Analysis of target molecules in a sample via hybridization chain reaction

Abstract

This paper provides a method for analyzing samples using a hybridization chain reaction (HCR). [Solution] A sample analysis method using HCR comprises one, two, or all three of the following: 1) repeated signal detection, 2) overlap binding sites, and 3) catalytic reporter deposition (CARD). Compositions and kits relating to these are also provided. Some embodiments include a method for repeated signal detection using a reporter-labeled HCR hairpin, comprising: preparing a sample containing a target and other non-target molecules; preparing a probe set containing either a) an HCR initiator-labeled probe, or b) a probe unit each containing two or more HCR split initiators; preparing an HCR amplifier (each labeled with a reporter); and detecting a signal from the reporter.

Description

[Technical Field]

[0001] Description of research and development funded by the federal government. This invention was made with government assistance, based on grant number R01EB006192 from the National Institutes of Health and grant number HR0011-17-2-0008 from DARPA. The U.S. Government has certain rights in this invention.

[0002] Priority rights and cross-references of related applications This application claims the benefit of U.S. Provisional Patent Application No. 62 / 986436, filed on 6 March 2020, which is incorporated herein by reference in its entirety.

[0003] Reference to electronic sequence listings This application is filed together with an electronic sequence listing. The electronic sequence listing is provided as a file named MOINS007WOSEQLIST.txt, created on March 4, 2021, with a size of 1,341 bytes. The information contained in the electronic sequence listing is incorporated herein by reference in its entirety. [Background technology]

[0004] field The present invention, as a whole, relates to compositions and methods relating to hybridization chain reactions.

[0005] Description of related technologies Hybridization chain reactions (HCRs) are elicit-induced hybridization methods of nucleic acid molecules that begin with metastable hairpin monomers or other metastable nucleic acid structures. HCRs do not require enzymes and can be performed isothermally.

[0006] HCR can include two or more metastable hairpin monomers. Each hairpin monomer has at least one single-stranded toehold, a single-stranded loop, and a double-stranded stem. The energy driving the self-assembly cascade is stored within the single-stranded loop segments and toehold segments of the hairpins.

[0007] Each monomer is trapped in a kinetic trap, preventing the system from rapidly equilibrating. That is, monomer pairs cannot hybridize with each other in the absence of an initiator. The introduction of an initiator strand causes the monomers to undergo a chain reaction of hybridization events to form a nicked double-stranded polymer. By utilizing HCR, for example, the presence of a target analyte in a sample can be detected by detecting the analyte with an initiator-labeled probe having an HCR initiator, and the HCR initiator induces HCR signal amplification. HCR signal amplification can increase the signal-to-background (SB) ratio for molecular detection and imaging applications by enhancing the signal over the background arising from the sample.

Prior Art Documents

Patent Documents

[0008]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Non-Patent Documents

[0009]

Non-Patent Document 1

[0010] Some embodiments provided herein yield a larger SB ratio using a probe unit comprising two or more split initiator probes, each containing a split initiator. If individual split initiator probes bind nonspecifically to the sample, the split initiator probes do not induce HCR. However, if a split initiator probe specifically binds to its cognitive target, the split initiators within the probe unit colocalize to form a complete HCR initiator, enabling the induction of HCR signal amplification. In some embodiments, splitting the initiator into two or more split initiators enables automatic background suppression during the detection step, as this is only effective when colocalized in the presence of the target. When combined with the automatic background suppression provided by HCR during amplification, this process enables automatic background suppression throughout the entire protocol (for example, if the reagent, whether an individual probe or an individual hairpin monomer, binds to the sample, it does not lead to the generation of amplified background).

[0011] In some embodiments provided herein, a probe unit comprises two split initiator probes, each containing a target binding region and a split initiator, wherein the target binding region within the probe unit is configured to bind to an adjacent binding site on the target and colocalize a complete HCR initiator, and the split initiator within each probe unit is configured to bind to an adjacent binding site on an HCR hairpin and induce HCR signal amplification. In some embodiments, the connection between the target, the split initiator probe, and / or the HCR hairpin is energetically unlikely. In some embodiments, the connection can be relaxed to an energetically more likely higher-order structure by configuring the target binding region within the probe unit to bind to an overlap binding site on the target, and / or configuring the split initiator within the probe unit to hybridize to an overlap binding site on an HCR hairpin, thereby increasing the intensity of HCR signal amplification.

[0012] In some embodiments provided herein, one or more targets can be analyzed in a sample by generating one or more HCR signals, detecting one or more HCR signals, removing one or more HCR signals, and repeating one or more of these steps.

[0013] In some embodiments provided herein, HCR signal amplification is used to mediate catalytic reporter deposition (CARD) and further increase the signal gain, enabling long-term archiving of stained samples for regulatory purposes.

[0014] In some embodiments, a repeat signal detection method using reporter-labeled hairpins is provided. In some embodiments, the method includes: preparing a sample which may contain up to N targets and other non-target molecules; preparing N probe sets (each targeting one of the N target species) each containing either i) one or more HCR initiator-labeled probes, or ii) one or more probe units, each containing two or more HCR splitting initiators; optionally washing the sample; preparing M HCR amplifiers corresponding to M of the N probe sets (if M ≤ N; each labeled with a different reporter); optionally washing the sample; detecting M signals corresponding to the M reporters; removing the M signals from the sample; optionally repeating one or more of the above steps until signal detection is performed for all N targets. The probe sets include either one or more HCR initiator-labeled probes, or one or more probe units. An HCR initiator-labeled probe includes one or more target-binding regions and one or more initiators. A probe unit includes two or more HCR split initiator probes. An HCR split initiator probe includes a target-binding region and a split initiator. An HCR amplifier includes two or more HCR hairpins. An HCR hairpin includes an input domain including a single-stranded toehold and a stem. An HCR hairpin further includes an output domain including a single-stranded loop and a complementary strand to the stem. An HCR hairpin further includes a reporter.

[0015] In some embodiments, a method for detecting repeated signals using reporter-labeled hairpins is provided. In some embodiments, the method comprises: preparing a sample which may contain one or more targets and other non-target molecules; preparing one or more probe sets, each containing i) one or more HCR initiator-labeled probes, or ii) one or more probe units, each containing two or more HCR splitting initiators; optionally washing the sample; preparing one or more HCR amplifiers (each labeled with one or more reporters); optionally washing the sample; detecting one or more signals from one or more reporters; optionally removing one or more probe sets from the sample; optionally removing one or more HCR amplifiers from the sample; optionally removing one or more reporters from the sample; and optionally removing one or more signals from the sample. A probe set includes either one or more HCR initiator-labeled probes, or one or more probe units. An HCR initiator-labeled probe includes one or more target-binding regions and one or more initiators. A probe unit includes two or more HCR split-initiator probes. An HCR split-initiator probe includes a target-binding region and split-initiators. An HCR amplifier includes two or more HCR hairpins. An HCR hairpin includes an input domain including a single-stranded toehold and a stem. An HCR hairpin further includes an output domain including a single-stranded loop and a complementary strand to the stem. An HCR hairpin further includes one or more reporters.

[0016] In some embodiments, a method for detecting repeated signals using substrate-labeled hairpins is provided. In some embodiments, the method comprises: a) preparing a sample which may contain up to N targets and other non-target molecules; b) preparing N probe sets, each containing i) one or more HCR initiator-labeled probes, or ii) one or more probe units, each containing two or more HCR splitting initiators; c) optionally washing the sample; d) preparing N HCR amplifiers corresponding to the N probe sets (each labeled with a different substrate); e) optionally washing the sample; f) preparing M labeled probes corresponding to M of the N substrates (if M ≤ N; each bound to a different reporter); g) optionally washing the sample; h) detecting M signals corresponding to the M separate reporters; i) removing the M signals from the sample; and j) optionally repeating one or more of steps f to i until signal detection is performed for all N targets. A probe set includes either one or more HCR initiator-labeled probes, or one or more probe units. An HCR initiator-labeled probe includes one or more target-binding regions and one or more initiators. A probe unit includes two or more HCR split-initiator probes. An HCR split-initiator probe includes a target-binding region and split initiators. An HCR amplifier includes two or more HCR hairpins. An HCR hairpin includes an input domain including a single-stranded toehold and a stem. An HCR hairpin further includes an output domain including a single-stranded loop and a complementary strand to the stem. An HCR hairpin further includes a substrate. A labeled probe includes a substrate-binding region and a reporter.

[0017] In some embodiments, a repeat signal detection method using substrate-labeled hairpins is provided. In some embodiments, the method comprises: a) preparing a sample which may contain one or more targets and other non-target molecules; b) preparing one or more probe sets, each containing i) one or more HCR initiator-labeled probes, or ii) one or more probe units, each containing two or more HCR splitting initiators; c) optionally washing the sample; d) preparing one or more HCR amplifiers (each labeled with a substrate) corresponding to one or more probe sets; e) optionally washing the sample; f) preparing one or more labeled probes (each bound to a reporter) corresponding to one or more substrates; g) optionally washing the sample; h) detecting one or more signals corresponding to one or more reporters; i) removing one or more signals from the sample; and j) optionally repeating any of steps b-i one or more times in any order. A probe set includes either one or more HCR initiator-labeled probes, or one or more probe units. An HCR initiator-labeled probe includes one or more target-binding regions and one or more initiators. A probe unit includes two or more HCR split-initiator probes. An HCR split-initiator probe includes a target-binding region and split initiators. An HCR amplifier includes two or more HCR hairpins. An HCR hairpin includes an input domain including a single-stranded toehold and a stem. An HCR hairpin further includes an output domain including a single-stranded loop and a complementary strand to the stem. An HCR hairpin further includes a substrate. A labeled probe includes a substrate-binding region and a reporter.

[0018] In some embodiments, a repeat signal detection method using reporter and / or substrate-labeled hairpins is provided. In some embodiments, the method involves preparing a sample which may contain one or more targets and other non-target molecules; preparing one or more HCR probe sets, each containing i) one or more HCR initiator-labeled probes, or ii) one or more probe units, each containing two or more HCR splitting initiators; preparing one or more HCR amplifiers corresponding to one or more probe sets (each labeled with one or more reporters and / or one or more substrates); and optionally, one or more corresponding to one or more substrates. The process includes preparing labeled probes (each bound to one or more reporters), detecting one or more signals, optionally washing the sample, optionally removing one or more signals from the sample, optionally removing one or more reporters from the sample, optionally removing one or more labeled probes from the sample, optionally removing one or more HCR amplifiers from the sample, optionally removing one or more probe sets from the sample, and optionally repeating any of the above steps in any order. A probe set includes either one or more HCR initiator-labeled probes or one or more probe units. An HCR initiator-labeled probe includes one or more target-binding regions and one or more initiators. A probe unit includes two or more HCR splitting initiator probes. An HCR splitting initiator probe includes a target-binding region and splitting initiators. An HCR amplifier comprises two or more HCR hairpins. Each HCR hairpin comprises an input domain including a single-stranded toehold and a stem. Each HCR hairpin further comprises an output domain including a single-stranded loop and a complementary strand to the stem. Each HCR hairpin further comprises one or more reporters and / or one or more substrates.The labeled probe includes a substrate-binding region and one or more reporters.

[0019] In some embodiments, a repeat signal detection method is provided using reporter and / or substrate-labeled hairpins. In some embodiments, the method comprises: preparing a sample which may contain one or more targets and other non-target molecules; performing any of steps c-g once or more times in any order; preparing one or more HCR probe sets, each comprising i) one or more HCR initiator-labeled probes, or ii) one or more probe units, each comprising two or more HCR splitting initiators; preparing one or more HCR amplifiers which directly or indirectly produce one or more signals; optionally washing the sample; detecting one or more signals; and optionally removing one or more signals, wherein the probe sets comprise either one or more HCR initiator-labeled probes or one or more probe units. The HCR initiator-labeled probes comprise one or more target-binding regions and one or more initiators. The probe unit comprises two or more HCR splitting initiator probes. Each HCR splitting initiator probe comprises a target binding region and a splitting initiator. The HCR amplifier comprises two or more HCR hairpins. Each HCR hairpin comprises an input domain comprising a single-stranded toehold and a stem. Each HCR hairpin further comprises an output domain comprising a single-stranded loop and a complementary strand to the stem. Each HCR hairpin further comprises one or more reporters and / or one or more substrates.

[0020] In some embodiments, an HCR method is provided that includes an overlapping binding site. In some embodiments, the method may include a target and other non-target molecules. The procedure includes preparing a sample, preparing a probe set comprising one or more probe units, each containing two or more HCR splitting initiators, wherein the target binding region on the probe within each probe unit is configured to bind to the overlap binding site on the target, optionally washing the sample, preparing an HCR amplifier labeled with a reporter and / or substrate, optionally washing the sample, optionally preparing a labeled probe (bound to the reporter) corresponding to the substrate, optionally washing the sample, and detecting a signal from the reporter. The probe set comprises one or more probe units. Each probe unit contains two or more HCR splitting initiator probes. Each HCR splitting initiator probe contains a target binding region and a splitting initiator. The target binding region on the probe within each probe unit is configured to bind to the overlap binding site on the target. The HCR amplifier contains two or more HCR hairpins. The HCR hairpin comprises an input domain including a single-stranded toehold and a stem. The HCR hairpin further comprises an output domain including a single-stranded loop and a complementary strand to the stem. The HCR hairpin further comprises a reporter and / or substrate. The labeled probe comprises a substrate-binding region and a reporter.

[0021] In some embodiments, an HCR method including overlap binding sites is provided. In some embodiments, the method includes: preparing a sample which may contain a target and other non-target molecules; preparing a probe set which includes one or more probe units each containing two or more HCR splitting initiators, and which is configured such that the splitting initiators on the probes in each probe unit bind to overlap binding sites on HCR hairpins; optionally washing the sample; preparing an HCR amplifier labeled with a reporter and / or substrate; optionally washing the sample; optionally preparing a labeled probe (bound to the reporter) corresponding to the substrate; optionally washing the sample; and detecting a signal from the reporter. The probe set includes one or more probe units. Each probe unit contains two or more HCR splitting initiator probes. Each HCR splitting initiator probe includes a target binding region and splitting initiators. The splitting initiators on the probes in each probe unit are configured to bind to overlap binding sites on HCR hairpins. An HCR amplifier comprises two or more HCR hairpins. Each HCR hairpin comprises an input domain including a single-stranded toehold and a stem. Each HCR hairpin further comprises an output domain including a single-stranded loop and a complementary strand to the stem. Each HCR hairpin further comprises a reporter and / or substrate. A labeled probe comprises a substrate-binding region and a reporter.

[0022] In some embodiments, an HCR method is provided that includes overlap junction sites by repeated signal detection. In some embodiments, the method comprises: preparing a sample which may contain one or more targets and other non-target molecules; preparing one or more probe sets which each include i) one or more HCR initiator-labeled probes, or ii) one or more probe units which each include two or more HCR split initiators, wherein the target-binding region on the probe in each probe unit is configured to bind to overlapping or non-overlapping binding sites on the target, and the split initiators on the probe in each probe unit are configured to bind to overlapping or non-overlapping binding sites on HCR hairpins; optionally washing the sample; preparing one or more HCR amplifiers each labeled with one or more reporters and / or substrates; optionally washing the sample; optionally preparing one or more labeled probes corresponding to one or more substrates (each bound to one or more reporters); optionally washing the sample; detecting signals from one or more reporters; The process optionally includes removing one or more signals from the sample, optionally removing one or more reporters from the sample, optionally removing one or more labeled probes from the sample, optionally removing one or more amplifiers from the sample, optionally removing one or more probe sets from the sample, and optionally repeating any of the above steps in any order. A probe set includes either one or more HCR initiator-labeled probes or one or more probe units. An HCR initiator-labeled probe includes one or more target-binding regions and one or more initiators. A probe unit includes two or more HCR split-initiator probes. An HCR split-initiator probe includes a target-binding region and split initiators. The target-binding regions within a probe unit are configured to bind to overlapping or non-overlapping binding sites on the target. The splitting initiator on the probe within each probe unit is configured to bind to either an overlapping or non-overlapping binding site on the HCR hairpin. The HCR amplifier includes two or more HCR hairpins. The HCR hairpin includes an input domain comprising a single-stranded toehold and a stem portion. The HCR hairpin further includes an output domain comprising a single-stranded loop and a complementary strand to the stem portion. The HCR hairpin further includes one or more reporters and / or one or more substrates. The labeled probe includes a substrate-binding region and one or more reporters.

[0023] In some embodiments, the method includes preparing a target molecule, a first split initiator probe including a first split initiator, a second split initiator probe including a second split initiator, a first hairpin monomer including a first input domain including a first toehold and a first stem portion, a first output domain including a first hairpin loop and a complementary chain to the first stem portion, and a first hapten molecule, a second hairpin monomer including a second input domain including a second toehold and a second stem portion, a second hairpin loop and a complementary chain to the second stem portion, and a second hapten molecule, and incubating the first split initiator probe and the second split initiator probe together with the target.

[0024] In some embodiments, the method includes preparing a target molecule, a first hairpin monomer comprising a first initiator-labeled probe containing at least one initiator, a first input domain comprising a first toehold and a first stem portion, a first output domain comprising a first hairpin loop and a complementary chain to the first stem portion, and a first hapten molecule, a second hairpin monomer comprising a second input domain comprising a second toehold and a second stem portion, a second output domain comprising a second hairpin loop and a complementary chain to the second stem portion, and a second hapten molecule, and incubating the at least one initiator-labeled probe containing the at least one initiator together with the target.

[0025] In some embodiments, the method includes preparing a target molecule, a first split initiator probe including a first split initiator, a second split initiator probe including a second split initiator, a first hairpin monomer including a first input domain including a first toehold and a first stem portion, a first output domain including a first hairpin loop and a complementary chain to the first stem portion, and a substrate, a second hairpin monomer including a second input domain including a second toehold and a second stem portion, a second output domain including a second hairpin loop and a complementary chain to the second stem portion, and a substrate, and incubating the first split initiator probe and the second split initiator probe together with the target.

