N HCl, 10% acetic acid, 10 mM HCl) 4. Other chemicals (e.g., hydrogen peroxide (HO), hydrogen peroxide + phenol, sodium azide, DEPC, MAB + 10 mM EDTA)

[0080] In some embodiments, HRP is inactivated using H2O2. In some embodiments, AP is inactivated using a combination of heat and acid. In some embodiments, AP is inactivated by fixation. In some embodiments, inactivation of the enzyme mediating CARD allows repeated CARD using the same enzyme in combination with different substrates for different targets, allowing multiplexed target analysis using HCR-mediated CARD. In some embodiments, inactivation of the enzyme mediating CARD allows repeated CARD using different enzymes in combination with different CARD substrates for different targets, allowing multiplexed target analysis using HCR-mediated CARD.

[0081] In some embodiments, CARD allows for storage of stained samples for 10 years or more, allowing for reanalysis in compliance with regulatory requirements. In some embodiments, CARD-stained samples are sufficiently stable for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 years or more and still comply with regulatory requirements. In some embodiments, CARD staining provides for long-term storage of archived samples.

[0082] Target type. In some embodiments, the fragmental initiator probe comprises a target binding domain, a fragmental initiator, and a proximity domain. In some embodiments, a probe unit comprises two or more fragmental initiator probes, and when their target binding domains bind to adjacent cognate targets and their proximity domains bind to a cognate proximity probe (or probes), the fragmental initiators co-localize to form a complete HCR initiator capable of triggering HCR.

[0083] In some embodiments, the probe units are capable of detecting targets comprising any molecule, including, but not limited to, an RNA molecule (e.g., mRNA, rRNA, lncRNA, 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, or a non-biological molecule.

[0084] In some embodiments, the probe units can detect targets comprising any complex of molecules (e.g., RNA:RNA complexes, RNA:protein complexes, DNA:protein complexes, RNA:DNA:protein complexes, protein:protein complexes), complexes of two or more molecules, including any combination of RNA, DNA, proteins, small molecules, biological molecules, and / or non-biological molecules.

[0085] In some embodiments, the probe units can detect targets comprising any collection of molecules or complexes in close proximity, and fragment initiator probes comprising the probe units. When the probes are bound to their respective targets and also to their cognate proximity probe(s) within an assembly of nearby molecules or complexes, the fragmentary initiators in the probe unit can colocalize to form a complete HCR initiator.

[0086] In some embodiments, the target binding domain in the probe unit is configured to bind to overlapping or non-overlapping regions of the target. In some embodiments, the proximity domain in the probe unit is configured to bind to overlapping or non-overlapping regions of the proximity probe. In some embodiments, the fragmentary initiator in the probe unit is designed to hybridize to overlapping or non-overlapping regions of the HCR hairpin. In some embodiments, non-specific binding of individual fragmentary initiator probes prevents complete HCR initiators from co-localizing, thereby suppressing the generation of false amplified HCR background. In some embodiments, all of the fragmentary initiator probes in the probe unit must bind to one or more proximity probes to trigger HCR signal amplification.

[0087] In any of the embodiments provided herein, the fragment initiators within the probe units are designed to be complementary to (or are complementary to) non-overlapping regions of the HCR hairpin (e.g., regions separated by 0, 1, or 2 or more nucleotides), or are designed to be complementary to (or are complementary to) overlapping regions of the HCR hairpin (e.g., regions overlapping by 1 or 2 or more nucleotides), or are designed to be substantially complementary to (e.g., complementary except for 0, 1, 2, a few, or several mismatches) the HCR hairpin, or are configured to bind to the HCR hairpin.

[0088] In any of the embodiments provided herein, the target binding regions in the probe units are configured to bind to non-overlapping regions of the targets (e.g., regions separated by 0, 1, or 2 or more nucleotides or regions separated by 0, 1, or 2 or more nanometers), or to bind to overlapping regions of the targets (e.g., regions that overlap by 1 or 2 or more nucleotides or regions that overlap by 1 or 2 or more nanometers).

[0089] In any of the embodiments provided herein, the proximity domains within a probe unit are configured to bind to non-overlapping regions of one or more proximity probes (e.g. regions separated by 0, 1, or 2 or more nucleotides or regions separated by 0, 1, or 2 or more nanometers), or to bind to overlapping regions of one or more proximity probes (e.g. regions that overlap by 1 or 2 or more nucleotides or regions that overlap by 1 or 2 or more nanometers).

[0090] In some embodiments, the fragmentary initiator probe comprises a kissing domain. In some embodiments, a probe unit comprises two fragmentary 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, a probe unit comprises two or more fragmentary initiator probes, each comprising a kissing domain. In some embodiments, the kissing domains within a probe unit are configured to bind to each other.

[0091] Fragmentary initiator probes for multiplexing. In some embodiments, fragmentary initiator probes are designed for multiplex experiments, in which 2, 3, 4, 5, 10, 20, or 100 or more fragmentary initiator probes are used to bind to different targets in the same sample. In some embodiments, multiplex experiments using fragmentary initiator probes can be used to detect multiple target complexes or multiple targets that are close to each other in a sample.

[0092] Materials and Composition of Initiator-Labeled Probes. In some embodiments, initiator-labeled probes comprise one or more target-binding domains and one or more HCR initiators (see, e.g., Figures 28A-28N and 29A-29F). In some embodiments, each domain may 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 molecules suitable for the purpose of the domain. For example:

[0093] In some embodiments, an initiator-labeled probe may include one or more initiators made from DNA and a target binding domain made from DNA.

[0094] In some embodiments, the initiator-labeled probe may include one or more initiators made from DNA, a chemical linker, and a target binding domain (e.g., an antibody or nanobody or antibody fragment) made from amino acids.

[0095] In some embodiments, an initiator-labeled probe may comprise an initiator made from a synthetic nucleic acid analog and a target binding domain made from a combination of DNA and 2'OMe-RNA.

[0096] In some embodiments, an initiator-labeled probe may include an initiator made from 2'OMe-RNA and a target binding domain made from a combination of RNA and protein.

[0097] In some embodiments, an initiator-labeled probe may comprise an initiator made from DNA and a target binding domain made from PNA.

[0098] In some embodiments, an initiator-labeled probe may comprise one or more initiators made from any nucleic acid or nucleic acid analog and one or more target binding domains made from any combination of materials suitable for binding to a target molecule.

[0099] In some embodiments, the initiator-labeled probe may comprise an antibody or nanobody conjugated to one or more oligonucleotides, each of which comprises one or more initiators.

[0100] In some embodiments, the initiator-labeled probe may comprise a single covalently linked molecule, or may comprise two or more molecules (each covalently linked) that interact non-covalently to form a complex. For example:

[0101] In some embodiments, the initiator-labeled probe may comprise a DNA-based initiator covalently linked to a DNA-based target binding domain.

[0102] In some embodiments, the initiator-labeled probe is a nucleic acid or nucleic acid that is covalently or non-covalently linked to a target binding domain that comprises one or more molecules. It may also include initiators made from analogs.

[0103] In some embodiments, the initiator-labeled probe may comprise one or more fragment initiators, antibodies or nanobodies conjugated to one or more oligonucleotides each comprising a proximity binding domain, where the antibodies or nanobodies are secondary probes that bind to a primary probe that binds to a target.