[0026] In some embodiments, the method includes preparing a target molecule, a first hairpin monomer comprising a first split initiator probe comprising a first split initiator, a second split initiator probe comprising a second split initiator, a first input domain comprising a first toehold and a first stem portion, a first hairpin loop and a first output domain comprising a complementary chain to the first stem portion, and a first split substrate, a second hairpin monomer comprising a second input domain comprising a second toehold and a second stem portion, a second hairpin loop and a complementary chain to the second stem portion, and a second split substrate, and incubating the first split initiator probe and the second split initiator probe together with the target molecule. [Brief explanation of the drawing]

[0027] [Figure 1A] Figure 1A shows several embodiments of insight amplification via hybridization chain reaction (HCR). [Figure 1B] Figure 1B shows several embodiments of insight amplification via hybridization chain reaction (HCR). [Figure 2A]Figure 2A shows a schematic diagram of Insights HCR using either a two-step or three-step protocol. [Figure 2B] Figure 2B shows a schematic diagram of Insights HCR using either a two-step or three-step protocol. [Figure 3A] Figure 3A shows several configurations of two adjacent target sections and two split initiator probes that hybridize to these adjacent target sections to colocalize two split initiators. [Figure 3B] Figure 3B shows several configurations of two adjacent target sections and two splitting initiator probes that hybridize to these adjacent target sections to colocalize two splitting initiators. [Figure 4A] Figure 4A shows several embodiments of segmented initiator probes co-localized by the target molecule. [Figure 4B] Figure 4B shows several embodiments of segmented initiator probes co-localized by the target molecule. [Figure 4C] Figure 4C shows several embodiments of segmented initiator probes co-localized by the target molecule. [Figure 4D] Figure 4D shows several embodiments of segmented initiator probes colocalized by the target molecule. [Figure 5A] Figure 5A shows several embodiments of fragmented initiator probes colocalized by the target complex. [Figure 5B] Figure 5B shows several embodiments of fragmented initiator probes colocalized by the target complex. [Figure 5C] Figure 5C shows several embodiments of fragmented initiator probes colocalized by the target complex. [Figure 5D] Figure 5D shows several embodiments of fragmented initiator probes colocalized by the target complex. [Figure 5E] Figure 5E shows several embodiments of fragmented initiator probes colocalized by the target complex. [Figure 6] Figure 6 shows imaging of target mRNA in whole chicken embryos using unoptimized standard probes and split initiator probes. [Figure 7A] Figure 7A shows several embodiments of target-colocalized segmented initiator probes and presents test tube data demonstrating the induction of HCR using target-colocalized segmented initiator probes. [Figure 7B] Figure 7B shows several embodiments of target-colocalized segmented initiator probes and presents test tube data demonstrating the induction of HCR using target-colocalized segmented initiator probes. [Figure 7C] Figure 7C shows several embodiments of target-colocalized segmented initiator probes and presents test tube data demonstrating the induction of HCR using target-colocalized segmented initiator probes. [Figure 8A] Figure 8A shows several embodiments of Insights HCR using a segmented initiator probe. [Figure 8B] Figure 8B shows several embodiments of Insights HCR using a segmented initiator probe. [Figure 9A] Figure 9A shows the background and SB ratio using a standard probe and a segmented initiator probe. [Figure 9B] Figure 9B shows the background and SB ratio using a standard probe and a segmented initiator probe. [Figure 9C] Figure 9C shows the background and SB ratio using a standard probe and a split initiator probe. [Figure 9D]Figure 9D shows the background and SB ratio using a standard probe and a split initiator probe. [Figure 10A] Figure 10A shows multiplex imaging of mRNA expression with a high SB ratio in fixed, whole-float chicken embryos using a split initiator probe that has not undergone probe set optimization. [Figure 10B] Figure 10B shows multiplex imaging of mRNA expression with a high SB ratio in fixed, whole-float chicken embryos using a split initiator probe that has not undergone probe set optimization. [Figure 10C] Figure 10C shows multiplex imaging of mRNA expression with a high SB ratio in fixed, whole-float chicken embryos using a split initiator probe that has not undergone probe set optimization. [Figure 10D] Figure 10D shows multiplex imaging of mRNA expression with a high SB ratio in fixed, whole-float chicken embryos using a split initiator probe that has not undergone probe set optimization. [Figure 11A] Figure 11A shows quantitative imaging of mRNA expression at intracellular resolution in a fully fixed chicken embryo using a split initiator probe. [Figure 11B] Figure 11B shows quantitative imaging of mRNA expression at intracellular resolution in a fully fixed chicken embryo using a split initiator probe. [Figure 12] Figure 12 shows several embodiments of hybridizing the first and second segmented initiator probes to a target molecule. [Figure 13]Figure 13 shows several embodiments of inducing self-assembly of HCR-amplified polymers from hairpin monomers after initiation by an HCR initiator. In Figure 13, 1050 shows a complete initiator consisting of two parts. Only a portion of the split initiator probe is shown. I1(1050) represents a complete initiator formed by two split initiator probes colocalized by a target. [Figure 14] Figure 14 shows several embodiments of HCR amplification using hairpin monomers induced by an HCR initiator. Only a portion of the split initiator probe is shown. I1 represents the complete initiator formed by two split initiator probes colocalized by the target. [Figure 15] Figure 15 shows several embodiments of the HCR mechanism using a simplified HCR hairpin monomer. [Figure 16A] Figure 16A shows several embodiments of a probe set, which includes one or more probe units and optionally one or more helper probes. [Figure 16B] Figure 16B shows several embodiments of a probe set, which includes one or more probe units and optionally one or more helper probes. [Figure 16C] Figure 16C shows several embodiments of a probe set, which includes one or more probe units and optionally one or more helper probes. [Figure 16D] Figure 16D shows several embodiments of a probe set, which includes one or more probe units and optionally one or more helper probes. [Figure 17A] Figure 17A shows several embodiments of a probe unit including a segmented initiator probe. [Figure 17B] Figure 17B shows several embodiments of a probe unit including a segmented initiator probe. [Figure 17C]Figure 17C shows several embodiments of a probe unit including a segmented initiator probe. [Figure 18A] Figure 18A shows several embodiments of the HCR amplifier. [Figure 18B] Figure 18B shows several embodiments of the HCR amplifier. [Figure 18C] Figure 18C shows several embodiments of the HCR amplifier. [Figure 18D] Figure 18D shows several embodiments of the HCR amplifier. [Figure 18E] Figure 18E shows several embodiments of the HCR amplifier. [Figure 18F] Figure 18F shows several embodiments of the HCR amplifier. [Figure 19A] Figure 19A shows several embodiments of an HCR amplifier including four HCR hairpins. [Figure 19B] Figure 19B shows several embodiments of an HCR amplifier including four HCR hairpins. [Figure 20A] Figure 20A shows several embodiments of the labeled probe. [Figure 20B] Figure 20B shows several embodiments of the labeled probe. [Figure 20C] Figure 20C shows several embodiments of the labeled probe. [Figure 20D] Figure 20D shows several embodiments of the labeled probe. [Figure 20E] Figure 20E shows several embodiments of the labeled probe. [Figure 20F] Figure 20F shows several embodiments of the labeled probe. [Figure 21A] Figure 21A shows several embodiments of a split initiator probe designed to be complementary to the overlapping region of the HCR hairpin. [Figure 21B]Figure 21B shows several embodiments of a split initiator probe designed to be complementary to the overlapping region of the HCR hairpin. [Figure 22] Figure 22 shows several embodiments of a segmented initiator probe designed to be complementary to the overlapping region of the target. [Figure 23A] Figures 23A to 23O illustrate several embodiments of removing the HCR signal from a sample. In some embodiments, any one or more steps of these methods can be combined with other methods in Figures 23A to 23O and 40A to 40N. In some embodiments, any one or more steps of the methods in Figures 23A to 23O can be combined with other methods in Figures 26A to 26T and 40A to 40N. [Figure 23B] Same as above. [Figure 23C] Same as above. [Figure 23D] Same as above. [Figure 23E] Same as above. [Figure 23F] Same as above. [Figure 23G] Same as above. [Figure 23H] Same as above. [Figure 23I] Same as above. [Figure 23J] Same as above. [Figure 23K] Same as above. [Figure 23L] Same as above. [Figure 23M] Same as above. [Figure 23N] Same as above. [Figure 23O] Same as above. [Figure 24A] Figure 24A shows several embodiments of increasing HCR signal intensity using a split initiator probe designed to be complementary to the overlapping region of the HCR hairpin. [Figure 24B]Figure 24B shows several embodiments of increasing HCR signal intensity using a split initiator probe designed to be complementary to the overlapping region of the HCR hairpin. [Figure 25A] Figure 25A shows several embodiments of multiple insight hybridization in cultured human cells via repeated reporter detection. [Figure 25B] Figure 25B shows several embodiments of multiple insight hybridization in cultured human cells via repeated reporter detection. [Figure 26A] Figures 26A–26T illustrate several embodiments of various methods for detecting one or more targets in a sample, using an HCR initiator-labeled probe and / or an HCR-divided initiator probe in combination with an HCR amplifier. In some embodiments, any one or more steps of these methods can be combined with other methods shown in Figures 26A–26T. [Figure 26B] Same as above. [Figure 26C] Same as above. [Figure 26D] Same as above. [Figure 26E] Same as above. [Figure 26F] Same as above. [Figure 26G] Same as above. [Figure 26H] Same as above. [Figure 26I] Same as above. [Figure 26J] Same as above. [Figure 26K] Same as above. [Figure 26L] Same as above. [Figure 26M] Same as above. [Figure 26N] Same as above. [Figure 26O] Same as above. [Figure 26P] Same as above. [Figure 26Q] Same as above. [Figure 26R] Same as above. [Figure 26S] Same as above. [Figure 26T] Same as above. [Figure 27] Figure 27 shows several embodiments of a segmented initiator probe designed to be complementary to the overlapping region of the HCR hairpin and to the overlapping region of the target. [Figure 28A] Figure 28A shows several embodiments of in vitro optimization examples of cooperative probe junctions that enhance split initiator HCR suppression (OFF state) and conversion (ON state). [Figure 28B] Figure 28B shows several embodiments of in vitro optimization examples of cooperative probe junctions that enhance split initiator HCR suppression (OFF state) and conversion (ON state). [Figure 28C] Figure 28C shows several embodiments of in vitro optimization examples of cooperative probe junctions that enhance split initiator HCR suppression (OFF state) and conversion (ON state). [Figure 29A] Figure 29A shows several embodiments of multiplexed HCR immunohistochemistry for imaging protein targets in formalin-fixed paraffin-embedded mouse brain sections using initiator-labeled primary antibody probes. [Figure 29B] Figure 29B shows several embodiments of multiplexed HCR immunohistochemistry for imaging protein targets in formalin-fixed paraffin-embedded mouse brain sections using initiator-labeled primary antibody probes. [Figure 29C] Figure 29C shows several embodiments of multiplexed HCR immunohistochemistry for imaging protein targets in formalin-fixed paraffin-embedded mouse brain sections using initiator-labeled primary antibody probes. [Figure 29D] Figure 29D shows several embodiments of multiplexed HCR immunohistochemistry for imaging protein targets in formalin-fixed paraffin-embedded mouse brain sections using initiator-labeled primary antibody probes. [Figure 30A]Figure 30A shows several embodiments of multiplex HCR immunohistochemistry for imaging protein targets in formalin-fixed, paraffin-embedded mouse brain sections using unlabeled primary antibody probes and initiator-labeled secondary antibody probes. [Figure 30B] Figure 30B shows several embodiments of multiplexed HCR immunohistochemistry for imaging protein targets in formalin-fixed, paraffin-embedded mouse brain sections using unlabeled primary antibody probes and initiator-labeled secondary antibody probes. [Figure 30C] Figure 30C shows several embodiments of multiplexed HCR immunohistochemistry for imaging protein targets in formalin-fixed, paraffin-embedded mouse brain sections using unlabeled primary antibody probes and initiator-labeled secondary antibody probes. [Figure 30D] Figure 30D shows several embodiments of multiplexed HCR immunohistochemistry for imaging protein targets in formalin-fixed, paraffin-embedded mouse brain sections using unlabeled primary antibody probes and initiator-labeled secondary antibody probes. [Figure 31A] Figure 31A shows several embodiments of RNA insight hybridization and protein immunohistochemistry by simultaneous HCR in formalin-fixed paraffin-embedded mouse brain sections, where HCR signal amplification was performed simultaneously across all targets using a DNA splitting initiator probe for mRNA targets and an initiator-labeled primary antibody probe for protein targets. [Figure 31B] Figure 31B shows several embodiments of RNA insight hybridization and protein immunohistochemistry by simultaneous HCR in formalin-fixed paraffin-embedded mouse brain sections, where HCR signal amplification was performed simultaneously across all targets using a DNA splitting initiator probe for mRNA targets and an initiator-labeled primary antibody probe for protein targets. [Figure 31C]Figure 31C shows several embodiments of RNA insight hybridization and protein immunohistochemistry by simultaneous HCR in formalin-fixed paraffin-embedded mouse brain sections, where HCR signal amplification was performed simultaneously across all targets using a DNA splitting initiator probe for mRNA targets and an initiator-labeled primary antibody probe for protein targets. [Figure 32A] Figure 32A shows several embodiments of RNA insight hybridization and protein immunohistochemistry by simultaneous HCR in formalin-fixed paraffin-embedded mouse brain sections, where HCR signal amplification was performed simultaneously across all targets using a DNA splitting initiator probe for mRNA targets and an unlabeled primary antibody probe and an initiator-labeled secondary antibody probe for protein targets. [Figure 32B] Figure 32B shows several embodiments of RNA insight hybridization and protein immunohistochemistry by simultaneous HCR in formalin-fixed paraffin-embedded mouse brain sections, where HCR signal amplification was performed simultaneously across all targets using a DNA splitting initiator probe for mRNA targets and an unlabeled primary antibody probe and an initiator-labeled secondary antibody probe for protein targets. [Figure 32C] Figure 32C shows several embodiments of RNA insight hybridization and protein immunohistochemistry by simultaneous HCR in formalin-fixed paraffin-embedded mouse brain sections, where HCR signal amplification was performed simultaneously across all targets using a DNA splitting initiator probe for mRNA targets and an unlabeled primary antibody probe and an initiator-labeled secondary antibody probe for protein targets. [Figure 33A] Figure 33A shows several embodiments of using HCR to mediate CARD signal amplification for various target species. [Figure 33B] Figure 33B shows several embodiments of using HCR to mediate CARD signal amplification for various target species. [Figure 33C] Figure 33C shows several embodiments of using HCR to mediate CARD signal amplification for various target species. [Figure 33D] Figure 33D shows several embodiments of using HCR to mediate CARD signal amplification for various target species. [Figure 33E] Figure 33E shows several embodiments of using HCR to mediate CARD signal amplification for various target species. [Figure 34A] Figure 34A shows several embodiments of using HCR to mediate CARD signal amplification in the case of generic target molecules and target complexes. [Figure 34B] Figure 34B shows several embodiments of using HCR to mediate CARD signal amplification in the case of generic target molecules and target complexes. [Figure 34C] Figure 34C shows several embodiments of using HCR to mediate CARD signal amplification in the case of generic target molecules and target complexes. [Figure 35-1] Figure 35-1 shows several embodiments of hapten placement in conjunction with HCR CARD signal amplification. [Figure 35-2] Figure 35-2 shows several embodiments of hapten placement in conjunction with HCR CARD signal amplification. [Figure 36A] Figure 36A shows several embodiments of HCR CARD signal amplification using substrate-labeled HCR hairpins or split substrate HCR hairpins. [Figure 36B] Figure 36B shows several embodiments of HCR CARD signal amplification using substrate-labeled HCR hairpins or split-substrate HCR hairpins. [Figure 37A]Figure 37A shows several embodiments of imaging RNA targets in formalin-fixed, paraffin-embedded human kidney sections and formalin-fixed, paraffin-embedded human liver sections using HCR-mediated CARD signal amplification. [Figure 37B] Figure 37B shows several embodiments of imaging RNA targets in formalin-fixed, paraffin-embedded human kidney sections and formalin-fixed, paraffin-embedded human liver sections using HCR-mediated CARD signal amplification. [Figure 38-1] Figure 38-1 shows several embodiments of fragmented initiator probes that are colocalized directly or indirectly by a target molecule or target complex. [Figure 38-2] Figure 38-2 shows several embodiments of fragmented initiator probes that are colocalized directly or indirectly by a target molecule or target complex. [Figure 39A] Figure 39A shows several embodiments of initiator-labeled probes that are directly or indirectly bound to a target molecule or target complex. [Figure 39B] Figure 39B shows several embodiments of initiator-labeled probes that are directly or indirectly bound to a target molecule or target complex. [Figure 39C] Figure 39C shows several embodiments of initiator-labeled probes that are directly or indirectly bound to a target molecule or target complex. [Figure 39D] Figure 39D shows several embodiments of initiator-labeled probes that are directly or indirectly bound to a target molecule or target complex. [Figure 39E] Figure 39E shows several embodiments of initiator-labeled probes that are directly or indirectly bound to a target molecule or target complex. [Figure 39F] Figure 39F shows several embodiments of initiator-labeled probes that are directly or indirectly bound to a target molecule or target complex. [Figure 39G]Figure 39G shows several embodiments of initiator-labeled probes that are directly or indirectly bound to a target molecule or target complex. [Figure 39H] Figure 39H shows several embodiments of initiator-labeled probes that are directly or indirectly bound to a target molecule or target complex. [Figure 39I] Figure 39I shows several embodiments of initiator-labeled probes that are directly or indirectly bound to a target molecule or target complex. [Figure 39J] Figure 39J shows several embodiments of initiator-labeled probes that are directly or indirectly bound to a target molecule or target complex. [Figure 39K] Figure 39K shows several embodiments of initiator-labeled probes that are directly or indirectly bound to a target molecule or target complex. [Figure 39L] Figure 39L shows several embodiments of initiator-labeled probes that are directly or indirectly bound to a target molecule or target complex. [Figure 39M] Figure 39M shows several embodiments of initiator-labeled probes that are directly or indirectly bound to a target molecule or target complex. [Figure 39N] Figure 39N shows several embodiments of initiator-labeled probes that are directly or indirectly bound to a target molecule or target complex. [Figure 40A] Figures 40A to 40N show several embodiments of removing the HCR signal from a sample. In some embodiments, any one or more steps of these methods can be combined with other methods in Figures 23A to 23O and 40A to 40N. In some embodiments, any one or more steps of the methods in Figures 40A to 40N can be combined with other methods in Figures 23A to 23O and 26A to 26T. [Figure 40B] Same as above. [Figure 40C] Same as above. [Figure 40D] Same as above. [Figure 40E] Same as above. [Figure 40F] Same as above. [Figure 40G] Same as above. [Figure 40H] Same as above. [Figure 40I] Same as above. [Figure 40J] Same as above. [Figure 40K] Same as above. [Figure 40L] Same as above. [Figure 40M] Same as above. [Figure 40N] Same as above. [Figure 41A] Figure 41A shows several embodiments of quantitative imaging of mRNA expression at intracellular resolution in formalin-fixed, paraffin-embedded mouse brain sections using a split initiator probe. [Figure 41B] Figure 41B shows several embodiments of quantitative imaging of mRNA expression at intracellular resolution in formalin-fixed, paraffin-embedded mouse brain sections using a split initiator probe. [Figure 41C] Figure 41C shows several embodiments of quantitative imaging of mRNA expression at intracellular resolution in formalin-fixed, paraffin-embedded mouse brain sections using a split initiator probe. [Figure 42A] Figure 42A shows several embodiments of initiator-labeled probes that include one or more HCR initiators. [Figure 42B] Figure 42B shows several embodiments of initiator-labeled probes that include one or more HCR initiators. [Figure 42C] Figure 42C shows several embodiments of initiator-labeled probes that include one or more HCR initiators. [Figure 42D] Figure 42D shows several embodiments of initiator-labeled probes that include one or more HCR initiators. [Figure 42E]Figure 42E shows several embodiments of initiator-labeled probes that include one or more HCR initiators. [Figure 42F] Figure 42F shows several embodiments of initiator-labeled probes that include one or more HCR initiators. [Figure 43A] Figure 43A shows several embodiments of imaging target microRNAs and mRNAs in whole-load zebrafish embryos using initiator-labeled probes. [Figure 43B] Figure 43B shows several embodiments of imaging target microRNAs and mRNAs in whole-load zebrafish embryos using initiator-labeled probes. [Figure 43C] Figure 43C shows several embodiments of imaging target microRNAs and mRNAs in whole-load zebrafish embryos using initiator-labeled probes. [Figure 44A] Figure 44A shows several embodiments of a shielding initiator-labeled probe that includes one or more shielding HCR initiators. [Figure 44B] Figure 44B shows several embodiments of a shielding initiator-labeled probe that includes one or more shielding HCR initiators. [Figure 44C] Figure 44C shows several embodiments of a shielding initiator-labeled probe that includes one or more shielding HCR initiators. [Figure 44D] Figure 44D shows several embodiments of a shielding initiator-labeled probe that includes one or more shielding HCR initiators. [Figure 44E] Figure 44E shows several embodiments of a shielding initiator-labeled probe that includes one or more shielding HCR initiators. [Figure 44F] Figure 44F shows several embodiments of a shielding initiator-labeled probe that includes one or more shielding HCR initiators. [Figure 44G]Figure 44G shows several embodiments of a shielding initiator-labeled probe that includes one or more shielding HCR initiators. [Figure 44H] Figure 44H shows several embodiments of a shielding initiator-labeled probe that includes one or more shielding HCR initiators. [Figure 44I] Figure 44I shows several embodiments of a shielding initiator-labeled probe that includes one or more shielding HCR initiators. [Figure 44J] Figure 44J shows several embodiments of a shielding initiator-labeled probe that includes one or more shielding HCR initiators. [Figure 44K] Figure 44K shows several embodiments of a shielding initiator-labeled probe that includes one or more shielding HCR initiators. [Figure 44L] Figure 44L shows several embodiments of a shielding initiator-labeled probe that includes one or more shielding HCR initiators. [Figure 44M] Figure 44M shows several embodiments of a shielding initiator-labeled probe that includes one or more shielding HCR initiators. [Figure 44N] Figure 44N shows several embodiments of a shielding initiator-labeled probe that includes one or more shielding HCR initiators. [Figure 44O] Figure 44O shows several embodiments of a shielding initiator-labeled probe that includes one or more shielding HCR initiators. [Figure 44P] Figure 44P shows several embodiments of a shielding initiator-labeled probe that includes one or more shielding HCR initiators. [Figure 44Q] Figure 44Q shows several embodiments of a shielding initiator-labeled probe that includes one or more shielding HCR initiators. [Figure 44R]Figure 44R shows several embodiments of a shielding initiator-labeled probe that includes one or more shielding HCR initiators. [Figure 44S] Figure 44S shows several embodiments of a shielding initiator-labeled probe that includes one or more shielding HCR initiators. [Figure 44T] Figure 44T shows several embodiments of a shielding initiator-labeled probe that includes one or more shielding HCR initiators. [Figure 44U] Figure 44U shows several embodiments of a shielding initiator-labeled probe that includes one or more shielding HCR initiators. [Figure 44V] Figure 44V shows several embodiments of a shielding initiator-labeled probe that includes one or more shielding HCR initiators. [Figure 44W] Figure 44W shows several embodiments of a shielding initiator-labeled probe that includes one or more shielding HCR initiators. [Figure 44X] Figure 44X shows several embodiments of a shielding initiator-labeled probe that includes one or more shielding HCR initiators. [Figure 44Y] Figure 44Y shows several embodiments of a shielding initiator-labeled probe that includes one or more shielding HCR initiators. [Figure 44Z] Figure 44Z shows several embodiments of a shielding initiator-labeled probe that includes one or more shielding HCR initiators. [Figure 45A] Figure 45A shows several embodiments of an example of using a shielding initiator-labeled probe to reduce background noise. [Figure 45B] Figure 45B shows several embodiments of an example of using a shielding initiator-labeled probe to reduce background noise. [Figure 46A]Figure 46A shows several embodiments of the shielding initiator when used in conjunction with a shielding initiator labeled probe. [Figure 46B] Figure 46B shows several embodiments of the shielding initiator when used in conjunction with a shielding initiator labeled probe. [Figure 46C] Figure 46C shows several embodiments of the shielding initiator when used in conjunction with a shielding initiator labeled probe. [Figure 46D] Figure 46D shows several embodiments of the shielding initiator when used in conjunction with a shielding initiator labeled probe. [Figure 46E] Figure 46E shows several embodiments of the shielding initiator when used in conjunction with a shielding initiator labeled probe. [Figure 46F] Figure 46F shows several embodiments of the shielding initiator when used in conjunction with a shielding initiator labeled probe. [Figure 46G] Figure 46G shows several embodiments of the shielding initiator when used in conjunction with a shielding initiator labeled probe. [Figure 46H] Figure 46H shows several embodiments of the shielding initiator when used in conjunction with a shielding initiator labeled probe. [Figure 46I] Figure 46I shows several embodiments of the shielding initiator when used in conjunction with a shielding initiator labeled probe. [Figure 46J] Figure 46J shows several embodiments of the shielding initiator when used in conjunction with a shielding initiator labeled probe. [Figure 46K] Figure 46K shows several embodiments of the shielding initiator when used in conjunction with a shielding initiator labeled probe. [Figure 46L] Figure 46L shows several embodiments of the shielding initiator when used in conjunction with a shielding initiator labeled probe. [Figure 46M] Figure 46M shows several embodiments of the shielding initiator when used in conjunction with a shielding initiator labeled probe. [Figure 47] Figure 47 shows an embodiment of the sequence used for dehybridizing HCR hairpins from HCR polymers in Figure 25A (Table 2). [Modes for carrying out the invention]