[0104] Materials and Composition of Fragmentary Initiator Probes. In some embodiments, a fragmentary initiator probe comprises one or more target binding domains, one or more fragmentary initiators, and optionally one or more proximity domains (see, e.g., Figures 6, 7, 8, 9, 10, 11, 12, 13, 14, 31A-31E, 32A-32D, 33A-33E, 36, 46A). In some embodiments, each domain may 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:

[0105] In some embodiments, a fragmentary initiator probe may include one or more fragmentary initiators made from DNA, an optional proximity domain made from DNA, and a target binding domain made from DNA.

[0106] In some embodiments, a fragmentary initiator probe may include one or more fragmentary initiators made from DNA, an optional proximity domain made from DNA, a chemical linker, and a target binding domain (e.g., an antibody or nanobody or antibody fragment) made from amino acids.

[0107] In some embodiments, a fragmentary initiator probe may comprise a fragmentary initiator made from a synthetic nucleic acid analog, an optional proximity domain made from a synthetic nucleic acid analog, and a target binding domain made from a combination of DNA and 2'OMe-RNA.

[0108] In some embodiments, a fragmentary initiator probe may include a fragmentary initiator made from 2'OMe-RNA, an optional proximity domain made from 2'OMe-RNA, and a target binding domain made from a combination of RNA and protein.

[0109] In some embodiments, a fragmentary initiator probe may comprise a fragmentary initiator made from DNA, an optional proximity domain made from DNA, and a target binding domain made from PNA.

[0110] In some embodiments, a fragmentary initiator probe may include one or more fragmentary initiators made from any nucleic acid or nucleic acid analog, one or more optional proximity domains made from a nucleic acid or nucleic acid analog, and one or more target binding domains made from any combination of materials suitable for binding to a target molecule.

[0111] In some embodiments, the fragmentary initiator probe may comprise an antibody or nanobody conjugated to an oligonucleotide comprising a fragmentary initiator and a proximity domain.

[0112] In some embodiments, a fragmentary initiator probe may comprise a target binding domain, a fragmentary initiator, and a proximity domain.

[0113] In some embodiments, the fragmentary initiator probe comprises a single covalently linked molecule. It may comprise a molecule, or it may comprise two or more molecules (each covalently linked) that interact non-covalently to form a complex. For example:

[0114] In some embodiments, a fragmentary initiator probe may comprise a DNA-based fragmentary initiator and optional proximity domain covalently linked to a DNA-based target binding domain.

[0115] In some embodiments, a fragmentary initiator probe may comprise one or more fragmentary initiators and one or more optional proximity domains made from DNA covalently linked to a guide RNA (gRNA) (or another Cas) that is non-covalently bound to a gRNA such that the target binding domain comprises a gRNA:dCas9 complex (or a gRNA:Cas complex using another Cas).

[0116] In some embodiments, a fragmentary initiator probe may comprise one or more fragmentary initiator and one or more optional proximity domains made from DNA covalently linked to a gRNA that is non-covalently bound to dCas9 (or another Cas) such that the target binding domain comprises a gRNA:dCas9 complex (or a gRNA:Cas complex using another Cas).

[0117] In some embodiments, a fragmentary initiator probe may comprise a fragmentary initiator made from a nucleic acid or nucleic acid analog that is covalently or non-covalently linked to a target binding domain comprising one or more molecules, and an optional proximity domain. Each fragmentary initiator probe within a probe unit may have the same or different material composition as the other fragmentary initiator probes in the probe unit. Each fragmentary initiator probe within a probe unit may have a target binding region that binds to a different detection site on the same target molecule, or to a different detection site within a target complex, or to a different detection site within a target collection of adjacent molecules or complexes.

[0118] In some embodiments, the fragmentary initiator probe may comprise an antibody or nanobody conjugated to one or more oligonucleotides each comprising a fragmentary initiator and optionally a proximity domain, where the antibody or nanobody is a secondary probe that binds to a primary probe that binds to a target.

[0119] In some embodiments, a fragmentary initiator probe may comprise a target binding domain, a proximity domain, and a fragmentary initiator, where the target is a primary probe that binds to a primary target.

[0120] Removal of signal from sample. In some embodiments, the HCR signal is removed from the target after detecting the signal. In some embodiments, the signal is removed by any method that reduces the number of signal-generating reporters and / or auxiliary reporters, such as photobleaching of fluorescent reporter molecules using light and / or chemical reactions, chemical cleavage of reporters from the HCR hairpin (e.g., TCEP), chemical cleavage of hairpins to fragment HCR amplified polymers, chemical cleavage of probes to unlink HCR amplified polymers from targets, use of auxiliary strands to dehybridize hairpins from HCR amplified polymers, use of auxiliary strands to dehybridize probes from targets, use of chemical denaturants and / or elevated temperatures to destabilize HCR amplified polymers, use of chemical denaturants and / or elevated temperatures to destabilize probes and targets, use of enzymes to degrade HCR amplified polymers, use of enzymes to degrade probes from targets, use of DNase to degrade DNA amplified polymers, use of DNase to degrade DNA probes, use of DNase to degrade DNA targets, degrade RNA targets. The signal may be removed by the use of RNase to remove the target signal, by the use of two or more of the above methods, or by any other method to remove the signal from the target simultaneously or at different times.

[0121] Assay formats. In some embodiments, the signal can be measured in different assay formats, including but not limited to blots, Northern blots, Western blots, Southern blots, spot blots, paper assays, flow cytometry assays, fluorescence 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 human eyes, assays using cell phone cameras, 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 the signal generated by the HCR amplified polymer.

[0122] Sample Type: In some embodiments, initiator-labeled probes and / or fragmentary initiator probes can be used with HCR amplification hairpins to detect targets in a sample, where the targets include molecules, complexes, or collections of nearby molecules or complexes. Target molecules may be, 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, nematodes, fruit fly embryos, model organisms, non-model organisms, multi-species mixtures of organisms, environmental samples containing unknown organisms, organismal communities (e.g., a mixture of protists and bacteria in the gut of another organism), termites, microbiomes, clinical specimens, diagnostic samples, sputum samples, tumor biopsies, research samples, samples containing material from humans, samples containing material from pets (e.g., dogs, cats, rabbits, lizards, snakes, or fish), material from wild animals (e.g., cheetahs, elephants, rhinos, or chimpanzees), extinct animals (e.g., woolly mammoths, dodos, giant auks), and the like. auk, Triceratops, or passenger pigeon), live cells (e.g., bacteria or cultured mammalian cells), or live organisms (e.g., live mice or live humans).

[0123] In some embodiments, the target may be free in solution within a sample, for example, a test tube, a cell, an embryo, an organism, a tissue section, a biological specimen, or other sample.

[0124] In some embodiments, the target may be covalently crosslinked 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 capture probes that are covalently linked to a microarray or bead.

[0125] In some embodiments, the target may be directly or indirectly immobilized, covalently crosslinked, or non-covalently bound to a solid support. For example, the target may be bound, immobilized, or covalently crosslinked to a slide, blot, membrane, paper substrate, or any other substrate. The target may be immobilized or covalently crosslinked to a cell, embryo, organism, tissue section, biological specimen, or any other sample. The target may be covalently linked within a sample that is fixed and permeabilized, fixed but not permeabilized, or not fixed but permeabilized.