[0028] Hybridization chain reactions (HCRs) are elicited hybridization methods for nucleic acid molecules, initiated from metastable hairpin monomers or other metastable nucleic acid structures. See, for example, Non-Patent Document 1, each incorporated herein by reference in its entirety, as well as Patent Documents 1 (filed March 22, 2005), 2 (filed January 31, 2012), 3 (filed August 13, 2013), and Patent Document 4 (filed June 30, 2017). In a simplified version of this process, when elicited by a nucleic acid initiator strand, a metastable hairpin monomer undergoes a chain reaction of hybridization events to form a nicked double-stranded polymer. The hairpin monomer stores energy to drive the polymerization process in its single-stranded loops and toeholds.

[0029] HCR may contain two or more metastable hairpin monomers. Each hairpin monomer has at least one single-stranded toehold, single-stranded loop, and double-stranded stem. The energy that drives the self-assembly cascade is stored within the single-stranded loop and toehold segments of the hairpin.

[0030] Each monomer is trapped in a kinetic trap, preventing the system from rapidly reaching equilibrium. That is, monomer pairs cannot hybridize with each other in the absence of an initiator. The introduction of an initiator chain causes the monomers to undergo a chain reaction of hybridization events, forming a nicked double-stranded polymer. By utilizing HCR, for example, the presence of a target analyte in a sample can be detected by detecting the analyte with a probe containing an HCR initiator, where the HCR initiator induces HCR signal amplification. HCR signal amplification increases the signal level above the background generated from the sample, making it possible to increase the SB ratio for molecular detection and imaging applications.

[0031] Embodiments of HCR are provided herein. Sample analysis methods using hybridization chain reaction (HCR) may include one, two, or all three of the following embodiments: 1) repeated signal detection, 2) overlapping binding sites, and 3) catalytic reporter deposition (CARD).

[0032] In some embodiments of the method, a sample that may contain up to N targets and other non-target molecules is combined with N probe sets, each containing one or more probe units, each containing two or more HCR splitting initiators (each targeting one of the N target species). M HCR amplifiers (for M ≤ N; each labeled with a separate reporter) corresponding to M of the N probe sets are added. Then, M signals corresponding to the M reporters are detected. Each step of the method is repeated until a signal has been detected for all N targets.

[0033] In some embodiments of the method, a sample that may contain one or more targets and other non-target molecules is combined with one or more probe sets, each containing one or more probe units, each containing two or more HCR splitting initiators. One or more HCR amplifiers (each labeled with one or more reporters) Add (a substance) and detect one or more signals from one or more reporters.

[0034] In some embodiments of the method, a sample that may contain up to N targets and other non-target molecules is combined with N probe sets, each containing one or more probe units, each containing two or more HCR splitting initiators. N HCR amplifiers (each labeled with a different substrate) corresponding to the N probe sets, and M labeled probes (if M ≤ N; each bound to a different reporter) corresponding to M of the N separate substrates are added. Then, M signals corresponding to the M separate reporters are detected. Each step of the above method is repeated until signal detection has been performed for all N targets. In some embodiments, a repeated signal detection method is provided. The above method involves combining a sample, which may contain one or more targets and other non-target molecules, with a substrate-labeled hairpin, one or more probe sets, each containing one or more probe units, each containing two or more HCR splitting initiators, one or more HCR amplifiers corresponding to one or more probe sets (each labeled with a substrate), and one or more labeled probes corresponding to one or more substrates (each bound to a reporter). Then, one or more signals corresponding to one or more reporters are detected. Each step of the above method is repeated once or more times in any order.

[0035] In some embodiments of the reporter and / or substrate-labeled hairpin repeat signal detection method, a sample that may contain one or more targets and other non-target molecules is combined with one or more HCR probe sets, each containing one or more probe units, each containing two or more HCR splitting initiators; one or more HCR amplifiers corresponding to one or more probe sets (each labeled with one or more reporters and / or one or more substrates); and one or more labeled probes corresponding to one or more substrates (each bound to one or more reporters). One or more signals are then detected. Each step of the above method is repeated in any order.

[0036] In some embodiments of the reporter and / or substrate-labeled hairpin repeat signal detection method, a sample that may contain one or more targets and other non-target molecules is combined with one or more HCR probe sets, each containing one or more probe units, each containing two or more HCR splitting initiators. One or more HCR amplifiers that directly or indirectly produce one or more signals are added, and one or more signals are detected. Each step of the above method can be performed once or multiple times in any order.

[0037] In some embodiments of the method, a sample that may contain a target and other non-target molecules is combined with a probe set comprising one or more probe units, each containing two or more HCR splitting initiators. The target-binding region on the probe within each probe unit is configured to bind to the overlap-binding site on the target. Subsequently, an HCR amplifier labeled with a reporter and / or substrate, along with a labeled probe (bound to the reporter) corresponding to the substrate as desired, is added, and the signal from the reporter is detected. In some embodiments, the amplifier may be labeled with the reporter instead, and labeling on the probe may be optional.

[0038] In some embodiments of the method, a sample that may contain a target and other non-target molecules is combined with a probe set comprising one or more probe units, each containing two or more HCR splitting initiators. The splitting initiators on the probe within each probe unit are configured to bind to overlapping binding sites on HCR hairpins. An HCR amplifier labeled with a reporter and / or substrate, and optionally a labeled probe (bound to the reporter) corresponding to the substrate, are added. The signal from the reporter is then detected. In some embodiments, the amplifier may be labeled with the reporter instead, and labeling on the probe may be optional.

[0039] In some embodiments of the method, a sample that may contain one or more targets and other non-target molecules is combined with one or more probe sets, each containing one or more probe units, each containing two or more HCR splitting initiators. The target-binding region on the probe within each probe unit is configured to bind to overlapping or non-overlapping binding sites on the target. The splitting initiator on the probe within each probe unit is configured to bind to overlapping or non-overlapping binding sites on the HCR hairpin. One or more HCR amplifiers, each labeled with one or more reporters and / or substrates, and one or more optionally labeled probes corresponding to (optional) one or more substrates (each bound to one or more reporters) are added. Signals from one or more reporters are detected. Each step of the above method can be repeated in any order. In some embodiments, the amplifiers are labeled with reporters instead, and labeling on the probes is optional.

[0040] In some embodiments of the method, a first split initiator probe containing a first split initiator, a second split initiator probe containing a second split initiator, a first hairpin monomer labeled with 0, 1, or more haptens, a second hairpin monomer labeled with 0, 1, or more haptens, and a target molecule are combined. As a result, the first split initiator probe binds to the target molecule, and the second split initiator probe binds to the target molecule. The first hairpin monomer binds to both the first and second split initiators, and the second hairpin monomer binds to the first hairpin monomer. An anti-hapten molecule labeled with one or more reporter compounds is prepared. The reporter compounds are CARD-mediated enzymes. One or more CARD substrates are prepared, and the signals from one or more deposited reporters generated from the CARD substrates by the CARD-mediated enzymes are measured.

[0041] In some embodiments of the method, a combination is made of at least one initiator-labeled probe containing at least one initiator, a first hairpin monomer labeled with 0, 1, or more haptens, a second hairpin monomer labeled with 0, 1, or more haptens, and 0, 1, or more target molecules. As a result, the at least one initiator-labeled probe containing at least one initiator binds to the 0, 1, or more target molecules. The first hairpin monomer binds to the at least one initiator, and the second hairpin monomer binds to the first hairpin monomer. An anti-hapten molecule labeled with one or more reporter compounds is prepared. The reporter compounds are CARD-mediated enzymes. One or more CARD substrates are prepared, and the signal from one or more deposited reporters generated from the CARD substrates by the CARD-mediated enzymes is measured.

[0042] In some embodiments of the method, a first split initiator probe containing a first split initiator, a second split initiator probe containing a second split initiator, a first hairpin monomer containing 0, 1, or more substrates, a second hairpin monomer containing 0, 1, or more substrates, and a target molecule are combined. As a result, the first split initiator probe binds to the target molecule, and the second split initiator probe binds to the target molecule. The first hairpin monomer binds to both the first and second split initiators, and the second hairpin monomer binds to the first hairpin monomer. A substrate-binding region labeled with one or more reporter compounds is prepared. The substrate-binding region binds to the substrate. The reporter compound is a CARD-mediated enzyme. One or more CARD substrates are prepared, and the signal from one or more deposited reporters generated from the CARD substrates by the CARD-mediated enzyme is measured.

[0043] In some embodiments of the method, a first split initiator probe containing a first split initiator, a second split initiator probe containing a second split initiator, a first hairpin monomer containing a first split substrate, a second hairpin monomer containing a second split substrate, and a target molecule are combined. As a result, the first split initiator probe binds to the target molecule, and the second split initiator probe binds to the target molecule. The first hairpin monomer binds to both the first and second split initiators, and the second hairpin monomer binds to the first hairpin monomer, resulting in a complete substrate containing the first and second split substrates. A substrate-binding region labeled with one or more reporter compounds is added. The substrate-binding region binds to the complete substrate. The reporter compound is an enzyme that mediates CARD. Prepare one or more CARD substrates and measure the signals from one or more deposition reporters generated from the CARD substrates by a CARD-mediated enzyme.

[0044] In some embodiments, the HCR process can be a split HCR process. In some embodiments, split initiator probe pairs or split initiator probe sets may have a small amount of overlap between them. In some embodiments, the initiator may be shorter or longer than the input domain of the HCR hairpin and / or incompletely complementary to the input domain of the hairpin, but may hybridize to the input domain of the hairpin, opening the hairpin and initiating the HCR polymerization cascade. In some embodiments provided herein, the split initiator in the probe unit is complementary to the overlapping region of the HCR hairpin (e.g., a region overlapping by one, two, or more nucleotides) or substantially complementary to the HCR hairpin (e.g., complementary except for 0, 1, 2, a few, or several mismatches).

[0045] In some embodiments, the initiator on the initiator-labeled probe can be shielded to enhance penetration into the sample (to increase the signal) and / or to reduce nonspecific binding of the probe in the sample (to reduce background). In some embodiments, the HCR process may include hairpin labeling that includes a substrate that serves to recruit a reporter body containing an enzyme that mediates catalytic reporter deposition (CARD).

[0046] In some embodiments, the labeled probe bound to the reporter molecule (or reporter body) does not strongly bind to the divided substrate on individual hairpins, but after HCR polymerization, the HCR hairpin label can be made to include the divided substrate so that the colocalized complete substrate strongly binds to the reporter molecule (or reporter body containing the enzyme that mediates CARD signal amplification) and the labeled probe bound to it, and adjacent hairpins in the HCR amplification polymer colocalize the complete substrate.

[0047] In some embodiments, the HCR process may include one or more haptens (see, for example, Figures 33A-33E, 34A-34C, 35-1-35-2, and 36A-36B) that mediate an additional layer of signal amplification via catalytic reporter deposition (CARD).

[0048] In some embodiments, a repeat signal detection method using reporter-labeled hairpins is provided. In some embodiments, the method comprises: a) preparing a sample which may contain up to N targets and other non-target molecules; b) preparing N probe sets, each containing one or more probe units, each containing two or more HCR splitting initiators (each targeting one of the N target species); c) optionally washing the sample; d) preparing M HCR amplifiers corresponding to M of the N probe sets (if M ≤ N; each labeled with a different reporter); e) optionally washing the sample; f) detecting M signals corresponding to the M reporters; g) removing the M signals from the sample; and h) optionally repeating one or more of steps b to g until signal detection is performed for all N targets.

[0049] In some embodiments, a repeat signal detection method using reporter-labeled hairpins is provided. In some embodiments, the method comprises: a) preparing a sample which may contain up to N targets and other non-target molecules; b) preparing N probe sets, each containing one or more initiator-labeled probes, each containing one or more initiators (each targeting one of the N target species); c) optionally washing the sample; d) preparing M HCR amplifiers corresponding to M of the N probe sets (if M ≤ N; each labeled with a different reporter); e) optionally washing the sample; f) detecting M signals corresponding to the M reporters; g) removing the M signals from the sample; and h) optionally repeating one or more of steps b to g until signal detection is performed for all N targets.

[0050] In some embodiments, a repeat signal detection method using reporter-labeled hairpins is provided. In some embodiments, the method comprises: a) preparing a sample which may contain up to N targets and other non-target molecules; b) preparing N probe sets (each targeting one of the N target species), each containing either 1) one or more HCR initiator-labeled probes, each containing one or more initiators, or 2) one or more probe units, each containing two or more HCR splitting initiators; c) optionally washing the sample; d) preparing M HCR amplifiers corresponding to M of the N probe sets (if M ≤ N; each is labeled with a different reporter); e) optionally washing the sample; f) detecting M signals corresponding to the M reporters; g) removing the M signals from the sample; and h) optionally repeating one or more of steps b to g until signal detection is performed for all N targets.

[0051] In some embodiments, the method comprises: a) preparing a sample that may contain up to N targets and other non-target molecules; b) preparing N probe sets, each containing one or more probe units, each containing two or more HCR splitting initiators (each targeting one of the N target species); c) washing the sample; d) preparing M HCR amplifiers corresponding to M of the N probe sets (if M ≤ N; each labeled with a separate reporter); e) washing the sample; f) detecting M signals corresponding to the M reporters; g) removing the M signals from the sample; and h) repeating one or more of steps b to g until signal detection is performed for all N targets.

[0052] In some embodiments, the method includes: a) preparing a sample which may contain one or more targets and other non-target molecules; b) preparing one or more probe sets which each contain one or more probe units which each contain two or more HCR splitting initiators; c) optionally washing the sample; d) preparing one or more HCR amplifiers (each labeled with one or more reporters); e) optionally washing the sample; f) detecting one or more signals from one or more reporters; g) optionally removing one or more probe sets from the sample; h) optionally removing one or more HCR amplifiers from the sample; i) optionally removing one or more reporters from the sample; and j) optionally removing one or more signals from the sample. The above process (all or a subset of the above steps) can be repeated as needed.

[0053] Definitions and Embodiments As used herein, “nucleic acid” encompasses any form of DNA oligomer and / or RNA oligomer. Nucleic acid also encompasses analogues of DNA or RNA modified in either the base or the backbone. For example, as used herein, nucleic acid encompasses the use of peptide nucleic acid (PNA). The term “nucleic acid” also encompasses chimeric molecules. This expression includes not only artificial constructs but also derivatives, etc. This expression includes, for example, any one or more of DNA, RNA, 2'OMe-RNA, LNA, XNA, synthetic nucleic acid analogues, and PNA.

[0054] The term "sticky end" refers to a nucleic acid sequence that can hybridize with a complementary nucleic acid sequence. The secondary structure of a sticky end is such that it can hybridize with a complementary nucleic acid under appropriate reaction conditions without undergoing a conformational change. Typically, sticky ends are single-stranded nucleic acids.

[0055] A "monomer" is an individual nucleic acid oligomer. Typically, at least two monomers are used in a hybridization chain reaction, but three, four, five, six, or more monomers may also be used. Typically, each monomer contains at least one region that is complementary to at least one other monomer used in the HCR reaction.

[0056] The composition may include a first hairpin monomer (1510) comprising a) a first input domain (1852) including a first toehold (1851) and a first stem portion, b) a first output domain (1854) including a first hairpin loop (1853) and a complementary chain to the first stem portion, and c) a first reporter molecule (1850) (see, for example, Figure 13). The composition may further include a second hairpin monomer (1610) comprising a) a second input domain (1952) including a second toehold (1951) and a second stem portion, b) a second output domain (1954) including a second hairpin loop (1953) and a complementary chain to the second stem portion, and c) a second reporter molecule (1950).

[0057] In some embodiments, the monomers are "metastable," meaning that in the absence of an initiator, it is kinetically unlikely that a monomer will bind to another monomer containing a complementary region. "HCR" monomers are monomers that can aggregate to form polymers when exposed to an initiator nucleic acid.

[0058] As used herein, "polymerization" means the bonding of two or more monomers to form a polymer. A "polymer" may include covalent, non-covalent, or both. For example, in some embodiments, two monomers alternately hybridize to form a polymer. Polymers containing double-chain polymers can be formed. In this specification, the polymer is also referred to as the "HCR product".

[0059] An "initiator-labeled probe" includes one or more target-binding domains and one or more HCR initiators (e.g., Figures 39A-39N and 42A-42F).