[0126] In some embodiments, the target may be free within a living cell, a living embryo, a living organism, a living ecosystem, or a community of organisms (e.g., the microbiome in a mammalian gut). The target may be associated with but external to a cell or organism, or may be contained within a cell or organism. The target may be covalently crosslinked within a living cell, a living embryo, a living organism, a living ecosystem, or a living community of organisms. The target may or may not be present within one or more cell types within the sample. The target may or may not be present within one or more biological species within the sample. The target may be present in a sample containing one or more off-targets with different degrees of similarity to the target molecule. The target may be present in an expanded sample. The target may be present in a compressed sample. The sample may be expanded prior to target detection to increase the spatial separation between molecules. The sample may be compressed prior to target detection to decrease the spatial separation between molecules. The target and / or other molecules may be crosslinked to the sample being expanded to maintain the relative positions of the molecules in the sample as the sample expands. The target and / or other molecules may be crosslinked to a gel, matrix, or other reagent introduced to the sample to expand the sample while maintaining the relative positions and / or orientation of the molecules in the sample as the sample expands. The sample may be differentially expanded and / or compressed at different expansion and / or compression factors in different tissues and / or organs within the sample.

[0127] Fixation of the sample. In some embodiments, the target molecule can be crosslinked to the sample so that it is retained during subsequent steps in the experiment. For example, the target molecule can be crosslinked to the sample using a chemical reagent (e.g., formaldehyde, paraformaldehyde, EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide)).

[0128] Sample permeabilization. In some embodiments, samples can be treated to enhance the accessibility of target molecules to HCR probes and amplification agents. For example, samples (e.g., cells, tissue sections, or whole-mount embryos) can be permeabilized using chemical reagents (e.g., methanol, ethanol, detergents) or enzymes (e.g., proteinase K). Target accessibility can also be enhanced through sample 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). Another option is to use chemical transfection reagents to deliver the HCR probe and amplification agent across the cell membrane.

[0129] Sample Washing to Remove Unbound Reagents from the Sample. In some embodiments, background can be reduced by washing unused imaging reagents from the sample. For example, washing can be used to remove probes, initiator-labeled probes, fragmented initiator probes, HCR amplification hairpins, amplification reagents, labeled probes, antibodies, and / or other imaging reagents from the sample. Washing can be performed using chemical reagents at a temperature such that the majority of specifically bound imaging reagents are not removed (retaining signal) and the majority of non-specifically bound imaging reagents are removed (reducing background). For example, the wash buffer can contain denaturants (e.g., formamide, urea), salt buffers (e.g., sodium chloride-sodium citrate (SSC), phosphate-buffered saline (PBS)), acids (e.g., citric acid), detergents (e.g., Tween 20, Triton-X, SDS), or blocking agents (e.g., tRNA, salmon sperm DNA, BSA). Wash buffers may include cellulose, Ficoll, polyvinylpyrrolidone, heparin, etc. Wash buffers may be combined with wash temperatures (e.g., 25-80°C) to optimize wash stringency.

[0130] Illustrative Embodiments Figure 6 depicts the detection of a target complex containing two target molecules using a fragmentary initiator probe and a proximity probe, where the fragmentary initiator probes bind directly to their targets. Fragmentary initiator probe 1, which includes target binding domain anti-target 1, binds to target 1, and fragmentary initiator probe 2, which includes target binding domain anti-target 2, binds to target 2. With both fragmentary initiator probes bound to their targets and in proximity to each other, the proximity probes can bind to proximity domains 1 and 2, allowing fragmentary initiators 1 and 2 to co-localize and form a complete initiator capable of initiating HCR.

[0131] Figure 7 depicts the detection of a target complex containing two target molecules using a fragmentary initiator probe and a proximity probe, where the fragmentary initiator probes indirectly bind to their targets. Primary probe anti-target 1 binds to target 1, and primary probe anti-target 2 binds to target 2. Fragmentary initiator probe 1, which contains target binding domain anti-anti-target 1, binds to primary probe anti-target 1, and fragmentary initiator probe 2, which contains target binding domain anti-anti-target 2, binds to primary probe anti-target 2. With both fragmentary initiator probes bound to their targets and in proximity to each other, the proximity probes can bind to proximity domains 1 and 2, allowing fragmentary initiators 1 and 2 to co-localize and form a complete initiator capable of initiating HCR.

[0132] Figure 8 depicts the detection of a target molecule using two fragmentary initiator probes and a proximity probe, where the fragmentary initiator probes indirectly bind to different sites on the target. Two separate sites on the target are bound by anti-target primary probes. Fragmentary initiator probe 1 binds to one anti-target primary probe using its anti-anti-target domain, and fragmentary initiator probe 2 binds to the other anti-target primary probe using its anti-anti-target domain. With both fragmentary initiator probes bound to their targets and in proximity to each other, the proximity probes can bind to proximity domains 1 and 2, allowing fragmentary initiators 1 and 2 to co-localize and form a complete initiator capable of initiating HCR.

[0133] Figure 9 depicts the detection of a first target molecule and a second target molecule using two fragmentary initiator probes and a proximity probe, where the first and second target molecules are not in complex with each other but are in complex with a third target molecule. Primary probe anti-target 1 binds to target 1, and primary probe anti-target 2 binds to target 2; targets 1 and 2 are not directly bound to each other but both bind to target 3. Fragmentary initiator probe 1 binds to anti-target 1 using its anti-anti-target 1 domain, and fragmentary initiator probe 2 binds to anti-target 2 using its anti-anti-target 2 domain. With both fragmentary initiator probes bound to their targets and in proximity to each other, the proximity probes can bind to proximity domains 1 and 2, allowing fragmentary initiators 1 and 2 to co-localize and form a complete initiator capable of initiating HCR.

[0134] Figure 10 depicts the detection of a first target and a second target using two fragmentary initiator probes and a proximity probe, where the first and second targets are in proximity to each other but are not in a complex. Targets 1 and 2 of the target complex are bound by anti-targets 1 and 2, respectively, and targets 1 and 2 are not directly bound to each other. Fragmentary initiator probe 1 is bound by its anti-anti-target Proximity probes bind to anti-target 1 using its anti-anti-target 2 domain, and fragmental initiator probe 2 binds to anti-target 2 using its anti-anti-target 2 domain. With both fragmental initiator probes bound to their targets and in proximity to each other, the proximity probes can bind to proximity domains 1 and 2, allowing fragmental initiators 1 and 2 to co-localize and form a complete initiator capable of initiating HCR.

[0135] Figure 11 depicts target detection using two fragmentary initiator probes and a proximity probe, where the fragmentary initiator probe indirectly binds to the target via binding to a different site on a primary probe that directly binds to the target. The primary probe anti-target binds to the target. Fragmentary initiator probe 1 and fragmentary initiator probe 2 bind to different sites on the anti-target primary probe using their respective anti-anti-target domains. With both fragmentary initiator probes bound to their targets and in proximity to each other, the proximity probes can bind to proximity domains 1 and 2, allowing fragmentary initiators 1 and 2 to co-localize and form a complete initiator capable of initiating HCR.