[0060] A segmented initiator probe comprises one or more target-binding domains and one or more segmented initiator domains (e.g., Figures 3A-3B, 5A-5E, and 38-1-38-2). As used herein, the terms "segmented initiator probe" and "segmented initiator probe" are interchangeable.

[0061] The term "complete initiator" derives from the fact that a combination of two or more fragmented initiators can initiate HCR polymerization when they colocalize. Some initiators contain a nucleic acid region complementary to the initiator complementary region of the HCR monomer. Fractionated initiators are insufficient on their own to induce HCR polymerization, but can induce HCR polymerization when they colocalize with one (or more) other fragmented initiators to form a complete initiator.

[0062] A "probe unit" includes two or more segmented initiator probes so that segmented initiator domains on segmented initiator probes within the probe unit can colocalize to form a complete initiator.

[0063] In Table 1, the following terms are presented as alternative options (whose scope may vary depending on the context) for terms used herein. The broadest term disclosure represents not only the broadest concept but also narrower concepts as used herein. This approach and table are used simply as abbreviations to more concisely illustrate both options.

[0064] [Table 1]

[0065] As used herein, “substrate” may refer to 1) a substrate domain on a hairpin that functions as a binding site for the substrate binding region of a labeled probe (e.g., domain “e” in Figures 18C, 18E, and 36A), or 2) a CARD substrate that acts as a deposition reporter mediated by the CARD enzyme (e.g., Figures 33A–33E, 34A–34C, 36A–36B). To avoid confusion, usage (2) substrate is intended to be written as “CARD substrate” so that the word CARD is attached to “substrate” for the context.

[0066] The section headings used herein are for structural purposes only and should be understood not to limit the subject matter of the inventions described. All cited and / or similar documents in this application, including but not limited to patents, patent applications, articles, books, papers, and internet web pages, are expressly incorporated by reference in their entirety for any purpose. If the definition of a word in an cited reference appears to differ from the definition provided in this instruction, the definition provided in this instruction shall prevail. It should be understood that there is an implicit “approximately” before the temperature, concentration, time, etc., discussed in this instruction, so that even minor and insubstantial deviations are within the scope of this instruction as described herein. In this application, unless otherwise specified, the use of the singular includes the plural. Also, the use of “comprise,” “comprises,” “comprising,” “contain,” “contains,” “containing,” “include,” “includes,” and “including” is not intended to be limiting. Please understand that the above summary and the following detailed description are for illustrative and illustrative purposes only, and not limiting. Unless otherwise noted, the technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art in which the invention pertains. See, for example, Non-Patent Document 2; Non-Patent Document 3. Please understand that the above summary and the following detailed description are for illustrative and illustrative purposes only, and not limiting the invention as claimed. In this application, unless otherwise noted, the use of the singular includes the plural. In this application, unless otherwise noted, the use of "or" means "and / or". Furthermore, the use of the word "including," as well as other forms such as "includes" and "included," is non-limiting.Furthermore, unless otherwise specified, terms such as "element" or "component" encompass both elements and components consisting of one unit and elements and components consisting of two or more subunits. Also, the use of the term "portion" may include a part of a portion or the entirety of a portion.

[0067] In some embodiments, one or more optional elements of any one or more figures in this specification may be combined with one or more other optional steps of any one or more figures in this specification.

[0068] In some embodiments, one or more elements, as shown in one or more of the figures herein, can be combined with one or more other steps, as shown in one or more of the figures herein.

[0069] In some embodiments, one or more optional elements, as shown in one or more figures herein, can be combined with one or more other optional steps, as shown in one or more figures herein.

[0070] The term “process” refers to an action that occurs and / or can be performed. Note that multiple “processes” may occur simultaneously, in overlapping periods, and / or sequentially. Unless otherwise stated (expressly or implicitly in the context of this disclosure), all options are as intended herein. Furthermore, unless otherwise stated, processes may be performed in different orders, repeated, or have additional intervening or overlapping processes added.

[0071] Analysis of target molecules in a sample via hybridization chain reaction In some embodiments, the target is detected using a probe set comprising one or more initiator-labeled probes, each containing one or more HCR initiators (e.g., Figures 39A-39N, 42A-42F, and 43A). In some embodiments, the target in the sample is detected using a probe set comprising one or more probe units (e.g., see the probe sets in Figures 8A-8B and 16A-16D), where the probe unit comprises two or more HCR split initiator probes (e.g., see the probe units in Figures 3A-5E and 17A-17C), each HCR split initiator probe comprising a target-binding region and a split initiator (e.g., see the split initiator probes in Figures 12 and 17A-17C). In some embodiments, each probe within the probe unit binds to adjacent cognitive binding sites on the target, resulting in colocalization of segmented initiators and the formation of a complete HCR initiator capable of hybridizing to an HCR hairpin to induce HCR signal amplification (see, for example, the complete HCR initiators in Figures 3A–5E, 8A–8B, 16A–16D, 21A–22, 27, and 38-1–38-2).

[0072] In some embodiments, HCR signal amplification increases the SB ratio for molecular detection and imaging applications by increasing the signal above the background generated from the sample by approximately 5x, 10x, 15x, 20x, 25x, 30x, 40x, 50x, 75x, 100x, 500x, 1000x, or 2000x, or by a range defined by any two of the above values.

[0073] In some embodiments, the target binding region within the probe unit is configured to bind to the overlapping or non-overlapping region of the target (see, for example, Figures 8A-8B, 21A-21B, 22, and 27). In some embodiments, the segmented initiator within the probe unit is designed to hybridize to the overlapping or non-overlapping region of the HCR hairpin (see, for example, Figures 8A-8B, 21A-22, 27, and 28). Individual probes that bind nonspecifically in the sample do not cause the formation of a complete HCR initiator through colocalization, thus suppressing HCR signal amplification. The HCR amplifier includes two or more HCR hairpins (see, for example, the HCR amplifiers in Figures 8A-8B, 18A-18F, and 19A-19B). In some embodiments, each HCR hairpin includes an input domain having a single-strand toehold and a stem, and an output domain having a single-strand loop and a complementary chain to the stem (see, for example, the HCR hairpins in Figures 8A-8B, 13, 14, 18A-18F, and 19A-19B). In some embodiments, one or more HCR initiators on an initiator-labeled probe initiate a chain reaction of polymerization steps, where the initiator hybridizes to the input domain of a first HCR hairpin, opening the first hairpin and exposing its output domain, which then hybridizes to the input domain of a second HCR hairpin, opening the second hairpin and exposing its output domain, and so on, until the hairpins polymerize to form an HCR-amplifying polymer anchored to the target (see, for example, the amplified polymers in Figures 13, 14, 19A-19B, 29A, 30A, 33A, 33C-33E, 34A, and 41A). In the absence of complete HCR initiators, the HCR hairpins are kinetically trapped and do not polymerize, suppressing background noise.However, if a segmented initiator probe within the probe unit binds to a nearby cognitive binding site on the target and forms a complete HCR initiator through colocalization, the complete HCR initiator initiates a chain reaction of polymerization steps, in which the complete initiator hybridizes to the input domain of the first HCR hairpin, opening the first hairpin and exposing its output domain, and this output domain hybridizes to the input domain of the second HCR hairpin, opening the second hairpin and exposing its output domain, and so on, until the hairpin polymerizes and becomes an HCR amplified polymer anchored to the target (see, for example, the amplified polymers in Figures 8A, 13, 14, 18C-18F, and 19A-19B). In some embodiments, the HCR hairpins further include one or more labels, each containing a reporter, or each containing a substrate (or split substrate) that recruits a labeled probe containing one or more reporters (see, for example, the reporter-labeled HCR hairpins, substrate-labeled HCR hairpins, and split substrate-labeled HCR hairpins in Figures 18A–18F). In some embodiments, the 0, 1, or more substrates on the HCR hairpins may include haptens (see, for example, Figures 35-1–35-2) that mediate an additional layer of signal amplification via catalytic reporter deposition (CARD) (see, for example, Figures 33A–33E, 34A–34D, and 37). In some embodiments, the labeled probe includes one or more reporters and further includes a substrate-binding region complementary to the substrate on the HCR hairpin or to the colocalized complete substrate within the HCR amplified polymer (see, for example, the labeled probes in Figures 20A–20F and 36A–36B). In some embodiments, a signal is generated by one or more reporters bound to an HCR amplified polymer anchored to a target in the sample. In some embodiments, the signal is removed from the sample (see, for example, Figure 23). In some embodiments, the HCR signal is generated, detected, and removed from the same sample one or more times (see, for example, Figures 23A–23O and 40A–40N).

[0074] In some embodiments, any one or more steps provided in any of the figures provided herein can be combined to form one of the other methods provided herein. Where used herein, unless otherwise specified, a general designation for a series of figures (e.g., Figure 18) refers to all the different figures contained within that number (e.g., Figures 18A to 18F), each of them combined, one or more of them, or each of them selectively.

[0075] HCR Initiator The initiator-labeled probe comprises one or more HCR initiators capable of initiating an HCR polymerization cascade. In some embodiments, the initiator is fully complementary to the input domain of the HCR hairpin so as to hybridize to the input domain of the hairpin, opening the hairpin and initiating the HCR polymerization cascade. In some embodiments, the initiator is partially complementary to the input domain of the HCR hairpin, but sufficiently complementary so as to hybridize to the input domain of the hairpin, opening the hairpin and initiating the HCR polymerization cascade. In some embodiments, the 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 can still hybridize to the input domain of the hairpin, opening the hairpin and initiating the HCR polymerization cascade. In some embodiments, the HCR initiator may have 60%, 70%, 80%, 90%, or 100% complementarity with the input domain of the HCR hairpin so as to hybridize to the input domain of the hairpin, opening the hairpin and initiating the HCR polymerization cascade. In some situations, initiator-labeled probes containing one or more initiators may have reduced penetration into the sample because the initiators nonspecifically bind to the surface of the sample. In some situations, initiator-labeled probes containing one or more initiators may cause increased background because the initiators nonspecifically bind to DNA, RNA, proteins, or other molecules in the sample. In some embodiments, the initiator on the initiator-labeled probe is shielded by base pairing to enhance penetration into the sample (to increase the signal) and / or to reduce nonspecific binding of the probe in the sample (to reduce background) (see, for example, Figures 44A–44Z, 45A–45B, and 46A–46M).In some embodiments, the initiator can be shielded by a hairpin structure (e.g., Figures 44A-44E, 44K-44L, 44O-44P, 44S-44T, 44W-44X, 46A-46E, and 46M). In some embodiments, the initiator can be shielded by one or more auxiliary oligos (e.g., Figures 44F-44J, 44M-44N, 44Q-44R, 44U-44V, 44Y-44Z, 46F-46J). In some embodiments, the initiator can be shielded by self-complementarity within the oligo containing the initiator, and / or complementarity to one or more auxiliary chains (e.g., Figures 44A-44Z and 46A-46M).

[0076] Automated background suppression using HCR splitting initiator probes In some embodiments, the HCR initiator (I1 or I2) is split into a pair of probes (see, for example, Figures 8 and 12), so the split initiator probes automatically suppress background noise. In some embodiments, if the probes specifically bind to the target at adjacent cognitive binding sites, the target colocalizes the two probes within the probe pair to form a complete HCR initiator. In some embodiments, individual probes that bind nonspecifically do not induce HCR because each probe only contains a portion of the HCR initiator, and HCR signal amplification is not induced unless the complete HCR initiator is colocalized.

[0077] Automatic background suppression using HCR hairpin In some embodiments, HCR hairpins automatically suppress background because they do not polymerize in the absence of an HCR initiator (I1 or I2) due to kinetic trapping. In some embodiments, when both probes in a split initiator probe pair specifically bind to adjacent cognitive binding sites on the target, the resulting colocalized complete HCR initiator (I1 or I2) induces the growth of an anchored HCR-amplified polymer (see, e.g., Figure 8). Individual HCR hairpins that bind nonspecifically within the sample do not induce HCR because they are kinetically trapped.

[0078] Automated background suppression using HCR split initiator probes and HCR hairpins By combining an HCR splitting initiator probe for target detection with an HCR amplification hairpin for signal amplification, automated background suppression becomes possible throughout the protocol, preventing background amplification even if the reagent binds nonspecifically within the sample.

[0079] A complete HCR initiator divided into two or more split initiator probes. It has been stated that each split initiator probe set generates a complete HCR initiator as a probe unit (see, for example, FIG. 17). In some embodiments, a complete HCR initiator (I1 or I2) is generated by split initiator probe pairs each having a part of the complete HCR initiator such that (fractions f1 for probe P1 and f2 for probe P2 such that f1 + f2 = 1); in this case, the probe unit is two split initiator probes (see, for example, FIG. 17A). The fractions f1 and f2 are sufficiently small compared to the complete HCR initiator so that HCR signal amplification is suppressed if the complete HCR initiators are not co-localized by the target (for example, f1 = 0.5, f2 = 0.5; or f1 = 0.45, f2 = 0.55; or f1 = 0.4, f2 = 0.6).

[0080] In some embodiments, the HCR initiator (I1 or I2) is split into three split initiator probes (fractions f1 for probe P1, f2 for probe P2, and f3 for probe 3 such that f1 + f2 + f3 = 1); in this case, the probe unit consists of three split initiator probes. In some embodiments, the HCR initiator (I1 or I2) is split into N split initiator probes (fractions f1 for probe P1, f2 for probe P2, ..., fN for probe PN such that f1 + f2 +... fN = 1; see, for example, FIG. 17B), where N = 2, 3, 4, or more; in this case, the probe unit consists of N split initiator probes. For any of these values of N, HCR signal amplification is suppressed if the complete HCR initiators are not co-localized by the target.

[0081] In some embodiments, a complete HCR initiator is generated by a probe pair (or set) each having a portion of an HCR initiator such that the sum of fraction f1 for probe P1 and fraction f2 for probe P2 (f1+f2) is sufficiently close to 1 (e.g., f1=0.47, f2=0.47, f1+f2=0.94), so that HCR signal amplification is induced by a colocalized complete initiator obtained from the binding of the probe pair to adjacent cognitive binding sites on the target. In some embodiments, a segmented initiator probe within a probe unit generates a complete HCR initiator equivalent to 100% HCR initiator. In some embodiments, a segmented initiator probe within a probe unit generates a proportion of HCR initiators sufficient to provide efficient HCR signal amplification compared to the rate of signal amplification when no segmented initiator probes are present or when individual segmented initiator probes are present but not colocalized by the target. In some embodiments, the percentage of complete HCR initiators generated by colocalized probes within a probe unit is 99%, 95%, 90%, 80%, or 60%, and includes any range of complete HCR initiators greater than one of the above values ​​or defined between any two of the above values. In some embodiments, a probe unit includes two, three, four, five, or more split initiator probes. In some embodiments, the split initiators within a probe unit are sufficient to function as HCR initiators when colocalized by the probes within the probe unit binding to adjacent cognitive binding sites on the target. In some embodiments, an HCR initiator may have a sequence of a specific length (e.g., 15 nucleotides), but the split initiators within a probe unit do not need to be exactly the same length. For example, in some embodiments, the combined length of the split initiators may be 14 or 13 nucleotides if they still function as an HCR initiator when colocalized.

[0082] In some embodiments, any two or more split initiators can be used as long as they together perform the function of the HCR initiator.

[0083] In some embodiments, the total (f1 + f2) of fraction f1 for probe P1 and fraction f2 for probe P2 is close enough to 1 (e.g., f1 = 0.45, f2 = 0.47, f1 + f2 = 0.92) such that HCR signal amplification is induced by the co-localized complete initiator obtained from the binding of probe pairs to adjacent cognate binding sites on the target. A complete HCR initiator is generated by a probe pair, each having a portion of the HCR initiator and further including one or a few or several sequence modifications. In some embodiments, the split initiator probes within a probe unit generate a complete HCR initiator that has 100% sequence identity with the HCR initiator. In some embodiments, the split initiator probes within a probe unit generate sequence identity to the HCR initiator sufficient to enable efficient HCR signal amplification as compared to the rate of signal amplification when no split initiator probes are present or when individual split initiator probes are present but not co-localized by the target. In some embodiments, the complete HCR initiator generated by the co-localized probes within a probe unit has 99%, 95%, 90%, 80%, or 60% sequence identity with the HCR initiator and includes any range defined between any two of the above values or exceeding any one of the above values.

[0084] Use of large probe sets to enhance the SB ratio Since the signal increases monotonically with the number of probe units, in some embodiments, it is advantageous to use a larger probe set containing multiple probe units, where the length of the target allows (e.g., mRNA targets, lncRNA targets, gDNA targets), in order to increase the number of signals generated per target molecule. Because automated background suppression ensures that only probe units that specifically bind to the target induce HCR signal amplification, it is usually unnecessary to inspect individual probes in the probe set to remove non-specifically binding probes. As a result, automated background suppression increases ease of use by eliminating the need to optimize the probe set when using a new probe set for a novel target. Since the signal increases monotonically with the size of the probe set and background is automatically suppressed for all probe pairs, increasing the size of the probe set is usually advantageous to increase the SB ratio. A probe set may contain one or more probe units, each containing two or more segmented initiator probes (see, for example, Figures 8 and 16). For example, the number of probe units in a probe set can be in the range of 1 to 1000, or 10 to 100, or 20 to 50, or 30 to 40. By increasing the number of probe units in a probe set, the SB ratio can be increased by 2, 5, 10, 100, or more. Based on the number of probe units in the probe set, the sample-specific background level, and the abundance of the target in the sample, the SB ratio can be increased to 2, 5, 10, 50, 100, 200, 500, 1000, or more by increasing the number of probe units in the probe set.

[0085] The size of the probe set is limited by the transcriptome and / or genome in the sample. In some cases, it may be desirable to distinguish one target in a sample from one or more other targets in a sample that are sequence-related. For example: 1) A target mRNA in an organism may have a sequence similar to another mRNA in the same organism, and this may include the possibility that the target mRNA is a splice variant that needs to be distinguished from other splice variants; 2) A target mRNA may contain multiple regions, each having sequence similarity to one or more other RNAs in the transcriptome of the organism; 3) A target mRNA in an organism may be present in multiple samples, such that the target mRNA has regions sequence-similar to one or more RNAs in the transcriptome of another species; and 4) A target rRNA or gDNA in an organism may be sequence-similar to rRNA or gDNA in other organisms present in the sample. One way to distinguish a target nucleic acid from all other nucleic acids present in a sample is to design a probe set for the target nucleic acid that contains only probe pairs that are selective for the target nucleic acid compared to the transcriptome and / or genome present in the sample. In some cases, this selectivity requirement can significantly limit the size of the probe set. For example, the target nucleic acid sequence may be very similar to one or more other nucleic acids in the sample, so the probe set may contain only one split initiator probe pair.

[0086] Cooperative target binding using a probe set containing multiple split initiator probe pairs. A probe that binds to a target nucleic acid (e.g., RNA or DNA) energetically competes with the native secondary structure (i.e., base-paired structures) within the target nucleic acid. For example, a single probe may not be able to bind to a domain in the target nucleic acid that is primarily base-paired to another domain within the target. The binding efficiency of a given probe is the proportion of the target molecule to which the probe is bound at its cognitive probe-binding site, and varies between 0 and 1 depending on factors such as the accessibility of the target region and the degree of affinity between the probe and its cognitive probe-binding site. Multiple split initiator probes Using a probe set containing pairs, the binding of one probe to a target can improve the binding efficiency of one or more other probes to that target, potentially resulting in a synergistic effect where the probes in the probe set collectively improve the binding efficiency of the other probes in the set. As a result, a probe set containing N+M probes can generate more signal than a probe set containing N probes, not only because the M new probes generate signal, but also because the N original probes have higher binding efficiency to the target, generating more signal per target molecule on average.