[0136] Figure 12 depicts the detection of three targets using a fragment initiator probe and a proximity probe, where the three targets are in a complex together. Targets 1, 2, and 3 of the target complex are bound by primary probes anti-targets 1, 2, and 3, respectively. Fragment initiator probe 1 binds to anti-target 1 using its anti-anti-target 1 domain, fragment initiator probe 2 binds to anti-target 2 using its anti-anti-target 2 domain, and fragment initiator probe 3 binds to anti-target 3 using its anti-anti-target 3 domain. With all the fragment initiator probes bound to their targets and in proximity to each other, proximity probes can bind to proximity domains 1, 2, and 3, allowing fragment initiators 1, 2, and 3 to co-localize and form complete initiators capable of initiating HCR.

[0137] Figure 13 depicts the detection of three targets using a fragmentary initiator probe and two proximity probes, where the three targets are in a complex together. Targets 1, 2, and 3 of the target complex are bound by primary probes anti-targets 1, 2, and 3, respectively. Fragmentary initiator probe 1 binds to anti-target 1 using its anti-anti-target 1 domain, fragmentary initiator probe 2 binds to anti-target 2 using its anti-anti-target 2 domain, and fragmentary initiator probe 3 binds to anti-target 3 using its anti-anti-target 3 domain. With all the fragmentary initiator probes bound to their targets and in close proximity 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 fragmentary initiators 1, 2, and 3 to co-localize and form complete initiators capable of initiating HCR.

[0138] Figure 14 depicts the detection of two protein targets using a fragmentary initiator probe 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 probes anti-protein 1 and anti-protein 2, respectively. Fragmentary initiator probe 1 binds to anti-protein 1 using its secondary antibody domain, and fragmentary initiator probe 2 binds to anti-protein 2 using its secondary antibody domain. With both fragmentary initiator probes bound to their targets and in proximity to each other, the proximity probes can bind to proximity domains 1 and 2, allowing fragmentary initiators 1 and 2 to co-localize and form a complete initiator capable of initiating HCR.

[0139] Additional Embodiments Any of the embodiments, compositions, and / or methods provided herein can be used in conjunction with or in alternative forms of any of the following. Thus, for example, a composition and / or method described above can employ any of the compositions or methods described below. Likewise, it should be understood that a composition and / or method described above also provides a method that employs or is part of the method described below.

[0140] Likewise, it should be understood that the embodiments and / or methods provided herein also provide embodiments involving methods, e.g., compositions, method components, kits, etc. In some embodiments, any of the components in one or more of the methods and / or steps provided herein may be provided as a kit including one or more of the described components (and optionally a target or target sequence or sample).

[0141] composition Some embodiments of the composition are outlined in Figures 46A and 46B as well as other figures provided herein.In some embodiments, a composition is provided, comprising: a first fragmentary initiator probe, comprising a first target binding domain configured to directly or indirectly bind to a first target, a first proximity domain, and a first fragmentary initiator; a second target binding domain configured to directly or indirectly bind to a second target, a second proximity domain, and a second fragmentary initiator; a proximity probe configured to bind to the first proximity domain and the second proximity domain; and an HCR amplification agent comprising two or more HCR hairpins.Optionally, the composition can comprise additional fragmentary initiator probes or proximity probes.

[0142] In some embodiments, when the first and second fragmental initiators are bound by the proximity probe, they together form a complete HCR initiator, which has the ability to bind to and open the first HCR hairpin. In some embodiments, the first fragmental initiator probe further comprises a first target binding domain, and the second fragmental initiator probe further comprises a second target binding domain, wherein the first target binding domain is configured to bind to the first target, and the second target binding domain is configured to bind to the second target. In some embodiments, these target binding domains are positioned adjacent to each other after binding to their respective targets, such that when both fragmental initiator probes are bound to both target domains, they can be bound by the proximity probes at their respective proximity domains, and the first and second fragmental initiators (within the fragmental initiator probes) can be co-localized to form a complete HCR initiator capable of binding to and opening the first HCR hairpin.

[0143] Some embodiments of the composition of HCR monomers are outlined in Figures 39 and 40 as well as other figures provided herein.

[0144] In some embodiments, a composition is provided comprising a) a first input domain (1852) comprising a first toehold (1851) and a first stem section (1755), b) a first output domain (1854) comprising a first loop (1853) and a complement to the first stem section (1756), and optionally c) a first HCR hairpin (1510) comprising 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 section (1855); b) a second output domain (1954) comprising a second loop (1953) and a complement to the second stem section (1856); and optionally c) one or more reporters (1950).

[0145] 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 to 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.

[0146] method In some embodiments, a method is provided that includes: A) providing a sample containing a first target and a second target; a first fragmentary initiator probe comprising a first target binding domain configured to bind directly or indirectly to the first target, a first proximity domain, and a first fragmentary initiator; a second fragmentary initiator probe comprising a second target binding domain configured to bind directly or indirectly to the second target, a second proximity domain, and a second fragmentary initiator; a proximity probe configured to bind to the first proximity domain and the second proximity domain; and an HCR amplification agent comprising two or more HCR hairpins; B) incubating the first fragmentary initiator probe and the second fragmentary initiator probe in the sample to allow them to bind; C) incubating the proximity probes in the sample to allow them to bind; D) incubating the HCR amplification agent in the sample; and E) detecting a signal. In some embodiments, a first fragmentary initiator probe binds to a first target and a second fragmentary initiator probe binds to a second target, and when the first and second targets are bound to and / or in proximity to each other, the proximity probe binds to 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.

[0147] In some embodiments, the method further comprises providing a third fragmentary initiator probe comprising a third target binding domain configured to directly or indirectly bind to a third target, a third proximity domain, and a third fragmentary initiator. In some embodiments, the proximity probe is configured to bind to the first proximity domain, the second proximity domain, and the third proximity domain. In some embodiments, a wash step is performed after B) to remove unbound fragmentary initiator probe. In some embodiments, a wash step is performed after C) to remove unbound proximity probe. In some embodiments, a wash step is performed after D) to remove unbound HCR hairpin. In some embodiments, the signal is removed after E). In some embodiments, any of the above steps are repeated to detect a signal for the same or a different target.

[0148] In some embodiments, 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 radiation scanner. In some embodiments, the method further includes providing a helper probe to maximize signal generation. In some embodiments, the helper probe does not include a fragment initiator. In some embodiments, the helper probe does not include a proximity domain. In some embodiments, binding of one or more helper probes to a target increases target accessibility, promoting binding of one or more fragment initiator probes to the target (see, e.g., Figure 30D). In some embodiments, the first target is a protein, a nucleic acid, a molecule, or a combination thereof, and the second target is a protein, a nucleic acid, a molecule, or a combination thereof. In some embodiments, the first target and the second target are bound to each other. In some embodiments, the first target and the second target are the same molecule. In some embodiments, the proximity probe prevents the first target from binding when one target is not in proximity. The fragmentary initiator probe contains one or more clamp domains configured to bind to part or all of the fragmentary initiator and / or the second fragmentary initiator. In some embodiments, the first fragmentary initiator probe and / or the second fragmentary initiator probe comprise an antibody, a nanobody, and / or an 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 self-assembly of the metastable fluorophore-labeled HCR hairpin into a linked fluorescence-amplifying polymer to generate an amplified signal at the target site. In some embodiments, the co-localized complete initiator is utilized 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 RNAs, DNAs, and / or proteins or other molecules in a sample. In some embodiments, the non-specifically binding fragmentary initiator probe does not co-localize the complete initiator and does not initiate HCR. In some embodiments. In some embodiments, the three cooperative probe junctions comprise the oligonucleotide sequences shown in Table 1, and each cooperative probe junction comprises a fragmentary initiator probe P1 comprising a fragmentary initiator and a proximity domain, a fragmentary initiator probe P2 comprising a fragmentary initiator and a proximity domain, and a proximity probe.In some embodiments, signal generation includes the use of a fluorophore, a chromophore, a luminophore, 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'-omega-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), carbon nanotubes, magnetized carbon nanotubes, gold nanoparticles (AuNPs), gold nanoshells, gold nanorods, silver-coated gold nanoparticles, latex, magnetic nanoparticles, silica nanoparticles, a fluorophore, a fluorophore-loaded nanoparticle, a dye-loaded nanoparticle, an enzyme, or any combination thereof. In some embodiments, the method further includes providing a hapten, a ligand, an oligonucleotide, digoxigenin (DIG), fluorescein isothiocyanate (FITC), a fluorophore, biotin, dinitrophenol, aniline, or an enzyme utilized to facilitate signal generation.

[0149] In some embodiments, the target in the sample is blocked for about 1 hour before the fragmental initiator probe or anti-target molecule is incubated with the target. In some embodiments, one or more anti-target molecules are incubated with the target in the sample before two or more fragmental initiator probes are incubated with the target. In some embodiments, one or more anti-target molecules comprise an unlabeled primary antibody probe. In some embodiments, two or more fragmental initiator probes are incubated with the sample after incubation with one or more anti-target molecules. In some embodiments, two or more fragmental initiator probes comprise a fragmental initiator secondary antibody probe. In some embodiments, two or more fragmental initiator 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 with two or more fragmental initiator probes. In some embodiments, one or more proximity probes are incubated with the sample for about 4 hours. In some embodiments, an HCR amplification agent is added to the sample after incubation with the proximity probe. In some embodiments, an HCR amplification agent is incubated with the sample overnight or for about 20 hours. In some embodiments, a wash step is performed prior to detection.

[0150] In some embodiments, the targets in the sample are blocked for about 1 hour before one or more anti-target molecules are incubated with the targets. In some embodiments, some or all of the three types of targets are detected simultaneously in the same sample in a multiplex experiment ( For example, Figure 47 shows: 1) targets that are individual protein molecules, 2) targets that are two proteins that are complexed or in close proximity, and 3) targets that are individual RNA molecules. 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 fragment-initiator secondary probes are incubated in the sample, optionally followed by one or more wash steps. In some embodiments, the total incubation time for the primary probes plus the total incubation time for the secondary probes is about 4, 8, 16, or 24 hours. In some embodiments, the primary probe comprises a primary antibody or nanobody, and the secondary probe comprises a secondary antibody or nanobody. In some embodiments, in the proximity stage, one or more proximity probes are incubated in the sample, optionally followed by one or more wash steps. In some embodiments, one or more proximity probes are incubated with the sample for about 0.5, 1, 2, 4, 8, 16, or 24 hours. In some embodiments, in the RNA detection stage, two or more fragmented initiator DNA probes are incubated with the sample, and optionally one or more washing steps are performed.In some embodiments, the DNA probes are incubated in the sample for about 1, or 2, or 4, or 8, or 16, or 20, or 24 hours.In some embodiments, in the amplification stage, HCR amplification agent is added to the sample, and optionally one or more washing steps are performed.In some embodiments, the HCR amplification agent is incubated with the sample for about 0.5, or 1, or 2, or 10, or 16, or 24 hours.In some embodiments, after detecting the signal of one or more targets, the signal is removed from the sample, and one or more of the above steps are repeated to detect the signal of one or more new targets. [Example]

[0151] Example 1 – Imaging of protein:protein target complexes Figure 15 depicts imaging of protein:protein target complexes in fixed, adherent A-431 human cells using a pair of fragment initiator probes, a proximity probe, and an HCR amplifier. For each protein:protein complex, background was assessed by a technical control in which the antibody corresponding to one protein of the complex was omitted to simulate a scenario in which the two proteins were not in complex with each other. High signal-to-background ratios were obtained for each of the three protein:protein target complexes: 170 ± 10 (SC35:SON), 50 ± 10 (β-catenin:E-cadherin), and 140 ± 10 (α-tubulin:β-tubulin) (standard error of the mean, representative areas in N = 3 cells).

[0152] Figure 16 depicts imaging of β-catenin:E-cadherin target complexes in highly autofluorescent FFPE human breast tissue sections (5 μm thick) using a fragment initiator probe pair, a proximity probe, and an HCR amplifier. Background was assessed by fluorescence in areas of the sample where protein:protein complex abundance was low / zero. A high signal-to-background ratio was obtained (70±20) (standard error of the mean, N=3 representative areas).

[0153] Example 2 – Multiplexed imaging of protein:protein target complexes Figure 17 depicts three-plex imaging of protein:protein complexes in fixed, adherent A-431 human cells utilizing a different pair of fragment initiator probes, an orthogonal HCR amplifier, and a proximity probe for each target complex. The three orthogonal HCR amplifiers have spectrally distinct fluorophores to enable multiplex imaging. Imaging was performed on the nuclear SC35:SON complex, the membrane β-catenin:E-cadherin complex, and the cytoplasmic β-catenin:E-cadherin complex. The fluorescent signals from the α-tubulin:β-tubulin complex and the cytoskeletal α-tubulin:β-tubulin complex were revealed.

[0154] Example 3 - Simultaneous multiplexed imaging of protein:protein complexes, protein targets, and RNA targets Figure 18 depicts simultaneous HCR imaging of protein:protein complexes, protein targets, and RNA targets in fixed, adherent A-431 human cells. The reagents and protocol are depicted in Figure 47. Protein targets were detected using an unlabeled primary antibody probe, which then bound to an initiator-labeled secondary antibody probe. RNA targets were detected using a probe set containing two pairs of fragmentary initiator DNA probes. Protein:protein target complexes were detected using two unlabeled primary antibody probes, which then bound to fragmentary initiator secondary antibody probes, which then bound to a proximity probe to colocalize the complete HCR initiator. Each of the three target types (protein:protein, protein, RNA) triggered orthogonal HCR amplifiers with spectrally distinct fluorophores. The amplified HCR signals revealed membrane β-catenin:E-cadherin complexes, cytoskeletal β-tubulin protein, and nuclear U6 RNA.

[0155] Example 4 - Quantitative HCR (qHCR) imaging of protein:protein complexes Quantitative protein:protein complex imaging is demonstrated via a two-channel overlapping detection experiment in Figure 19A. Figure 19B depicts overlapping detection of membrane β-catenin:E-cadherin protein:protein complexes in fixed, adherent A-431 human cells. Figure 19C shows a two-channel scatter plot of voxel intensities for intracellular 2 x 2 x 0.8 μm voxels in the squared region of Figure 19B, revealing a tight linear distribution (Pearson correlation coefficient, r) corresponding to relative quantification of accuracy and precision. Accuracy corresponds to linearity with a zero intercept, and precision corresponds to scattering around the line.