[0087] Signal increase using helper probes In situations where one or more transcriptomes and / or genomes are present in the sample, the probe set size may be limited to one or fewer probe units, two or fewer probe units, or N or fewer probe units, to maintain selectivity for the target nucleic acid, where N is less than the number of probe units N+M, which are preferredly used based on sensitivity considerations and / or the length of the target nucleic acid. In this scenario where the probe set size is limited by selectivity considerations, the amount of signal generated may be less than when detecting the same target with N+M probe units, because the probe set may generate fewer than M complete initiators to induce HCR signal amplification, and because, without M additional probe units, synergistic effects may reduce the binding efficiency of the remaining N probe units.

[0088] In some embodiments, the probe has a splitting initiator as a signal probe (see, for example, Figure 16A). In some embodiments, the probe does not have a splitting initiator as a helper probe. To increase the signal without reducing selectivity, a probe set containing N probe units can be augmented with M helper probes (see, for example, Figure 16D).

[0089] For example: 1) To detect target RNA having a unique splice junction, the probe set can be made to include one probe unit that binds to the target spanning the splice junction, and further to include 30 (or any number) helper probes that bind to other locations on the target RNA and cooperatively improve the binding efficiency of the probe unit, thereby increasing the signal without reducing selectivity; 2) To detect target RNA or target DNA in which, due to selectivity considerations, the probe set would contain 5 probe units, the probe set could further contain 45 helper probes; 3) To detect target RNA or target DNA in which, due to selectivity considerations, the probe set would contain N probe units (where N is less than 40), the probe set could further contain 40-N helper probes; 4) To detect target nucleic acids in which, due to selectivity considerations, the probe set would contain N probe units and the total number of probes would be limited to 2N+M depending on the length of the target nucleic acid, the probe set could further contain M helper probes.

[0090] In some embodiments, the helper probe is designed for the target nucleic acid with less stringent selectivity requirements than the one used to design the signal probe for the same target nucleic acid. In some embodiments, the helper probe is used for the target nucleic acid even if it has equivalent selectivity to other off-target nucleic acids in the transcriptome and / or genome present in the sample. In some embodiments, the target binding region on the signal probe is the same length as the target binding region on the helper probe. In some embodiments, the target binding region on the signal probe is shorter or longer than the target binding region on the helper probe. In some embodiments, the target binding region may vary over a range of lengths (number of nucleotides) for each different signal probe and / or helper probe. In some embodiments, the affinity between the signal probe and the target nucleic acid is equivalent to the affinity between the helper probe and the target nucleic acid. In some embodiments, the affinity between the signal probe and the target nucleic acid is lower or higher than the affinity between the helper probe and the target nucleic acid.

[0091] Signal amplification using a signal probe with one or two split initiators In some embodiments, a probe unit includes two split initiator probes (fraction f1 for probe P1 and fraction f2 for probe P2, such that f1 + f2 = 1) that together form a complete HCR initiator. In this scenario, each probe participates in one probe unit. When both probes in a probe unit bind to a given target RNA molecule, they produce one complete HCR initiator and grow one HCR amplification polymer anchored to the probe unit. Consider a scenario where the length of the target RNA limits the size of the probe set to N probe units, corresponding to 2N signal probes. When each signal probe participates in only one probe unit, the maximum number of complete HCR initiators is N, corresponding to the case where the target mRNA binds to all signal probes in the probe set. Thus, the maximum number of HCR polymers anchored to the target RNA is N. To increase the signal per target molecule, we now consider the case where the probe can have two split initiators, which contributes to having two different probe units (see, for example, Figure 16C). In some embodiments, the following three possibilities are considered: 1. In an embodiment where all 2N signal probes hybridize to adjacent subsequences in the target RNA, the signal probes at the 5' and 3' ends of the target each have one adjacent signal probe, and all other signal probes in between have two adjacent signal probes. In this scenario, if each probe other than the 5' and 3' end signal probes has two splitting initiators contributing to two distinct probe units, the total number of probe units increases from N to (2N-1). As a result, the maximum number of complete HCR initiators increases from N to (2N-1), and the total number of HCR polymers anchored to the target RNA increases from N to (2N-1). This increases the signal generated per target molecule compared to the case where each signal probe has one splitting initiator. 2. In another embodiment, due to considerations of selectivity due to the transcriptome and / or genome present in the sample, the probe set may include 2N signal probes and N probe units in the target nucleic acid where the probe units are not in close proximity to each other. In this scenario, each signal probe has only one splitting initiator, contributing to only one probe unit, so the maximum number of complete HCR initiators is N and the maximum number of anchored HCR amplification polymers is N. 3. In another embodiment, which is an intermediate case, some signal probes are proximal to two or more adjacent signal probes, so 2N signal probes will participate in a number of probe units intermediate between N and 2N-1. For example, if there are 2N signal probes, M of which are tiled in one part of the target (the 5' and 3' signal probes among these have one splitting initiator, resulting in one probe unit, and the other M-2 signal probes have two splitting initiators, resulting in two probe units), and the remaining L probes (2N=M+L, where L is an even number) are each coupled in pairs to the target, and each signal probe is proximal to only one other signal probe (each of the L signal probes has one splitting initiator, resulting in one probe unit), then the total number of probe units is M-1+L / 2, which is intermediate between N and 2N-1. For example, if there are 40 signal probes (i.e., N=20), 20 of them are placed close to the target (i.e., M=20), and the other 20 are coupled to the target as proximity pairs (i.e., L=20), then the total number of probe units will be M-1 + L / 2 = 20 - 1 + 20 / 2 = 29.

[0092] In cases where a probe unit includes three or more signal probes, for example, three signal probes, or four or more signal probes, the number of probe units can be increased without increasing the number of signal probes by using several probes that include two split initiators and participate in two probe units (one split initiator per probe unit).

[0093] HCR amplifier with two hairpins In some embodiments, the HCR amplifier includes two hairpins (H1 and H2; see, for example, Figures 8 and 18). In some embodiments, each hairpin includes an input domain having a single-stranded toehold and a stem, and an output domain having a single-stranded loop and a complementary chain to the stem. In the absence of an HCR initiator (I1 or I2), hairpins H1 and H2 coexist metastable, i.e., are kinetically trapped and do not polymerize.

[0094] Initiated by Full Initiator I1 In some embodiments, initiator I1 includes a domain complementary to the toehold of hairpin H1 and a domain complementary to the stem of H1. When H1 hairpin encounters complete initiator I1, complete initiator I1 hybridizes to the input domain of hairpin H1 via chain displacement through the toehold, opening hairpin H1 and exposing its output domain to form complex I1-H1. The output domain of hairpin H1 includes a domain complementary to the toehold of hairpin H2 and a domain complementary to the stem of H2. When H2 hairpin encounters I1-H1 complex, the exposed output domain of H1 hybridizes to the input domain of hairpin H2 via chain displacement through the toehold, opening hairpin H2 and exposing its output domain to form complex I1-H1-H2. The output domain of hairpin H2 includes a domain complementary to the toehold of hairpin H1 and a domain complementary to the stem of H1. When H1 hairpin encounters the I1-H1-H2 complex, the exposed output domain of H2 hybridizes to the input domain of hairpin H1 via chain displacement through the toehold, opening hairpin H1 and exposing its output domain, forming the I1-H1-H2-H1 complex. This polymerization process can produce polymers in the form I1-H1-H2-H1-H2-H1-H2-… by alternately repeating the H1 polymerization step and the H2 polymerization step. In the case of a polymer incorporating N alternating copies of hairpin H1 and hairpin H2, the structure is I1-(H1-H2) N It can be expressed as follows. For example, a polymer may incorporate several H1 and H2 molecules, or tens of thousands 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 even more. Since a polymer can end in either H1 or H2, I1-(H1-H2) N -H1 and I1-(H1-H2) N -H1-H2 is possible for both, and the latter is I1-(H1-H2) N+1It is equal to.

[0095] Fully initiated by Initiator I2 In some embodiments, initiator I2 includes a domain complementary to the toehold of hairpin H2 and a domain complementary to the stem portion of H2. When the H2 hairpin encounters complete initiator I2, complete initiator I2 hybridizes to the input domain of hairpin H2 by chain displacement via the toehold, and the hairpin When the H2 hairpin encounters the I2-H2 complex, the exposed output domain of H2 hybridizes to the input domain of hairpin H1 via toehold-mediated chain displacement, opening hairpin H1 and exposing its output domain, forming the I2-H2-H1 complex. When the H2 hairpin encounters the I2-H2-H1 complex, the exposed output domain of H1 hybridizes to the input domain of hairpin H2 via toehold-mediated chain displacement, opening hairpin H2 and exposing its output domain, forming the I2-H2-H1-H2 complex. This polymerization process involves alternating between H1 polymerization steps and H2 polymerization steps to produce polymers in the form I2-H2-H1-H2-H1-H2-H1…, and in the case of a polymer incorporating N alternating copies of H2 and H1, the polymer is I2-(H2-H1) N It can be expressed as follows. For example, a polymer may incorporate several H1 and H2 molecules, or tens of thousands 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 even more. Since a polymer can end in either H1 or H2, I2-(H2-H1) N -H2 and I2-(H2-H1) N -H2-H1 is possible for both, and the latter is I2-(H2-H1) N+1 It is equal to.

[0096] HCR Amplifier with 4 HairpinsIn some embodiments, an HCR amplifier may include more than two hairpins. For example, an HCR amplifier may include four hairpins H1, H2, H3, and H4 (see, for example, Figure 19). Similar to the two-hairpin HCR, each hairpin includes an input domain having a single-stranded toehold and a stem, and an output domain having a single-stranded loop and a complementary chain to the stem. In the absence of an HCR initiator (I1, I2, I3, or I4), hairpins H1, H2, H3, and H4 coexist metastable, i.e., are kinetically trapped and do not polymerize. The output domain of hairpin H1 includes a domain complementary to the toehold of hairpin H2 and a domain complementary to the stem of H2. The output domain of hairpin H2 includes a domain complementary to the toehold of hairpin H3 and a domain complementary to the stem of H3. The output domain of hairpin H3 includes a domain complementary to the toehold of hairpin H4 and a domain complementary to the stem of H4. The output domain of hairpin H4 includes a domain complementary to the toehold of hairpin H1 and a domain complementary to the stem of H1. Initiator I1 includes a domain complementary to the toehold of hairpin H1 and a domain complementary to the stem of H1. Initiator I2 includes a domain complementary to the toehold of hairpin H2 and a domain complementary to the stem of H2. Initiator I3 includes a domain complementary to the toehold of hairpin H3 and a domain complementary to the stem of H3. Initiator I4 includes a domain complementary to the toehold of hairpin H4 and a domain complementary to the stem of H4.Similar to the case of 2 hairpin-type HCR, when hairpin H1 meets the complete HCR initiator I1, the complete initiator I1 opens hairpin H1 to form a complex I1-H1 with the exposed H1 output domain. This H1 output domain opens hairpin H2 to form a complex I1-H1-H2 with the exposed H2 output domain. This H2 output domain opens hairpin H3 to expose the output domain and form a complex I1-H1-H2-H3 with the exposed H3 output domain. This H3 output domain opens hairpin H4 to form a complex I1-H1-H2-H3-H4 with the exposed H4 output domain. This H4 output domain opens hairpin H1 to form a complex I1-H1-H2-H3-H4-H1 with the exposed H1 output domain, and so on. Polymerization occurs through the alternating polymerization process of H1, H2, H3, and H4, generating a polymer in the form of I1-H1-H2-H3-H4-H1-H2-H3-H4-H1-H2-H3-H4…. In the case of a polymer incorporating N alternating copies of H1, H2, H3, and H4, it can be represented as I1-(H1-H2-H3-H4). N It can be expressed as such. Since the polymer can also end with H1, H2, H3, or H4, 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, and finally it is equal to I1-(H1-H2-H3-H4) N+1 HCR polymerization can be induced by any of the cognate complete initiators (I1, I2, I3, or I4). For example, if it is an initiator by the complete initiator I3, it is I3-(H3-H4-H1-H2) NThis allows for the generation of polymers in this form. The HCR amplifier with four hairpins is advantageous when generating signals that do not exist in the monomeric state but do exist in the polymeric state (for example, Figure 19B illustrates a FRET pair that generates a FRET signal by colocalization only when the hairpins colocalize within the amplified polymer; unused hairpins that are not washed off from the sample do not participate in FRET, thus avoiding background generation and providing the basis for a no-wash method).

[0097] HCR amplifier with two or more hairpins More generally, in some embodiments, the HCR amplifier may contain M HCR hairpins (H1, H2, ..., HM), where M is an integer greater than or equal to 2. In the absence of an HCR initiator (I1, I2, ..., IM), the hairpins H1, H2, ..., HM coexist metastable, i.e., are kinetically trapped and do not polymerize. In the presence of a cognitive complete HCR initiator, polymerization occurs via an alternating polymerization process similar to that of a 2-hairpin or 4-hairpin HCR. For example, in the case of a polymer incorporating N alternating copies of H1, H2, ..., HM, initiator I1 is I1-(H1-H2-...-HM) N This results in the growth of polymers in the form I1-(H1-H2-...-HM). Since polymers can also end in H1, H2, ..., HM, the result is 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, and the last one is I1-(H1-H2-...-HM) N+1 This is equivalent to HCR polymerization being induced by any of the cognitive complete initiators (I1, I2, ..., IM). For example, if the initiator is complete initiator I3, then I3-(H3-...-HM-H1-H2) N This allows for the production of polymers in this form.

[0098] HCR hairpin label In a given HCR amplifier, each HCR hairpin contains zero, one, or multiple labels. Labels on different hairpins within the amplifier may be the same or different. For example, an amplifier containing hairpins H1 and H2 may have the following cases: 1) having the same label on H1 and H2, 2) having different labels on H1 and H2, 3) having a label on H1 but no label on H2, 4) having a label on H2 but no label on H1, 5) having no label on either H1 or H2, 6) having zero, one, or multiple labels on H1, where zero, one, or multiple of them may be the same as or different from the zero, one, or multiple labels on H2. Similarly, in the case of an HCR amplifier containing hairpins H1, H2, H3, and H4, each hairpin may contain zero, one, or multiple labels (e.g., 3, 5, or 10 labels), and zero, one, or multiple of them may be the same as the zero, one, or multiple labels on each of the other hairpins. In some embodiments, one or more of the labels of a given hairpin can be unique in the mixture of hairpins and / or hairpin labels. In some embodiments, there are 1, 10, 100, or more unique labels in the mixture (including any range defined between any two of the above numerical values).

[0099] HCR hairpin label as a reporter In some embodiments, the HCR hairpin label may include a reporter molecule that facilitates the measurement of a signal, for example, by generating a signal, by changing a signal, or by removing a signal. For example, the reporter can be a fluorophore, chromophore, luminophore, phosphor, FRET pair, several FRET pairs, quencher, fluorophore / quencher pair, rare earth element or compound, radioactive molecule, magnetic molecule, or any other molecule that promotes the measurement of a signal.

[0100] HCR hairpin labeling as a substrate In some embodiments, the HCR hairpin label may include a substrate that serves to recruit a reporter that directly or indirectly mediates the localization of the reporter near the hairpin label. for example: 1. The hairpin label may include digoxigenin (DIG) as the reporter, which recruits an anti-DIG antibody that is directly labeled with one or more reporters, or with one or more substrates or reporters that directly or indirectly mediate the localization of the reporter near the hairpin label. 2. Hairpin labeling can include nucleic acid domains that act as substrates having full or partial sequence complementarity to domains in a labeled probe having one or more reporters (e.g., Figure 18C). 3. The hairpin label may include a nucleic acid domain that acts as a substrate fully or partially complementary to the domain in the labeled probe, having one or more substrates that mediate the localization of the reporter near the hairpin label. 4. The hairpin label may include a nucleic acid domain that acts as a substrate for a reporter organism that directly or indirectly mediates the localization of the reporter near the hairpin label. 5. The hairpin label may include a substrate that recruits a reporter organism that indirectly mediates the localization of the reporter near the hairpin label. 6. Hairpin labeling may include a substrate that recruits a reporter body containing an enzyme that mediates catalytic reporter deposition (CARD) near the hairpin label (e.g., Figure 36A). 7. The hairpin label may include biotin, which recruits streptavidin directly labeled with one or more reporters as the reporter, or with one or more substrates or reporters that directly or indirectly mediate the localization of the reporter near the hairpin label. 8. The hairpin label may include a hapten that recruits an anti-hapten antibody or anti-hapten nanobody (registered trademark) that directly or indirectly mediates the localization of the reporter near the hairpin label via CARD signal amplification. For example, the anti-hapten antibody or nanobody may include a reporter body which is an enzyme that mediates CARD (e.g., Figures 33A-33E). 9. The hairpin label may include a hapten that recruits an anti-hapten that directly or indirectly mediates the localization of the reporter near the hairpin label. For example, the anti-hapten may include a reporter body which is an enzyme that mediates CARD (e.g., Figures 34A-34C). 10. Hairpin labeling may include an enzyme that mediates CARD signal amplification to deposit the reporter molecule near the hairpin. 11. Hairpin markers directly or indirectly localize reporters near hapten. It may contain zero, one, or more haptens that mediate the interaction (e.g., Figures 35-1 to 35-2).

[0101] In some embodiments, the hairpin labeling may include a hapten that recruits a reporter-labeled anti-hapten (e.g., an antibody, nanobody, streptavidin, or another molecule).

[0102] In some embodiments provided herein, HCR signal amplification is used to further increase the signal gain by mediating catalytic reporter deposition (CARD). In some embodiments, the further higher single gain is a value having a range defined by approximately 5x, approximately 10x, approximately 15x, approximately 20x, approximately 25x, approximately 30x, approximately 40x, approximately 50x, approximately 75x, approximately 100x, approximately 500x, approximately 1000x, approximately 2000x, approximately 5000x, or approximately 10,000x, or any two of the above values.

[0103] In some embodiments, the labeled probe molecule bound to the reporter molecule does not strongly bind to the divided substrate on individual hairpins, but after HCR polymerization, the divided substrate may be included in the HCR hairpin labeling so that adjacent hairpins in the HCR amplification polymer colocalize the complete substrate so that the colocalized complete substrate strongly binds to the labeled probe bound to the reporter molecule (e.g., Figure 18D) or to the reporter body containing the enzyme that mediates CARD signal amplification (e.g., Figure 36B).

[0104] Haptens and anti-haptens In some embodiments, the hairpin label, which is a hapten-containing substrate, can be, for example, digoxygenin (DIG), dinitrophenyl (DNP), fluorophores, biotin, or any small molecule, biomolecule, or non-biomolecule capable of recruiting an anti-hapten. Examples of anti-haptens include antibodies, nanobodies, streptavidins, aptamers, or any other molecule or molecular complex that selectively bind to the hapten.

[0105] Labeled probe In some embodiments, the labeled probe comprises a substrate-binding region and one or more reporters (e.g., see Figures 18C–18E and 20) or reporter bodies (e.g., see Figures 33A–33E and 34A–34D). In some embodiments, the labeled probe comprises an unstructured chain labeled with one reporter molecule (e.g., see Figure 20A). In some embodiments, the labeled probe comprises multiple reporter molecules (e.g., see Figure 20B). In some embodiments, the labeled probe comprises a labeled chain (bound to the reporter) hybridized to a blocker chain (bound to the quencher) (e.g., see Figure 20C). In some embodiments, the labeled probe has a hairpin structure (e.g., see the labeled probe in Figure 20D) or other intramolecular base pairings. In some embodiments, the hairpin labeled probe is bound to one or more reporters, quenchers, and / or FRET pairs (e.g., see Figures 20E and 20F). In some embodiments, the labeled probe comprises an anti-hapten antibody or nanobody that recognizes a hapten label on an HCR hairpin and further comprises a reporter body containing a CARD-mediated enzyme (e.g., Figures 33A-33E). In some embodiments, the labeled probe comprises an anti-hapten body that recognizes a hapten label on an HCR hairpin and further comprises a reporter body containing a CARD-mediated enzyme (e.g., Figures 34A-34D). In some embodiments, the labeled probe comprises a substrate complementary chain that recognizes a substrate label on an HCR hairpin or recognizes a complete substrate co-localized within an HCR amplified polymer and further comprises a reporter body containing a CARD-mediated enzyme (e.g., Figures 36A-36B).