[0156] Example 5 - Imaging protein:protein complexes with high signal to background Figure 45 demonstrates the performance of HCR protein:protein imaging by comparing fluorescence intensity between two pairs of biological sample types using the same imaging settings for both sample types. Positive samples contained the protein:protein complex of interest; negative samples did not contain the protein:protein complex of interest. For each pair of sample types, signal to background was calculated by estimating signal + background using the positive sample type and estimating background using the negative sample type.

[0157] The β-catenin:E-cadherin targeting complex was imaged using A-431 adherent human cells as a positive sample and HeLa adherent human cells as a negative sample. A-431 cells formed β-catenin:E-cadherin complexes at the cell membrane of cell-cell junctions, but 63 HeLa cells express N-cadherin but not E-cadherin. 64、65 A-431 cells (Figure 45A) showed strong signal at cell-cell junctions, and HeLa cells (Figure 45B) showed no visible staining, resulting in a signal-to-background ratio of 26±4 between the two cell lines (mean±SEM for a representative area of ​​N=3 replicate wells on a slide).

[0158] We detected β-catenin:E-cadherin targeting complexes in highly autofluorescent FFPE human breast tissue sections. The β-catenin:E-cadherin complex is tightly coupled to normal breast epithelial cells. Although β-catenin and E-cadherin protein expression and their interaction are disrupted when breast epithelial cells become cancerous in the invasive lobular carcinoma disease process. 66、67Paired normal and invasive lobular carcinoma FFPE breast tissue sections from the same patient were evaluated for β-catenin:E-cadherin targeting complexes, yielding a 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-background ratio of 30±3 between the two tissue types (mean±SEM for representative areas of N=3 replicate sections).

[0159] Example 6 – Cooperative probe junction sequences Table 1 shows the sequences of three cooperative probe junctions. Each cooperative probe junction contains a) a P1 oligonucleotide containing a fragmentary initiator (underlined sequence) and a proximity domain (bold sequence), b) a P2 oligonucleotide containing a fragmentary initiator (underlined sequence) and a proximity domain (bold sequence), and c) a proximity probe containing binding sites for the P1 and P2 proximity domains (bold sequence). It is noted that the fragmentary initiator probe P1 contains a P1 oligonucleotide, and the fragmentary initiator probe P2 contains a P2 oligonucleotide (see, e.g., the diagram in Figure 46A).

[0160] Each of the two 1-plex imaging studies in Figures 45A-45B and 45C-45D uses a fragmentary initiator-antibody probe pair (P1 and P2) and a proximity probe that form cooperative probe junction J1 (oligonucleotide sequence in Table 1).

[0161] The 3-plex imaging study in Figure 17 uses a fragmentary initiator probe pair and a proximity probe that form a different cooperative probe junction for each target complex: junction J1 for β-catenin:E-cadherin, junction J2 for α-tubulin:β-tubulin, and junction J3 for SC35:SON. [Table 1]

[0162] Additional configuration Composition 1: A composition comprising: a first fragmentary initiator probe comprising a first target binding domain configured to bind directly or indirectly to a first target, a first proximity domain, and a first fragmentary initiator; a second target binding domain configured to bind directly or indirectly to a second target, a second proximity domain, and a second fragmentary initiator; two or more proximity probes configured to bind to the first proximity domain and the second proximity domain; at least one of which comprises a reporter. The composition comprises a hybridization chain reaction (HCR) amplification agent comprising an HCR hairpin monomer; and wherein the first fragmentary initiator probe binds to the first target and the second fragmentary initiator probe binds to the second target; and wherein, when the first and second targets are bound to and / or in proximity to each other, the proximity probe binds to the first proximity domain and the second proximity domain to co-localize a complete initiator comprising the first fragmentary initiator and the second fragmentary initiator, and the co-localized complete initiator is configured to initiate HCR signal amplification, wherein the HCR hairpin monomer self-assembles into a linked HCR amplification polymer, thereby generating a signal.

[0163] Configuration 2: The composition of Configuration 1, wherein at least one of said HCR hairpin monomers comprises an input domain, and said first fragmentary initiator and said second fragmentary initiator together form a complete initiator configured to hybridize to said input domain.

[0164] Configuration 3: The composition of any one of Configurations 1-2, further comprising a third fragmentary initiator probe comprising a third target binding domain configured to bind directly or indirectly to a third target, a third proximity domain, a third fragmentary initiator; and a proximity probe further configured to bind to the third proximity domain.

[0165] Configuration 4: The composition of any one of configurations 1 to 3, 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.

[0166] Configuration 5: The composition of configuration 4, wherein said first target and said second target are bound to one another.

[0167] Configuration 6: The composition of configuration 4, wherein said first target and said second target are the same molecule.

[0168] Configuration 7: The composition of any one of Configurations 1-6, wherein the proximity probe contains one or more clamp domains configured to bind to some or all of the first fragmentary initiator and / or the second fragmentary initiator when one target is not in proximity.

[0169] Configuration 8: The composition of any one of Configurations 1-7, wherein the first fragmentary initiator probe and / or the second fragmentary initiator probe comprises an antibody, a nanobody, and / or an oligonucleotide.

[0170] Configuration 9: The composition of any one of configurations 1-8, wherein the co-localized intact initiator is used to directly or indirectly mediate the generation of a signal.

[0171] Configuration 10: The composition of any one of configurations 1 to 9, wherein the co-localized complete initiator triggers self-assembly of metastable fluorophore-labeled HCR hairpins into linked fluorescence-amplifying polymers to generate an amplified signal at the target site.

[0172] Configuration 11: The composition of any one of configurations 1-10, wherein the first target and the second target are selected from proteins, RNA, DNA, or other nucleic acids.

[0173] Configuration 12: The composition of any one of configurations 1 to 11, wherein the first target and the second target form a complex.

[0174] Configuration 13: The composition of configuration 12, comprising an additional fragmentary initiator for each of the N target complexes to be detected in the sample.

[0175] Configuration 14: The composition of Configuration 13, wherein each of the N target complexes is detected in the same sample using a different fragment initiator probe pair, proximity probe, and HCR amplifier for each target complex.

[0176] Configuration 15: The composition of any one of configurations 1-14, wherein the sequence of the first fragmentary initiator is selected from SEQ ID NO:1, SEQ ID NO:4, and SEQ ID NO:7; the sequence of the second fragmentary initiator is selected from SEQ ID NO:2, SEQ ID NO:5, and SEQ ID NO:8; and the sequence of the proximity probe is selected from SEQ ID NO:3, SEQ ID NO:6, and SEQ ID NO:9.

[0177] Configuration 16: The composition of any one of Configurations 1-15, wherein the at least one reporter comprises a fluorophore, a chromophore, a luminophore, 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), carbon nanotubes, magnetized carbon nanotubes, gold nanoparticles (AuNPs), gold nanoshells, gold nanorods, silver-coated gold nanoparticles, latex, magnetic nanoparticles, silica nanoparticles, fluorophore-loaded nanoparticles, dye-loaded nanoparticles, an enzyme, or any combination thereof.

[0178] Configuration 17: The composition of any one of configurations 1 to 16, wherein said at least one reporter directly or indirectly mediates the generation of said signal.

[0179] Configuration 18: The composition of any one of Configurations 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 utilized to facilitate generation of the signal.

[0180] Configuration 19: The composition of any one of configurations 1 to 18, wherein said HCR-amplifying polymer mediates catalyzed reporter deposition (CARD).

[0181] Configuration 20: A method comprising: providing a sample containing a first target and a second target; a first fragmentary initiator probe comprising a first target binding domain configured to bind directly or indirectly to the first target, a first proximity domain, and a first fragmentary initiator; a second fragmentary initiator probe comprising a second target binding domain configured to bind directly or indirectly to the second target, a second proximity domain, and a second fragmentary initiator; a proximity probe configured to bind to the first proximity domain and the second proximity domain; a hybridization chain reaction (HCR) amplifier comprising two or more HCR hairpin monomers, at least one of which comprises a reporter; adding the first fragmentary initiator probe and the second fragmentary initiator 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 an HCR polymer; wherein the first fragmentary initiator probe is bound to the first target and the second fragmentary initiator probe is bound to the second target. wherein when the first and second targets are bound to or in proximity to each other, the proximity probe binds to the first proximity domain and the second proximity domain to co-localize the complete initiator; and the co-localized complete initiator initiates polymerization of the HCR monomer, thereby generating a signal.

[0182] Configuration 21: The method of Configuration 20, further comprising providing a third fragmentary 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 fragmentary initiator.

[0183] Configuration 22: The method of Configuration 21, wherein the proximity probe is configured to bind to the first proximity domain, the second proximity domain, and the third proximity domain.

[0184] Configuration 23: The method of any one of Configurations 20-22, wherein a washing step is performed after b and before c to remove unbound fragmentary initiator probe.

[0185] Configuration 24: The method of any one of Configurations 20 to 23, wherein a washing step is performed after c and before d to remove unbound proximity probes.

[0186] 25. The method of any one of embodiments 20 to 24, wherein a washing step is performed after d to remove unbound HCR hairpins.

[0187] Configuration 26: The method of any one of Configurations 20 to 25, wherein the signal is removed after e.

[0188] Configuration 27: The method of Configuration 26, wherein any of the above steps are repeated to detect signals for the same or different targets.

[0189] Configuration 28: The method of any one of Configurations 20 to 27, 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 radiation scanner.

[0190] Configuration 29: The method of any one of Configurations 20 to 28, further comprising providing a helper probe to maximize signal generation.

[0191] Configuration 30: The method of any one of Configurations 20 to 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.

[0192] Configuration 31: The method of Configuration 30, wherein the first target and the second target are bound to each other.

[0193] Configuration 32: The method of Configuration 30, wherein the first target and the second target are the same molecule.

[0194] Configuration 33: The method of any one of Configurations 20-32, wherein the proximity probe contains one or more clamp domains configured to bind to some or all of the first fragmentary initiator and / or the second fragmentary initiator when one target is not in proximity.

[0195] Configuration 34: The method of any one of Configurations 20 to 33, wherein the first fragmentary initiator probe and / or the second fragmentary initiator probe comprises a target binding region comprising an antibody, a nanobody, and / or an oligonucleotide.

[0196] Configuration 35: The method of any one of Configurations 20 to 34, wherein the co-localized intact initiator is used to directly or indirectly mediate the generation of a signal.

[0197] Configuration 36: The method of any one of Configurations 20 to 34, wherein the co-localized complete initiator triggers self-assembly of metastable fluorophore-labeled HCR hairpins into linked fluorescence-amplifying polymers to generate an amplified signal at the site of the adjacent target.

[0198] Configuration 37: The method of any one of Configurations 20-36, wherein the co-localized intact initiator is utilized 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 RNAs, DNAs, and / or proteins or other molecules in a sample.

[0199] Configuration 38: The method of any one of Configurations 20 to 37, wherein non-specifically binding fragmentary initiator probes do not co-localize with the complete initiator and do not initiate HCR.

[0200] Configuration 39: The method of any one of Configurations 20 to 38, wherein each of the N target complexes is detected in the same sample using a different pair of fragmentary initiator probes, proximity probes, and HCR amplifiers for each target complex.

[0201] Configuration 40: The method of any one of Configurations 20 to 39, wherein the sequence of the first fragmentary initiator is selected from SEQ ID NO:1, SEQ ID NO:4, and SEQ ID NO:7; the sequence of the second fragmentary initiator is selected from SEQ ID NO:2, SEQ ID NO:5, and SEQ ID NO:8; and the sequence of the proximity probe is selected from SEQ ID NO:3, SEQ ID NO:6, and SEQ ID NO:9.

[0202] Aspect 41: The method of any one of Aspects 20-40, wherein the at least one reporter comprises a fluorophore, a chromophore, a luminophore, 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), carbon nanotubes, magnetized carbon nanotubes, gold nanoparticles (AuNPs), gold nanoshells, gold nanorods, silver-coated gold nanoparticles, latex, magnetic nanoparticles, silica nanoparticles, a fluorophore, a fluorophore-loaded nanoparticle, a dye-loaded nanoparticle, an enzyme, or any combination thereof.

[0203] Configuration 42: The method of any one of Configurations 20 to 41, wherein said at least one reporter directly or indirectly mediates the production of said signal.

[0204] Configuration 43: The method of any one of Configurations 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 utilized to promote generation of the signal.

[0205] Configuration 44: The method of any one of Configurations 20 to 43, wherein the HCR amplification polymer mediates catalyzed reporter deposition (CARD).

[0206] While the foregoing invention has been described in terms of certain preferred embodiments, other embodiments will be apparent to those skilled in the art. Additionally, other combinations, omissions, substitutions, and modifications will be apparent to those skilled in the art in view of the disclosure herein. Accordingly, the present invention is not intended to be limited by the description of the preferred embodiments, but rather is intended to be defined by reference to the appended claims.

[0207] Any of the embodiments, compositions, and / or methods provided herein can be used in conjunction with or in alternative forms of any of the following. Thus, for example, a composition and / or method described above can employ any of the compositions or methods described below. Likewise, it should be understood that a composition and / or method described above also provides a method that employs or is part of the method described below.

[0208] Likewise, it should be understood that the embodiments and / or methods provided herein also provide embodiments involving methods, e.g., compositions, method components, kits, etc. In some embodiments, any of the components in one or more of the methods and / or steps provided herein may be provided as a kit including one or more of the described components (and optionally a target or target sequence or sample).

[0209] The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described in any way. All literature and similar materials referenced in this application, including, but not limited to, patents, patent applications, papers, books, articles, and internet web pages, are expressly incorporated by reference in their entirety for any purpose. If the definition of a term in an incorporated reference appears to differ from the definition provided in the present teachings, the definition provided in the present teachings shall control. The word "about" is implied prior to temperatures, concentrations, times, etc. discussed in the present teachings, so that minor and insubstantial deviations will be understood to be within the scope of the teachings herein. In this application, the use of the singular includes the plural unless specifically stated otherwise. Also, the use of "comprise," "comprises," "comprising," "contain," "contains," containing, "include," "includes," and "including" is 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. Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. See, e.g., Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons, 1999. Sons (New York, NY 1994); Sambrook et See, e.g.,

[0010] al., Molecular Cloning, A Laboratory Manual, Cold Springs Harbor Press (Cold Springs Harbor, NY 1989). It is to be understood that both the general and detailed description are exemplary and explanatory only and are not limiting of the claimed invention. 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 specifically stated otherwise. Furthermore, the use of the term "including," as well as other forms such as "includes" and "included," is non-exclusive. Also, unless specifically stated otherwise, the term "element" ( Terms such as "element" or "component" encompass both elements and components that contain one unit and elements and components that contain more than one subunit. Also, use of the term "component" can include a portion or a part of a whole part.