[0106] Enzymes for catalytic reporter deposition (CARD) via HCR In some embodiments, the HCR hairpin mediates signal amplification via catalytic reporter deposition (CARD) by a reporter body containing an enzyme that catalyzes a CARD substrate to deposit the reporter near the hairpin (see, for example, Figures 33A-33E, 34A-34C, and 36A-36B). For example: 1. The above enzyme can be horseradish peroxidase (HRP) (or polymer HRP containing multiple HRP enzymes) that acts on a CARD substrate to catalyze the deposition of a pigment-producing reporter such as AEC, DAB, TMB, or Stay Yellow, or catalyzes a CARD substrate to catalyze the deposition of a fluorescent reporter such as a fluorophore-labeled tyramide, or catalyzes the deposition of a hapten-labeled CARD substrate such as a biotin-labeled tyramide, with the hapten mediating the localization of the reporter near the hairpin label. 2. The above enzyme may be an alkaline phosphatase (AP) (or a polymer AP containing multiple AP enzymes) that acts on a CARD substrate to catalyze the deposition of a reporter, such as, but not limited to, BCIP / NBT, BCIP / TNBT, naphthol AS-MX phosphate + Fast Blue BB, naphthol AS-MX phosphate + Fast Red TR, Stay Green, or other pigment-producing reporters. 3. The enzyme described above may be a glucose oxidase that acts on a CARD substrate to catalyze the deposition of a reporter, such as NBT. 4. The enzyme described above can be any molecule or complex that directly or indirectly mediates the localization of the reporter near the hairpin label.

[0107] In some embodiments, the CARD-mediated enzyme is deactivated (sometimes called inactivation) after reporter deposition (e.g., by chemical or thermal denaturation). For example, the CARD-mediated enzyme can be deactivated using any combination of the following: 1. Heat (e.g., above 65°C) 2. Fixative (e.g., 4% PFA) 3. Acid (e.g., 0.1M glycine hydrochloride + 1% Tween® 20 (pH 2.2), 0.2N HCl, 10% acetic acid, 10mM HCl) 4. Other chemical substances (e.g., hydrogen peroxide (H2O2), hydrogen peroxide + phenol, sodium azide, DEPC, MAB + 10mM EDTA)

[0108] 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 with a fixative. In some embodiments, by inactivating the CARD-mediated enzyme, it becomes possible to perform repeated CARD with the same enzyme in combination with different substrates for different targets, enabling multi-target analysis using HCR-mediated CARD. In some embodiments, by inactivating the CARD-mediated enzyme, it becomes possible to perform repeated CARD with different enzymes in combination with different substrates for different targets, enabling multi-target analysis using HCR-mediated CARD.

[0109] In some embodiments, CARD enables the storage of stained samples for more than 10 years, allowing for re-imaging in compliance with biopharmaceutical regulatory requirements. In some embodiments, CARD-based stained samples are sufficiently stable for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 years, or longer, and are also compliant with biopharmaceutical regulatory requirements. In some embodiments, CARD enables the storage of formalin-fixed paraffin-embedded (FFPE) samples for several decades. In some embodiments, CARD enables the storage of pathological samples for several decades, enabling retrospective scientific and medical research. In some embodiments, CARD staining enables the long-term storage of archival samples.

[0110] Types of targets In some embodiments, the initiator-labeled HCR probe includes one or more target-binding regions and further includes one or more HCR initiators. In some embodiments, the initiator-labeled probe can detect targets including: 1. Any molecule, including but not limited to RNA molecules (e.g., mRNA, rRNA, lncRNA, siRNA, shRNA, microRNA, non-coding RNA, synthetic RNA, or modified RNA), DNA molecules, non-natural nucleic acid molecules, protein molecules, small molecules, biomolecules, chemically modified biomolecules, and non-biomolecules. 2. Molecular complexes containing any combination of RNA, DNA, proteins, small molecules, biomolecules, and / or non-biomolecules (e.g., RNA / RNA complexes, RNA / protein complexes, DNA / protein complexes, and RNA / DNA / protein complexes, protein / protein complexes).

[0111] A segmented initiator HCR probe includes one or more target binding regions and further includes one or more segmented initiator regions. A probe unit includes two or more segmented initiator probes such that the segmented initiator probes within the probe unit combine to form a complete HCR initiator. A probe unit can detect targets including: 1. Any molecule, including but not limited to RNA molecules (e.g., mRNA, rRNA, lncRNA, siRNA, shRNA, microRNA, non-coding RNA, synthetic RNA, or modified RNA), DNA molecules, non-natural nucleic acid molecules, protein molecules, small molecules, biomolecules, chemically modified biomolecules, and non-biomolecules. 2. Molecular complexes containing any combination of RNA, DNA, proteins, small molecules, biomolecules, and / or non-biomolecules (e.g., RNA / RNA complexes, RNA / protein complexes, DNA / protein complexes, and RNA / DNA / protein complexes, protein / protein complexes) 3. Any assembly of proximal molecules or complexes such that when a divided initiator probe containing a probe unit binds to each target within the assembly of proximal molecules or complexes, the divided initiators within the probe unit can colocalize to form a complete HCR initiator.

[0112] In any embodiment provided herein, the splitting initiator in the probe unit is designed (or is complementary) to a non-overlapping region of the HCR hairpin (e.g., a region separated by zero, one, two, or more nucleotides), or to an overlapping region of the HCR hairpin (e.g., a region overlapping by one, two, or more nucleotides), or to be substantially complementary to the HCR hairpin (e.g., complementary except for zero, one, two, a few, or several mismatches).

[0113] In any of the embodiments provided herein, the target-binding region within the probe unit is configured to bind to a non-overlapping region of the target (e.g., a region separated by 0 nucleotides, 1 nucleotide, 2 nucleotides, or more, or a region separated by 0 nm, 1 nm, 2 nm, or more), or to an overlapping region of the target (e.g., 1 nucleotide, 2 nucleotides, or It is configured to bind to regions where only a minimum number of nucleotides overlap, or regions where there is an overlap of 1 nm, 2 nm, or more.

[0114] The signal probe is configured to bind to the overlapping or non-overlapping region of the target and / or is designed to have a splitting initiator that hybridizes to the overlapping or non-overlapping region of the HCR hairpin. In some embodiments, the probe unit includes two or more split initiator probes, each containing a target binding region and a split initiator. In some embodiments, the split initiators within the probe unit are: 1. Is it designed to be complementary to the adjacent area of ​​the HCR hairpin? 2. Or, it is designed to be complementary to the non-overlapping regions of the HCR hairpin (for example, regions separated by only 0, 1, 2, or more nucleotides), 3. Or, it is designed to be complementary to the overlapping region of the HCR hairpin (for example, a region where only one, two, or more nucleotides overlap), 4. Or, it is designed to be substantially complementary to the HCR hairpin (e.g., complementary except for 0, 1, 2, a few, or several mismatches), 5. Or, it is designed to hybridize in the vicinity of the HCR hairpin, 6. Or, it is designed to hybridize into the non-overlapping area of ​​the HCR hairpin, 7. Or, it is designed to hybridize into the overlapping area of ​​the HCR hairpin, 8. Or, it is designed to have an arrangement that is complementary to the adjacent region of the HCR hairpin, 9. Or, it is designed to have an arrangement complementary to the non-overlapping region of the HCR hairpin, 10. Or, it is designed to have an arrangement complementary to the overlapping region of the HCR hairpin, 11. Or, they are designed to have an arrangement that is substantially complementary to the adjacent region of the HCR hairpin, 12. Or, they are designed to have an arrangement that is substantially complementary to the non-overlapping region of the HCR hairpin, 13. Alternatively, it is designed to have an arrangement that is substantially complementary to the overlapping region of the HCR hairpin.

[0115] In some embodiments, the target binding region within the probe unit is: 1. Is it configured to bond to the vicinity of the target? 2. Or, it is configured to bind to a non-overlapping region of the target (for example, a region separated by only 0, 1, 2, or more nucleotides, or a region separated by 0 nm, 1 nm, 2 nm, or more), 3. Or, it is configured to bind to the overlapping region of the target (for example, a region where only 1, 2, or more nucleotides overlap, or a region where 1 nm, 2 nm, or more overlap), 4. Is it designed to bond to the vicinity of the target? 5. Or, it is designed to bind to non-overlapping regions of the target, 6. Or, it is designed to bond to the overlapping region of the target, 7. Or, it is designed to have a sequence that hybridizes to the adjacent region of the target, 8. Or, it is designed to have a sequence that hybridizes to a non-overlapping region of the target, 9. Alternatively, it is designed to have a sequence that hybridizes into the overlapping region of the target.

[0116] In some embodiments, the probe unit includes two split initiator probes. These two split initiator probes bind to a cognitive target to colocalize a complete HCR initiator. The colocalized complete HCR initiator binds to a cognitive HCR hairpin to induce HCR polymerization, with one split initiator hybridizing to the hairpin to form a first double helix and the other split initiator hybridizing to the hairpin to form a second double helix. In some embodiments, there is an energetically unlikely connection between these two double helixes. In some embodiments, by configuring a split initiator to bind to the overlapping region of a hairpin, the location of the connection within the hairpin, as well as the tertiary structure of the two probes and the hairpin near the connection, can be relaxed to create an energetically more readily occurring higher-order structure, increasing the affinity between the colocalized complete initiator and the HCR hairpin, and increasing the amount of amplified HCR signal generated during a given period (e.g., Figures 27 and 28). In some embodiments, the affinity between the two probes and a cognitive target can be increased by configuring the target-binding regions of the two probes to bind to the overlapping region of the target in order to relax the connection between these molecules to create an energetically more readily occurring higher-order structure.

[0117] In some embodiments, the probe set is designed for multiplex experiments in which two, three, four, five, ten, twenty, or 100 or more probe sets are used to bind to different targets in the same sample, and one, two, three, four, five, ten, twenty, or 100 or more probe sets contain one or more initiator-labeled probes. In some embodiments, the probe set is designed for multiplex experiments in which two, three, four, five, ten, twenty, or 100 or more probe sets are used in the same sample, and more than 1%, more than 2%, more than 5%, more than 10%, more than 30%, more than 50%, or more than 100% of the probe sets contain one or more initiator-labeled probes.

[0118] In some embodiments, the probe set is designed for multiplex experiments in which two, three, four, five, ten, twenty, or 100 or more probe sets are used to bind to different targets in the same sample, and one, two, three, four, five, ten, twenty, or 100 or more probe sets include one or more probe units that include split initiators designed to hybridize to the overlapping region of HCR hairpins. In some embodiments, the probe set is designed for multiplex experiments in which two, three, four, five, ten, twenty, or 100 or more probe sets are used in the same sample, and more than 1%, more than 2%, more than 5%, more than 10%, more than 30%, more than 50%, or more than 100% of the probe set includes one or more probe units that include split initiators having an arrangement designed to be complementary to the overlapping region of HCR hairpins.

[0119] In some embodiments, the probe set may consist of 2, 3, 4, 5, 10, 20, or 100 or more probe sets used to identify different labels in the same sample. Multiplex experiments for target binding are designed for multiplex experiments in which one, two, three, four, five, ten, twenty, or 100 or more probe sets include one or more probe units containing a target binding region designed to bind to the overlapping region of the target. In some embodiments, the probe sets are designed for multiplex experiments in which three, four, five, ten, twenty, or 100 or more probe sets are used in the same sample, and more than 1%, more than 2%, more than 5%, more than 10%, more than 30%, more than 50%, or more than 100% of the probe sets include one or more probe units containing a target binding region designed to bind to the overlapping region of the target.

[0120] In some embodiments, the probe sets are designed for multiplex experiments in which two, three, four, five, ten, twenty, or 100 or more probe sets are used to bind to different targets in the same sample, wherein one or more probe sets include one or more initiator-labeled probes, and one or more probe sets include one or more probe units, each containing two or more segmented initiator probes. In some embodiments, the probe sets are designed for multiplex experiments in which two, three, four, five, ten, twenty, or 100 or more probe sets are used in the same sample, wherein more than 0.1%, more than 1%, more than 2%, more than 5%, more than 10%, more than 30%, or more than 50% of the probe sets contain one or more initiator-labeled probes, and more than 0.1%, more than 1%, more than 2%, more than 5%, more than 10%, more than 30%, or more than 50% of the probe sets contain one or more probe units, each containing two or more segmented initiator probes.

[0121] In some embodiments, the probe unit includes a splitting initiator designed to bind to the overlapping region of an HCR hairpin, wherein the overlapping region overlaps by only one, two, three, four, five, or more bases.

[0122] In some embodiments, the probe unit includes a target binding region designed to bind to an overlapping region of a target, wherein the overlapping region overlaps by at least 0.1 nm, or at least 0.2 nm, or at least 0.3 nm, or at least 0.5 nm, or at least 1 nm, or at least 2 nm, or at least 3 nm, or at least 5 nm. In some embodiments, the probe unit includes a target binding region comprising a sequence designed to bind to an overlapping region of a target, wherein the overlapping region overlaps by at least 1 base, or 2 bases, or 3 bases, or 4 bases, or 5 bases, or more.

[0123] Material and composition of initiator-labeled probes In some embodiments, the initiator-labeled probe comprises one or more target-binding domains and one or more HCR initiators (e.g., Figures 39A-39N and 42A-42F). Each domain may consist of one or more materials, including DNA, RNA, 2'OMe-RNA, PNA, XNA, chemically modified nucleic acids, synthetic nucleic acid analogs, amino acids, synthetic amino acid analogs, and / or any other molecule suitable for the purpose of the domain. For example: 1. The initiator-labeled probe may include one or more initiators composed of DNA, and a target-binding domain composed of DNA. 2. The initiator-labeled probe may include one or more initiators composed of DNA, and a target-binding domain composed of amino acids (e.g., an antibody, nanobody, or antibody fragment). 3. The initiator-labeled probe is an initiator composed of a synthetic nucleic acid analog. It may also include a target-binding domain composed of a combination of DNA and 2'OMe-RNA. 4. The initiator-labeled probe may include an initiator composed of 2'OMe-RNA and a target-binding domain composed of an RNA-protein combination. 5. The initiator-labeled probe may include an initiator composed of DNA and a target-binding domain composed of PNA. 6. The initiator-labeled probe may comprise one or more initiators composed of any nucleic acid or nucleic acid analog, and one or more target-binding domains composed of any combination of materials suitable for binding to the target molecule.

[0124] In some embodiments, the initiator-labeled probe may contain one covalent molecule, or it may contain two or more molecules (each covalently linked) that interact non-covalently to form a complex. For example: 1. The initiator-labeled probe may contain an initiator composed of DNA covalently linked to a target-binding domain composed of DNA. 2. The initiator-labeled probe may contain one or more initiators made of DNA, covalently linked to a dCas9 (or another Cas) noncovalently bound to a guide RNA (gRNA), such that the target-binding domain contains a gRNA:dCas9 complex (or a gRNA:Cas complex using another Cas). 3. The initiator-labeled probe may contain one or more initiators made of DNA, covalently ligated to a gRNA noncovalently bound to dCas9 (or another Cas), such that the target-binding domain includes a gRNA:dCas9 complex (or a gRNA:Cas complex using another Cas). 4. The initiator-labeled probe may include an initiator, composed of a nucleic acid or nucleic acid analog, covalently or noncovalently bound to a target-binding domain containing one or more molecules.

[0125] Material and composition of the segmented initiator probe In some embodiments, the split initiator probe comprises one or more target-binding domains and one or more split initiator domains (e.g., Figures 3A-3B, 5A-5E, and 38-1-38-2). Each domain may consist of one or more materials, including DNA, RNA, 2'OMe-RNA, PNA, XNA, chemically modified nucleic acids, synthetic nucleic acid analogs, amino acids, synthetic amino acid analogs, and / or any other molecules suitable for the purpose of the domain. For example: 1. A split initiator probe may contain one or more split initiator domains composed of DNA, and a target-binding domain composed of DNA. 2. A split initiator probe may contain one or more split initiator domains composed of DNA, and a target-binding domain composed of amino acids (e.g., an antibody, or a nanobody, or an antibody fragment). 3. The split initiator probe may include a split initiator domain composed of a synthetic nucleic acid analog, and a target binding domain composed of a combination of DNA and 2'OMe-RNA. 4. The split initiator probe may include a split initiator domain composed of 2'OMe-RNA and a target binding domain composed of an RNA-protein combination. 5. The split initiator probe may include a split initiator domain composed of DNA and a target-binding domain composed of PNA. 6. A split initiator probe consists of one or more split initiator domains composed of any nucleic acid or nucleic acid analog, and a suitable material for binding to the target molecule. It may contain one or more target-binding domains composed of a combination of the above.

[0126] In some embodiments, the split initiator probe may contain one covalent molecule, or it may contain two or more molecules (each covalently linked) that interact non-covalently to form a complex. For example: 1. The splitting initiator probe may include a splitting initiator domain composed of DNA, covalently linked to a target-binding domain composed of DNA. 2. The splitting initiator probe may include one or more splitting initiator domains composed of DNA, covalently linked to a dCas9 (or another Cas) noncovalently bound to a guide RNA (gRNA), such that the target-binding domain contains a gRNA:dCas9 complex (or a gRNA:Cas complex using another Cas). 3. The splitting initiator probe may contain one or more splitting initiator domains made of DNA, covalently linked to a gRNA noncovalently bound to dCas9 (or another Cas), such that the target binding domain includes a gRNA:dCas9 complex (or a gRNA:Cas complex using another Cas). 4. A split initiator probe may include a split initiator domain composed of a nucleic acid or nucleic acid analog, which is covalently or noncovalently bound to a target-binding domain containing one or more molecules. Each split initiator probe in a probe unit may have the same or different material composition as other split initiator probes in the probe unit. Each split initiator probe in a probe unit may have a target-binding region that binds to different detection sites on the same target molecule, or to different detection sites within a target molecule complex, or to different detection sites within a target assembly of a proximal molecule or complex.

[0127] No-wash signal generation using split initiator probes and HCR signal amplification. In some embodiments, split initiator probes and HCR amplifiers are used to generate a signal using a no-wash protocol in which unused probes and amplifiers are not removed from the sample. In some embodiments, two or more split initiator probes in a probe unit each contain a split initiator and a target-binding region such that, although the individual split initiator probes are not sufficient to efficiently induce HCR signal amplification, when the target-binding domain of each probe binds to a cognitive detection site on the target, the split initiators colocalize to form a complete HCR initiator, inducing the growth of an anchored HCR amplification polymer. In some embodiments, each HCR hairpin is labeled with one or more reporters and / or one or more quenchers and / or one or more FRET pairs. In some embodiments, the HCR amplifier includes two HCR hairpins, H1 and H2, which undergo a polymerization cascade, a process of alternately polymerizing H1 and H2, to grow an anchored HCR amplification polymer. In some embodiments, the HCR amplifier includes four HCR hairpins, H1, H2, H3, and H4, which undergo a polymerization cascade, a process of alternately polymerizing H1, H2, H3, and H4, to grow an anchored HCR-amplified polymer. In some embodiments, one or more reporters on the HCR hairpins are quenched before polymerization and non-quenched after polymerization. In some embodiments, two reporters, including a FRET pair, are not close enough to perform efficient FRET before HCR polymerization but are close enough to perform efficient FRET after polymerization. In some embodiments, HCR signal generation is higher-order structure-dependent, such that the total HCR signal in the sample is lower before HCR polymerization than after HCR polymerization, due to a) steric changes that occur when HCR monomers open to bond with the HCR polymer, and / or b) colocalization of two or more HCR hairpins within the HCR polymer. In some embodiments, the HCR signal is enriched at the target site by the growth of an anchored HCR amplification polymer.In some embodiments, the split initiator probe and HCR amplifier are used to generate a signal using a no-wash protocol in which unused probes and amplifiers are not removed from the sample. In some embodiments, the split initiator probe and HCR amplifier are used to generate a signal using a no-wash protocol in a sample containing one or more targets within any of the following: living cells, living organisms, tissue sections, brain slices, bulk solution, fixed cells, fixed tissues, or fixed embryos. In some embodiments, the targets are not crosslinked and / or fixed in the sample and / or trapped in a solid phase. In some embodiments, the HCR hairpin includes a label that is a split substrate such that the complete substrate colocalizes upon HCR amplification (see, for example, Figure 18F). In some embodiments, the labeled probe includes a labeled chain (bound to the reporter) that is hybridized to a blocker chain (bound to the quencher) such that when the labeled chain hybridizes to a complete colocalized entity within the HCR amplified polymer, the blocker chain is removed from the labeled chain, the reporter is separated from the quencher, and a signal is generated.

[0128] Removal of signals from the sample In some embodiments, the HCR signal is removed from the sample after signal detection (see, for example, Figures 23A–23N and 40A–40N). The signal can be removed from the sample by any method that reduces the number of reporters generating the signal in the sample. For example: • Photobleaching of the fluorescent reporter molecule using light and / or chemical reagents (see, for example, Figures 23A and 40A), • Chemically cut the reporter from the HCR hairpin and wash it off the sample (e.g., TCEP) (see, for example, Figures 23B and 40B). • Chemically cleave the reporter from the labeled probe and wash it from the sample (see, for example, Figures 23C and 40C). • Chemically cleave the hairpin to fragment the HCR amplification polymer and wash the fragments away from the sample (see, for example, Figures 23D and 40D). • Chemically cleave the probe to release the HCR amplification polymer from the target, and wash the released amplification polymer from the sample (see, for example, Figures 23E and 40E). • Dehybridize the hairpins from the HCR amplification polymer using auxiliary chains and wash the hairpins from the sample (see, for example, Figures 23F, 40F, and 40N). • Dehybridize the labeled probe from the HCR amplification polymer using auxiliary chains and wash the labeled probe from the sample (see, for example, Figures 23G and 40G). • After destabilizing the HCR amplification polymer using a chemical modifier and / or high temperature, wash the hairpin from the sample (see, for example, Figures 23H and 40H). • After destabilizing the interaction between the probe and its target using a chemical modifier and / or high temperature, wash the unattached amplified polymer from the sample (see, for example, Figures 23I and 40I). • After destabilizing the interaction between the labeled probe and its substrate using a chemical denaturing agent and / or high temperature, wash the labeled probe from the sample (see, for example, Figures 23J and 40J). • Decompose the amplified polymer and / or probe using an enzyme, and wash the decomposed molecules from the sample (see, for example, Figures 23K and 40K-40M). • Degrade the DNA amplification polymer and / or DNA probe and / or DNA target using deoxyribonuclease, and wash the resulting molecules from the sample (see, for example, Figures 23K, 40K-40M). • After degrading the RNA target using RNase, wash the unattached amplified polymer from the sample (see, for example, Figures 23L and 40K-40M). After degrading the protein target using protease, the amplified polymer, whose anchorage has been released, is used as a sample. Wash from (see, for example, Figures 23M and 40K to 40M), • Degrading RNA targets using a combination of RNases, degrading DNA amplification polymers and / or DNA probes using deoxyribonucleases, and washing the resulting molecules from the sample (see, for example, Figures 23N and 40K-40M). • Degrading protein targets using a combination of proteases, and degrading DNA amplification polymers and / or DNA probes and / or DNA targets using deoxyribonucleases, followed by washing the resulting molecules from the sample (see, for example, Figures 23O and 40K-40M). • Using two or more of the above methods simultaneously or separately.

[0129] Assay format In some embodiments, the HCR signal can be measured in a variety of assay formats, including but not limited to: blot, Northern blot, Western blot, Southern blot, spot blot, paper assay, flow cytometry assay, fluorescence flow cytometry assay, cell sorting assay, fluorescently labeled cell sorting assay, magnetically activated cell sorting assay, microscopy assay, optical microscopy assay, epifluorescence microscopy assay, confocal microscopy assay, lightsheet microscopy assay, microarray assay, bead-based assay, mass spectrometry assay, fluorescence microscopy assay, mass spectrometry microscopy assay, mass spectrometry flow cytometry assay, fluorescence assay, chemiluminescence assay, bioluminescence assay, colorimetric analysis, electrochemical impedance assay, electrochemical chemiluminescence assay, energy dissipation assay, assay using the human eye, assay using a mobile phone camera, gel electrophoresis assay, insight hybridization (ISH) assay, RNA-ISH assay, DNA-ISH assay, immunohistochemistry (IHC) assay, autoradiography assay, or any assay capable of detecting the signal generated by the HCR amplification polymer.

[0130] Type of sample In some embodiments, HCR initiator-labeled probes and / or HCR-splitting initiator probes can be used together with HCR amplification hairpins to detect targets in a sample, including molecules, complexes, or aggregates of proximal molecules or complexes. Target molecules may be present in samples including, for example, bacteria, zebrafish embryos, chicken embryos, mouse embryos, human biopsy specimens, human tissue sections, FFPE tissue sections, urine samples, blood samples, stool samples, mouse tissue sections, brain slices, sea urchin embryos, nematode larvae, fruit fly embryos, model organisms, non-model organisms, mixed biotapes, environmental samples containing unknown organisms, communities of organisms (e.g., mixtures of intestinal protists and bacteria from another organism), termites, microbiomes, clinical specimens, diagnostic specimens, Sputum samples, tumor biopsy specimens, research samples, samples containing human-derived material, samples containing material from pets (e.g., dogs, cats, rabbits, lizards, snakes, or fish), samples containing material from wild animals (e.g., cheetahs, elephants, rhinos, or chimpanzees), samples containing material from extinct animals (e.g., mammoths, dodos, great auks, triceratops, or passenger pigeons), living cells (e.g., bacteria or cultured mammalian cells), or living organisms (e.g., living mice or living humans).

[0131] In some embodiments, the target may be free in a soluble state within the sample. For example, the target may be free in a soluble state within a sample such as a test tube, cells, embryos, organisms, tissue sections, or biological specimens.

[0132] In some embodiments, the target may be covalently crosslinked or non-covalently bound to one or more capture probes covalently or non-covalently attached to a solid phase. For example, it may be directly or indirectly bound to capture probes covalently linked to a microarray or beads.

[0133] In some embodiments, the target may be fixed, covalently crosslinked, or noncovalently bound to a solid phase directly or indirectly. For example, the target may be bound, fixed, or covalently crosslinked to a slide, blot, membrane, paper substrate, or any other substrate. The target may be fixed or covalently linked to cells, embryos, organisms, tissue sections, biological specimens, or any other sample. The target may be covalently bound within a sample that is fixed and permeabilized, fixed but not permeabilized, or not fixed but permeabilized.

[0134] In some embodiments, the target may be free within living cells, living embryos, organisms, living ecosystems, or communities of organisms (e.g., the intestinal microbiome of a mammal). The target may be attached to but outside of a cell or organism, or contained within a cell or organism. The target may be covalently cross-linked within living cells, living embryos, organisms, living ecosystems, or communities of living organisms. The target may be present or absent within one or more cell types in the sample. The target may be present or absent within one or more species of organisms in 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 a spread sample. The target may be present in a compressed sample. To increase spatial isolation between molecules, the sample may be spread before detecting the target. To decrease spatial isolation between molecules, the sample may be compressed before detecting the target. Target and / or other molecules may be crosslinked to the stretched sample to maintain the relative positions of molecules within the sample during stretching. Target and / or other molecules may be crosslinked to the gel, matrix, or other reagents introduced into the sample to stretch the sample while maintaining the relative positions and / or orientations of molecules within the sample during stretching. The sample may be stretched and / or compressed separately in different tissues and / or organs within the sample with different stretching and / or compressing coefficients.

[0135] Sample fixation In some embodiments, the target molecule can be crosslinked to the sample so that it is retained during subsequent experimental steps. 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)).

[0136] Permeabilization treatment of the sample In some embodiments, the sample can be processed to enhance the accessibility of the target molecule to the HCR probe and amplifier. For example, the sample (e.g., cells, tissue sections, or whole embryos) can be permeabilized using chemical reagents (e.g., methanol, ethanol, washing agents) or enzymes (e.g., protease K). Target accessibility can also be enhanced by homogenization, microdissection, electroporation, sectioning, heat treatment (e.g., Non-Patent Document 4), and / or microwave treatment (e.g., Non-Patent Document 5). Alternatively, the HCR probe and amplifier may be delivered by passing a chemical transfection reagent through the cell membrane.

[0137] Sample washing to remove unbound reagents from the sample. In some embodiments, background can be reduced by washing away unused imaging reagents from the sample. For example, washing can be used to remove probes, HCR initiator-labeled probes, HCR splitting initiator probes, HCR amplification hairpins, amplification reagents, labeled probes, antibodies, and / or other imaging reagents from the sample. Washing can be performed at a certain temperature using chemical reagents such that specifically bound imaging reagents are largely not removed (signal retention) and nonspecifically bound imaging reagents are largely removed (background reduction). For example, washing buffers can include denaturants (e.g., formamide, urea), salt buffers (e.g., sodium chloride sodium citrate (SSC), phosphate-buffered saline (PBS)), acids (e.g., citric acid), surfactants (e.g., Tween® 20, Triton-X, SDS), or blocking agents (e.g., tRNA, salmon sperm DNA, BSA, Ficol, polyvinilypyrrolidone, heparin). By combining the washing buffer with the washing temperature (e.g., 25-80°C), the precision of the washing process can be optimized.

[0138] Highly accurate and precise target quantification In some embodiments, HCR probes and HCR amplifiers enable quantitative analysis of target molecules in an anatomical context, generating a signal that changes approximately proportionally to the number of target molecules per imaging voxel. This quantitative characteristic is derived from the sum of signals occurring at three levels during imaging: 1) The sum of one or more initiator-labeled probes for each target molecule, or one or more probe units (each containing two or more segmented initiator probes) for each target molecule. 2) The sum of multiple HCR amplification hairpins for each amplified polymer, anchored to the initiator-labeled probe or to the probe unit of a segmented initiator probe that colocalizes the complete initiator. 3) The total number of target molecules (0, 1, or more) in the imaging pixel. By defining imaging voxels that average the intensity of adjacent pixels, quantitative accuracy can be further increased while maintaining intracellular resolution. For example, highly accurate and precise quantitative imaging at intracellular resolution is shown in Figures 11A-11B for mRNA targets and in Figures 41A-41C for protein targets. The quantitative properties of the HCR signal are derived from the binding properties of the HCR probe, the polymerization properties of the HCR amplification hairpin, and the central limit theorem, and by utilizing summation and averaging during and after image acquisition, a signal that changes approximately proportionally to the target amount is generated. The same quantitative properties apply to other assay formats in which summation and / or averaging are performed during and / or after data acquisition (e.g., by flow cytometry or blot scanner).

[0139] Multiplexing using initiator-labeled probes In some embodiments, an HCR probe set containing initiator-labeled probes and an HCR amplifier containing HCR hairpins can be used for multi-target analysis (e.g., targeted analysis via imaging, blotting, flow cytometry, mass cytometry, gel analysis, or any other analytical method) in which multiple targets are analyzed simultaneously within the same sample. Consider a sample containing some or all of the N target species and 0, 1, or more additional non-target off-target species. Each target can be detected using a probe set containing one or more initiator-labeled probes (each containing one or more HCR initiators) that selectively bind to a cognitive target. In some embodiments, the probe set for each of the N target species is labeled with a different HCR initiator for each HCR amplifier. For example, target 1 can be detected with probe set 1 labeled with an HCR initiator for HCR amplifier 1, and target 2 can be detected with probe set 2 labeled with an HCR initiator for HCR amplifier 2, and so on, with probe set N being labeled with an HCR initiator for HCR amplifier N.

[0140] In some embodiments, the N probe sets operate orthogonally so that each probe set selectively binds to its cognitive target, regardless of whether other probe sets and / or targets are present in the sample. Each amplifier operates orthogonally such that 1) the hairpins for each amplifier coexist metastable in the absence of a cognitive HCR initiator, and 2) each amplifier selectively induces polymerization if a cognitive initiator is present, regardless of whether other amplifiers are present in the sample.

[0141] In some embodiments, the labels supported by each HCR amplifier are orthogonal (e.g., fluorescent labels identifiable by fluorescence microscopy, or rare-earth labels identifiable by mass cytometry) so that the analytical method can measure the signal produced by each HCR amplifier regardless of the presence of other labels in the sample.

[0142] For example, multiplex imaging using an initiator-labeled probe and simultaneous HCR signal amplification for all targets is shown in Figures 29, 30, 41, and 43.

[0143] In some embodiments, multi-target analysis for N target species (type j=1, ..., N) can be achieved as follows: 1. All targets are detected simultaneously using N orthogonal HCR initiator-labeled probe sets (for target species j=1, ..., N, the initiator-labeled probe of probe set j binds to target j). 2. Using N orthogonal HCR amplifiers, the signals of all target species are amplified simultaneously (for j=1, ..., N, hairpins from amplifier j polymerize in response to initiator j, forming an amplified polymer j anchored to target type j). 3. In the case of target species j=1, ..., N, the sample is analyzed using a measuring device to detect reporter j, which is directly supported by one or more hairpins of amplifier j, or indirectly bound to one or more hairpins of amplifier j.

[0144] Multiplexing using split initiator probes In some embodiments, an HCR probe set including a segmented initiator probe and an HCR amplifier including an HCR hairpin can be used for multi-target analysis (e.g., targeted analysis via imaging, blotting, flow cytometry, mass cytometry, gel analysis, or any other analytical method) in which multiple targets are analyzed simultaneously within the same sample. Consider a sample containing some or all of the N target species and 0, 1, or more additional non-target off-target species. Each target can be detected using a probe set containing one or more probe units (each containing two or more segmented initiator probes) that selectively bind to a cognitive target such that each bound probe unit co-localizes a complete HCR initiator. In some embodiments, the probe set for each of the N target species co-localizes a different complete HCR initiator for each HCR amplifier. For example, target 1 can be detected by probe set 1 which colocalizes one or more complete HCR initiators for HCR amplifier 1, and target 2 can be detected by probe set 2 which colocalizes one or more complete HCR initiators for HCR amplifier 2, and so on, with probe set N colocalizing one or more complete HCR initiators for HCR amplifier N.

[0145] In some embodiments, N probe sets operate orthogonally so that each probe set selectively binds to its cognitive target, regardless of whether other probe sets and / or targets are present in the sample. In some embodiments, N amplifiers operate orthogonally so that 1) the hairpin for each amplifier coexists metastable in the absence of a cognitive complete initiator colocalized by the cognitive target, and 2) each amplifier selectively induces and polymerizes if a cognitive complete initiator is colocalized by its cognitive target, regardless of whether other amplifiers are present in the sample.

[0146] In some embodiments, the labels supported by each HCR amplifier are orthogonal (e.g., fluorescent labels identifiable by fluorescence microscopy, or rare-earth labels identifiable by mass cytometry) so that the analytical method can measure the signal produced by each HCR amplifier regardless of the presence of other labels in the sample.

[0147] For example, multiplex imaging using a segmented initiator probe and simultaneous HCR signal amplification for all targets is shown in Figures 10 and 11.

[0148] In some embodiments, multi-target analysis for N target species (type j=1, ..., N) can be achieved as follows: 1. All targets are detected simultaneously using N orthogonal HCR splitting initiator probe sets (for target species j=1, ..., N, the splitting initiator probes of probe set j bind to target j so that a complete HCR initiator j is colocalized for each probe unit of probe set j). 2. Using N orthogonal HCR amplifiers, the signals of all target species are amplified simultaneously (for j=1, ..., N, hairpins from amplifier j polymerize in response to the complete HCR initiator j, forming an amplified polymer j anchored to target type j). 3. In the case of target species j=1, ..., N, the sample is analyzed using a measuring device to detect reporter j, which is directly supported by one or more hairpins of amplifier j, or indirectly bound to one or more hairpins of amplifier j.

[0149] Multiplexing using a combination of initiator-labeled probes and segmented initiator probes. In some embodiments, multiplex analysis is performed on a sample using initiator-labeled probes to detect one or more targets (which may be of different types) and split initiator probes to detect one or more other targets (which may be of different types). For example, in the same sample, one or more protein targets and one or more small RNA targets can be detected with an orthogonal initiator-labeled probe, one or more mRNA targets and / or DNA targets can be detected with an orthogonal split initiator probe, and one or more complex targets (including complexes of two or more non-covalently linked molecules) can be detected with a split initiator probe. In some embodiments, a target-specific probe set (including one or more probe units, each containing one or more initiator-labeled probes or two or more split initiator probes) induces an orthogonal HCR amplifier that produces an orthogonal signal (directly or indirectly). In some embodiments, HCR signal amplification is performed simultaneously for all target species.

[0150] For example, Figures 31 and 32 show multiplex imaging using initiator-labeled probes for one or more targets, segmented initiator probes for one or more targets, and simultaneous HCR signal amplification for all targets.

[0151] Multiplexing using spectral imaging In some embodiments, spectral analysis can be used to increase the number of labels that can be distinguished from one another. For example, two fluorophores can be identified by their emission intensity using a band filter. If the measurements have overlapping emission spectra that prevent the identification of the two labels, they can be identified using spectral imaging, which uses multiple emission measurements at different wavelengths to identify signals from the two labels, even if the emission spectra of the labels overlap considerably. In some embodiments, spectral imaging can enable spectral identification of 10 fluorescent dyes, or 20 fluorescent dyes, or even 30 or more fluorescent dyes.

[0152] Multiplexing using hybrid spectra with multi-reporter polymers Since HCR polymerization proceeds by alternating H1 polymerization and H2 polymerization steps, the resulting HCR-amplified polymer will contain either 1) an equal number of H1 and H2 hairpins, 2) one more H1 hairpin, or 3) one more H2 hairpin. As the length of the polymer increases, the proportion of H1 hairpins in the polymer approaches 0.5, and the proportion of H2 hairpins in the polymer also approaches 0.5. In some embodiments, hairpin H1 is labeled with reporter R1 and hairpin H2 is labeled with reporter R2. The signal produced by the HCR-amplified polymer is a 1:1 blend signal produced by reporters R1 and R2, each having a novel hybrid spectrum. Consider N sets of reporters, each with a distinct spectrum. Using N reporters, it is possible to create N*(N-1) / 2 hybrid spectra, corresponding to the number of different reporter pairs that can be selected from the N sets of reporters. For example: 1) With 6 reporters, it is possible to create 6 * 5 / 2 = 15 hybrid reporter spectra; 2) With 8 reporters, it is possible to create 8 * 7 / 2 = 28 hybrid reporter spectra; 3) With 15 reporters, it is possible to create 15 * 14 / 2 = 105 hybrid reporter spectra; 4) With 50 reporters, it is possible to create 50 * 49 / 2 = 1225 hybrid reporter spectra; 5) With 100 reporters, it is possible to create 100 * 99 / 2 = 4950 hybrid reporter spectra.

[0153] Computer-aided sequencing of orthogonal HCR amplifiers using NUPACK In some embodiments, a series of orthogonal HCR amplifiers (with or without substrates and / or auxiliary chains) are designed using the reaction pathway designer included in the NUPACK software suite. 56、57In some embodiments, the sequence design is formulated as a multi-state optimization problem using a series of target test tubes to represent elemental steps in the reaction pathway and to model global crosstalk. 56 In some embodiments, each element step tube includes a series of desirable on-target complexes (each having a target secondary structure and target concentration) corresponding to the on-path hybridization product of a given step, as well as a series of undesirable off-target complexes (each having a disappearing target concentration) corresponding to the on-path reactants and off-path hybridization crosstalk of a given step. 56 In this scenario, these elemental step tubes facilitate the complete conversion of cognitive reactants to cognitive products and counteract local hybridization crosstalk between these same reactants. In some embodiments, elemental step tubes are specified for each orthogonal system in order to design N orthogonal systems simultaneously. In some embodiments, one global crosstalk tube is also specified to counteract extra-pathway interactions between systems. 56In some embodiments, in a global crosstalk tube, the on-target complex corresponds to all reactive species generated during all elemental steps of the system (e.g., the single-stranded output domain of the HCR hairpin opened via polymerization). In some embodiments, in a global crosstalk tube, the off-target complex corresponds to non-cognitive interactions between these reactive species. In some embodiments, the global crosstalk tube population excludes cognitive products (not visible as either on-target or off-target) that the reactive species are intended to form. In this scenario, all reactive species within the global crosstalk tube can be forced to either not react (remaining as desirable on-targets) or to undergo crosstalk reactions (forming undesirable off-targets), which is fundamental to minimizing global crosstalk during sequence optimization. In some embodiments, sequence design is constrained by complementarity specific to the reaction pathway (e.g., in Figure 1A, domain "a" is constrained by domain "a" * It is complementary to " and domain "b" is domain "b * (Complementary to ") 56 In some embodiments, the sequence is optimized by reducing ensemble defects, which quantify the average percentage of mispaired nucleotides across a multi-tube ensemble. 56 In some embodiments, the weight of the defect is applied within the ensemble defect, prioritizing design effort. 56 Ensemble defect optimization employs both a positive design paradigm, which explicitly designs element steps along the path, and a negative design paradigm, which explicitly designs to counter off-path crosstalk. 56 .