[0210] In some embodiments, any one or more of the optional elements of any one or more of the drawings herein can be combined with any one or more of the other optional elements of any one or more of the drawings herein. In some embodiments, any one or more of the compositions or steps provided in any of the drawings provided herein can be combined with any of the other compositions or steps provided herein. Unless otherwise indicated, as used herein, a general reference to a set of drawings (e.g., Figure 36) refers to all of the different drawings contained within that number (e.g., Figures 36A-36R), each in combination, with one or more of them, or each in the alternative.

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Claims

1. 1. A composition comprising: a. i. a first target binding domain configured to bind directly or indirectly to a first target; ii. a first proximity domain; iii. First Fractional Initiator a first fragmentary initiator probe comprising: b. i. a second target binding domain configured to bind directly or indirectly to a second target; ii. a second proximity domain; iii. Second Fragmentary Initiator a second fragmentary initiator probe comprising: c. a proximity probe configured to bind to the first proximity domain and the second proximity domain; and d. A hybridization chain reaction (HCR) amplification agent comprising two or more HCR hairpin monomers, at least one of which comprises a reporter. Including; the first fragmentary initiator probe is bound to the first target and the second fragmentary initiator probe is bound to the second target; and when the first and second targets are bound to and / or in proximity to each other, the proximity probe can bind to the first proximity domain and the second proximity domain to co-localize a complete initiator comprising the first fragmentary initiator and the second fragmentary initiator, and the co-localized complete initiator is configured to initiate HCR signal amplification, wherein the HCR hairpin monomers self-assemble into linked HCR amplification polymers, thereby generating a signal. The composition.

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

3. a. i. a third target binding domain configured to bind directly or indirectly to a third target; ii. a third proximal domain; iii. A third fragmentary initiator a third fragmentary initiator probe comprising: b. a proximity probe further configured to bind to said third proximity domain. The composition of claim 1 further comprising:

4. 10. The composition of claim 1, 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.

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

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

7. 2. The composition of claim 1, wherein the proximity probe contains one or more clamp domains configured to bind to some or all of the first fragmentary initiator and / or the second fragmentary initiator when a target is not in proximity.

8. 10. The composition of claim 1, wherein the first fragmentary initiator probe and / or the second fragmentary initiator probe comprises an antibody, a nanobody, and / or an oligonucleotide.

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 complete initiator triggers self-assembly of metastable fluorophore-labeled HCR hairpins into linked fluorescence-amplifying polymers to generate an amplified 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. 13. The composition of claim 12, comprising an additional fragmentary initiator for each of the N target complexes to be detected in the sample.

14. 14. The composition of claim 13, wherein each of the N target complexes is detected in the same sample using a different pair of fragmentary initiator probes, proximity probes, and HCR amplification agents for each target complex.

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

9.

16. 10. The composition of claim 1, wherein the at least one reporter comprises a fluorophore, a chromophore, a luminophore, 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-coated gold nanoparticle, latex, a magnetic nanoparticle, a silica nanoparticle, a fluorophore-loaded nanoparticle, a dye-loaded nanoparticle, an enzyme, or any combination thereof.

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

18. The at least one reporter may be a hapten, a ligand, an oligonucleotide, digoxigenin (DIG), fluorescein isothiocyanate (FITC), a fluorophore, biotin, dinitrophenol, aniline, or a nucleotide sequence that facilitates the generation of the signal. The composition of claim 1 comprising the enzyme to be utilized.

19. The composition of claim 1 , wherein the HCR-amplifying polymer mediates catalyzed reporter deposition (CARD).

20. 1. A method comprising: a. i. a sample containing a first target and a second target; ii.

1. a first target binding domain configured to bind directly or indirectly to said first target; 2. a first proximate domain; 3. First Fragmentary Initiator a first fragmentary initiator probe comprising: iii.

1. a second target binding domain configured to bind directly or indirectly to said second target; 2. a second adjacent domain; 3. Second Fragmentary Initiator a second fragmentary initiator probe comprising: iv. a proximity probe configured to bind to said first proximity domain and said second proximity domain; v. A hybridization chain reaction (HCR) amplification agent comprising two or more HCR hairpin monomers, at least one of which comprises a reporter. To prepare; b. adding the first fragmentary initiator probe and the second fragmentary initiator probe to the sample; c. adding the proximal probe to the sample; d. adding the HCR amplifier to the sample; e. Detecting a signal indicative of the presence of HCR polymer. Including; when the first fragmentary initiator probe is bound to the first target and the second fragmentary initiator probe is bound to the second target, and the first and second targets are bound to or in proximity to each other, the proximity probe binds to the first proximity domain and the second proximity domain to co-localize the complete initiator; and The co-localized intact initiator initiates polymerization of the HCR monomer, thereby generating a signal. The method.

21. 1. a third target binding domain configured to bind directly or indirectly to a third target; 2. a third adjacent domain, and 3. Third Fragmentary Initiator 21. The method of claim 20, further comprising providing a third fragmentary initiator probe comprising:

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

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

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

25. 21. The method of claim 20, wherein a washing step is performed after step d to remove unbound HCR hairpins.

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

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

28. 21. 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 radiation scanner.

29. 21. The method of claim 20, further comprising providing a helper probe to maximize signal generation.

30. 21. The method of claim 20, 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.

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

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

33. 21. The method of claim 20, wherein the proximity probe contains one or more clamp domains configured to bind to some or all of the first fragmentary initiator and / or the second fragmentary initiator when a target is not in proximity.

34. 21. The method of claim 20, wherein the first fragmentary initiator probe and / or the second fragmentary initiator probe comprise a target binding region comprising an antibody, a nanobody, and / or an oligonucleotide.

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

36. 21. The method of claim 20, wherein the co-localized complete initiator triggers self-assembly of metastable fluorophore-labeled HCR hairpins into linked fluorescence-amplifying polymers to generate an amplified signal at the site of the adjacent target.

37. 21. The method of claim 20, wherein the co-localized intact initiator is utilized 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 RNA, DNA, and / or protein or other molecules in a sample.

38. 21. The method of claim 20, wherein non-specifically binding fragmentary initiator probes do not co-localize with the complete initiator and do not initiate HCR.

39. 21. The method of claim 20, wherein each of the N target complexes is detected in the same sample using a different pair of fragmentary initiator probes, proximity probes, and HCR amplification agents for each target complex.

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

9.

41. 21. The method of claim 20, wherein the at least one reporter comprises a fluorophore, a chromophore, a luminophore, 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-coated gold nanoparticle, latex, a magnetic nanoparticle, a silica nanoparticle, a fluorophore, a fluorophore-loaded nanoparticle, a dye-loaded nanoparticle, an enzyme, or any combination thereof.

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

43. 21. The method of claim 20, 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 utilized to facilitate generation of the signal.

44. 21. The method of claim 20, wherein the HCR-amplifying polymer mediates catalyzed reporter deposition (CARD).

Images