[0154] Multiplexing using repeated reporter detection In some embodiments, the same N labels can be used to detect multiple targets in a series of analysis rounds, increasing the number of targets that can be analyzed within a sample. For example, after imaging N targets using N labels, the signal can be removed from the sample, and another set of N targets can be detected using the same N labels. This approach is applicable to imaging multiple target species in the same sample, regardless of whether 1) the expression levels of various target species are high, low, variable within target species, and / or variable between different target species, and regardless of whether the expression patterns of various target species spatially overlap or not within the sample. Examples of multiplex analysis methods using repeated reporter detection are, but are not limited to, those described in Methods A to T below. In some embodiments, in one, more, and / or all of the steps in Methods A to T below, the selection of probe species can be different for each different target and, if desired, can be mixed in any process. In some embodiments of Methods A to T, 1) all targets may be detected with initiator-labeled probes, or 2) all targets may be detected with segmented initiator probes, or 3) one or more targets may be detected with initiator-labeled probes and other targets may be detected with segmented initiator probes. Thus, disclosure of one option in this specification provides for the other option and combinations thereof. For example, the statement "prepare N probe sets, each containing either a) one or more HCR initiator-labeled probes, or b) one or more probe units, each containing two or more HCR segmented initiators" means that any one of the N probe sets may be of either type (a or b), including the possibility that all probe sets are of the same type (all type a or all type b) and the possibility that some probe sets are of one type and some probe sets are of the other type (some type a and some type b).In the embodiments of the disclosure herein in which "or..." is indicated in this context, it is understood that the above types may be mixed (unless otherwise specified).

[0155] In some embodiments, any one of the following methods A-T can be combined with CARD, enzyme deactivation, and / or repeated CARD. Where the expression "further modifications optionally with CARD, enzyme deactivation, and / or repeated CARD" is used, it indicates that any of the embodiments discussed may be combined with any one or more of the embodiments discussed, including CARD, enzyme deactivation, and / or repeated CARD. This explicitly allows for further combinations of the various methods provided herein. Similarly, where the expression "further modifications optionally with multiplexing, CARD, enzyme deactivation, repeated CARD, repeated reporter detection, and / or repeated signal removal" is used, it indicates that any of the embodiments discussed may be combined with any one or more of the embodiments discussed, including multiplexing, CARD, enzyme deactivation, repeated CARD, repeated reporter detection, and / or repeated signal removal. These expressions are used as abbreviations to simplify the disclosure by relying on reference rather than repeating other parts of the disclosure.

[0156] In some embodiments, one or more optional steps of any one or more methods described herein can be combined with one or more other optional steps of any one or more methods described herein.

[0157] In some embodiments, one or more steps different by any one or more methods described herein can be combined with one or more other steps different by any one or more methods described herein.

[0158] In some embodiments, one or more optional steps differentiating by one or more methods described herein can be combined with one or more other optional steps differentiating by one or more methods described herein.

[0159] In some embodiments, a multiplex analysis method using repeated reporter detection is Method A (Example 15) (which may be further modified optionally with CARD, enzyme deactivation, and / or repeated CARD): 1. Prepare a sample that may contain the target and other non-target molecules. 2. Fix the above sample if desired. 3. If desired, the above sample may be subjected to permeabilization treatment. 4. Prepare a probe set that includes either a) one or more HCR initiator-labeled probes, or b) one or more probe units, each containing two or more HCR split initiators. 5. Wash the above sample if desired. 6. Prepare an HCR amplifier labeled with a reporter. 7. Wash the above sample if desired. 8. Detect the signal from the above reporter.

[0160] In some embodiments, Method A (Example 15) involves: Step A1: preparing a sample that may contain the target and other non-target molecules; Step A2: optionally immobilizing the sample; Step A3: optionally permeabilizing the sample; Step A4: a) one or more HCR initiator-labeled probes (see, for example, the probes in Figures 39A-39N, 41A, 42A-42F, and 43A), or b) two or more HCR splitting initiator probes (see, for example, the probes in Figures 3, 4, 5, and 17). Step A5: Prepare a probe set including one or more probe units (see, for example, the probe sets in Figures 8 and 16), each containing a probe unit; Step A6: Wash the sample as desired; Step A7: Prepare a reporter-labeled HCR amplifier (see, for example, the reporter-labeled amplifiers in Figures 8 and 18); Step A8: Detect a signal from the reporter as desired (see Figure 26A).

[0161] In some embodiments, Method A (for example, one example is shown in Example 15) is: Step A1: Prepare a sample which may contain the target and other molecules that are not the target; Step A2: Fix the sample; Step A3: Permeabilize the sample; Step A4 Step A5: Prepare a probe set comprising either a) one or more HCR initiator-labeled probes (see, for example, probes in Figures 39A-39N, 41A, 42A-42F, and 43A), or b) one or more probe units each containing two or more HCR splitting initiator probes (see, for example, probe units in Figures 3, 4, 5, and 17) (see, for example, probe sets in Figures 8 and 16); Step A5: Wash the sample; Step A6: Prepare a reporter-labeled HCR amplifier (see, for example, reporter-labeled amplifiers in Figures 8 and 18); Step A7: Wash the sample; Step A8: Detect a signal from the reporter (see Figure 26A).

[0162] In some embodiments, a multiplex analysis method using repeated reporter detection includes Method B (e.g., Example 16) (which may be further modified optionally with CARDs, enzyme deactivation, and / or repeated CARDs): 1. Prepare a sample that may contain up to N targets and other non-target molecules. 2. Fix the above sample if desired. 3. If desired, the above sample may be subjected to permeabilization treatment. 4. Prepare N probe sets, each containing either a) one or more HCR initiator-labeled probes, or b) one or more probe units, each containing two or more HCR splitting initiators. 5. Wash the above sample if desired. 6. Prepare N HCR amplifiers corresponding to the N probe sets mentioned above (each labeled with a different reporter). 7. Wash the above sample if desired. 8. Detect N signals from the N separate reporters mentioned above.

[0163] In some embodiments, Method B (e.g., Example 16) involves: Step B1: preparing a sample that may contain up to N targets and other non-target molecules; Step B2: optionally immobilizing the sample; Step B3: optionally permeabilizing the sample; Step B4: a) one or more HCR initiator-labeled probes (see, for example, probes in Figures 39A-39N, 41A, 42A-42F, and 43A), or b) two or more HCR splitting initiator probes (see, for example, probe units in Figures 3, 4, 5, and 17). The process includes: preparing N probe sets, each containing either multiple probe units (see, for example, the probe sets in Figures 8 and 16); step B5: optionally washing the sample; step B6: preparing N HCR amplifiers corresponding to the N probe sets (each labeled with a separate reporter) (see, for example, the reporter-labeled HCR amplifiers in Figures 8 and 18); step B7: optionally washing the sample; and step B8: detecting N signals from the N separate reporters (see Figure 26B).

[0164] In some embodiments, Method B (e.g., Example 16) involves: Step B1: Preparing a sample that may contain up to N targets and other non-target molecules; Step B2: Immobilizing the sample; Step B3: Permeabilizing the sample; Step B4: a) Adding one or more HCR initiator-labeled probes (e.g., see probes in Figures 39A-39N, 41A, 42A-42F, and 43A), or b) Adding two or more HCR splitting initiator probes (e.g., see probe units in Figures 3, 4, 5, and 17). The process includes: preparing N probe sets, each containing one of several probe units (see, for example, the probe sets in Figures 8 and 16); step B5: washing the sample; step B6: preparing N HCR amplifiers corresponding to the N probe sets (each labeled with a different reporter) (see, for example, the reporter-labeled HCR amplifiers in Figures 8 and 18); step B7: washing the sample; and step B8: detecting N signals from the N separate reporters (see Figure 26B).

[0165] In some embodiments, the multiplex analysis method using repeated reporter detection is Method C (e.g., Example 17) (which may be further modified optionally with CARD, enzyme deactivation, and / or repeated CARD): 1. Prepare a sample that may contain up to N targets and other non-target molecules. 2. Fix the above sample if desired. 3. If desired, the above sample may be subjected to permeabilization treatment. 4. Prepare a probe set (targeting one of the N target species listed above) that includes either a) one or more HCR initiator-labeled probes, or b) one or more probe units, each containing two or more HCR-divided initiators. 5. Wash the above sample if desired. 6. Prepare an HCR amplifier (labeled with a reporter) that corresponds to the probe set prepared above. 7. Wash the above sample if desired. 8. Detect the signal from the above reporter. 9. Remove the above signal from the above sample. 10. If desired, repeat one or more of steps C4 to C9 until signal detection is performed for all N targets.

[0166] In some embodiments, Method C (e.g., Example 17) includes either step C1: preparing a sample that may contain up to N targets and other non-target molecules; step C2: optionally immobilizing the sample; step C3: optionally permeabilizing the sample; and step C4: a) one or more HCR initiator-labeled probes (see, for example, probes in Figures 39A-39N, 41A, 42A-42F, and 43A), or b) one or more probe units each containing two or more HCR splitting initiator probes (see, for example, probe units in Figures 3, 4, 5, and 17) (see, for example, probe sets in Figures 8 and 16). The process includes: preparing a probe set (targeting one of the N target species); step C5: optionally washing the sample; step C6: preparing an HCR amplifier (labeled with a reporter) corresponding to the prepared probe set (see, for example, the reporter-labeled HCR amplifiers in Figures 8 and 18); step C7: optionally washing the sample; step C8: detecting a signal from the reporter; step C9: removing the signal from the sample (see, for example, Figure 23); and step C10: optionally repeating one or more of steps C4-C9 until a signal has been detected for all N targets (see Figure 26C).

[0167] In some embodiments, Method C (e.g., Example 17) involves either step C1: preparing a sample that may contain up to N targets and other non-target molecules; step C2: immobilizing the sample; step C3: permeabilizing the sample; and step C4: a) one or more HCR initiator-labeled probes (see probes in Figures 39A-39N, 41A, 42A-42F, and 43A), or b) one or more probe units (see probe sets in Figures 8 and 16), each containing two or more HCR splitting initiator probes (see probe units in Figures 3, 4, 5, and 17). The process includes: preparing a probe set (targeting one of the N target species); step C5: washing the sample; step C6: preparing an HCR amplifier (labeled with a reporter) corresponding to the prepared probe set (see, for example, the reporter-labeled HCR amplifiers in Figures 8 and 18); step C7: washing the sample; step C8: detecting the signal from the reporter; step C9: removing the signal from the sample (see, for example, Figure 23); and step C10: repeating one or more of steps C4-C9 until a signal has been detected for all N targets (see Figure 26C).

[0168] In some embodiments, the multiplex analysis method using repeated reporter detection is Method D (e.g., Example 18) (which may be further modified optionally with CARD, enzyme deactivation, and / or repeated CARD): 1. Prepare a sample that may contain up to N targets and other non-target molecules. 2. Fix the above sample if desired. 3. If desired, the above sample may be subjected to permeabilization treatment. 4. Prepare M probe sets (for M ≤ N; each targeting one of the M target species) each containing either a) one or more HCR initiator-labeled probes, or b) one or more probe units each containing two or more HCR splitting initiators. 5. Wash the above sample if desired. 6. Prepare M HCR amplifiers (each labeled with a different reporter) corresponding to the M probe sets mentioned above. 7. Wash the above sample if desired. 8. Detect M signals corresponding to the M separate reporters mentioned above. 9. Remove the above M signals from the above sample. 10. If desired, repeat one or more of steps 4-9 until signal detection is performed for all N targets.

[0169] In some embodiments, Method D (e.g., Example 18) involves: Step D1: preparing a sample that may contain up to N targets and other non-target molecules; Step D2: optionally immobilizing the sample; Step D3: optionally permeabilizing the sample; Step D4: preparing M probe sets (M ≤ M) each containing either a) one or more HCR initiator-labeled probes (e.g., probes in Figures 39A-39N, 41A, 42A-42F, and 43A), or b) one or more probe units (e.g., probe sets in Figures 8 and 16) each containing two or more HCR splitting initiator probes (e.g., probe units in Figures 3, 4, 5, and 17). Step D5: Prepare the samples (each targeting one of the M target species); Step D6: Wash the samples as desired; Step D7: Prepare M HCR amplifiers (each labeled with a different reporter) corresponding to the M probe sets (see, for example, reporter-labeled HCR amplifiers in Figures 8 and 18); Step D8: Detect M signals corresponding to the M separate reporters; Step D9: Remove the M signals from the samples (see, for example, Figure 23); Step D10: Repeat one or more of steps 4-9 until signal detection is performed for all N targets (see Figure 26D).

[0170] In some embodiments, Method D (e.g., Example 18) involves: Step D1: preparing a sample that may contain up to N targets and other non-target molecules; Step D2: immobilizing the sample; Step D3: permeabilizing the sample; Step D4: preparing M probe sets, each containing either a) one or more HCR initiator-labeled probes (e.g., see probes in Figures 39A-39N, 41A, 42A-42F, and 43A), or b) one or more probe units (e.g., see probe sets in Figures 8 and 16) each containing two or more HCR splitting initiator probes (e.g., see probe units in Figures 3, 4, 5, and 17). For M ≤ N, prepare a probe set (each targeting one of the M target species); step D5: wash the sample; step D6: prepare M HCR amplifiers (each labeled with a different reporter) corresponding to the M probe sets (see, for example, reporter-labeled HCR amplifiers in Figures 8 and 18); step D7: wash the sample; step D8: detect M signals corresponding to the M separate reporters; step D9: remove the M signals from the sample (see, for example, Figure 23); step D10: repeat one or more of steps 4-9 until signal detection is performed for all N targets (see Figure 26D).

[0171] In some embodiments, the multiplex analysis method using repeated reporter detection is Method E (e.g., Example 19) (which may be further modified optionally with CARD, enzyme deactivation, and / or repeated CARD): 1. Prepare a sample that may contain up to N targets and other non-target molecules. 2. Fix the above sample if desired. 3. If desired, the above sample may be subjected to permeabilization treatment. 4. Prepare N probe sets, each containing either a) one or more HCR initiator-labeled probes, or b) one or more probe units, each containing two or more HCR-divided initiators (each targeting one of the N target species). 5. Wash the above sample if desired. 6. Prepare an HCR amplifier (labeled with a reporter) that corresponds to one of the probe sets mentioned above. 7. Wash the above sample if desired. 8. Detect the signal from the above reporter. 9. Remove the above signal from the above sample. 10. If desired, repeat one or more of steps 6-9 until signal detection is performed for all N targets.

[0172] In some embodiments, Method E (e.g., Example 19) involves: Step E1: preparing a sample that may contain up to N targets and other non-target molecules; Step E2: optionally immobilizing the sample; Step E3: optionally permeabilizing the sample; Step E4: a) one or more HCR initiator-labeled probes (see probes in Figures 39A-39N, 41A, 42A-42F, and 43A), or b) one or more probe units (see probe sets in Figures 8 and 16), each containing two or more HCR splitting initiator probes (see probe units in Figures 3, 4, 5, and 17). The process includes: preparing N probe sets (each targeting one of N target species); step E5: optionally washing the sample; step E6: preparing an HCR amplifier (labeled with a reporter) corresponding to one of the probe sets (see, for example, the reporter-labeled amplifiers in Figures 8 and 18); step E7: optionally washing the sample; step E8: detecting the signal from the reporter; step E9: removing the signal from the sample (see, for example, Figure 23); and step E10: optionally repeating one or more of steps 6-9 until signal detection is performed for all N targets (see Figure 26E).

[0173] In some embodiments, Method E (e.g., Example 19) involves: Step E1: preparing a sample that may contain up to N targets and other non-target molecules; Step E2: immobilizing the sample; Step E3: permeabilizing the sample; Step E4: a) one or more HCR initiator-labeled probes (see probes in Figures 39A-39N, 41A, 42A-42F, and 43A), or b) one or more probe units (see probe sets in Figures 8 and 16) each containing two or more HCR splitting initiator probes (see probe units in Figures 3, 4, 5, and 17). The process includes: preparing N probe sets (each targeting one of N target species); step E5: washing the sample; step E6: preparing an HCR amplifier (labeled with a reporter) corresponding to one of the probe sets (see, for example, the reporter-labeled amplifiers in Figures 8 and 18); step E7: washing the sample; step E8: detecting the signal from the reporter; step E9: removing the signal from the sample (see, for example, Figure 23); and step E10: repeating one or more of steps 6-9 until a signal has been detected for all N targets (see Figure 26E).

[0174] In some embodiments, the multiplex analysis method using repeated reporter detection is Method F (e.g., Example 20) (which may be further modified optionally with CARD, enzyme deactivation, and / or repeated CARD): 1. Prepare a sample that may contain up to N targets and other non-target molecules. 2. Fix the above sample if desired. 3. If desired, the above sample may be subjected to permeabilization treatment. 4. Prepare N probe sets, each containing either a) one or more HCR initiator-labeled probes, or b) one or more probe units, each containing two or more HCR-divided initiators (each targeting one of the N target species). 5. Wash the above sample if desired. 6. Prepare M HCR amplifiers (if M ≤ N; each is labeled with a separate reporter) corresponding to M of the N probe sets mentioned above. 7. Wash the above sample if desired. 8. Detect M signals corresponding to the M reporters mentioned above. 9. Remove the above M signals from the above sample. 10. If desired, repeat one or more of steps 6-9 until signal detection is performed for all N targets.

[0175] In some embodiments, Method F (e.g., Example 20) involves: Step F1: preparing a sample that may contain up to N targets and other non-target molecules; Step F2: optionally immobilizing the sample; Step F3: optionally permeabilizing the sample; Step F4: N probe sets, each containing either a) one or more HCR initiator-labeled probes (see probes in Figures 39A-39N, 41A, 42A-42F, and 43A), or b) one or more probe units (see probe sets in Figures 8 and 16) each containing two or more HCR splitting initiator probes (see probe units in Figures 3, 4, 5, and 17), respectively. Step F5: Prepare a sample (targeting one of N target species); optionally wash the sample; step F6: Prepare M HCR amplifiers corresponding to M of the N probe sets (if M ≤ N; each is labeled with a different reporter) (see, for example, reporter-labeled amplifiers in Figures 8 and 18); step F7: optionally wash the sample; step F8: detect M signals corresponding to the M reporters; step F9: remove the M signals from the sample (see, for example, Figure 23); step F10: optionally repeat one or more of steps 6-9 until signal detection is performed for all N targets (see Figure 26F).

[0176] In some embodiments, Method F (e.g., Example 20) involves step F1: preparing a sample which may contain up to N targets and other non-target molecules; step F2: the above Step F3: Fix the sample; Step F4: Permeabilize the sample; Step F4: Prepare N probe sets, each containing either a) one or more HCR initiator-labeled probes (see, for example, probes in Figures 39A-39N, 41A, 42A-42F, and 43A), or b) one or more probe units (see, for example, probe sets in Figures 8 and 16) each containing two or more HCR splitting initiator probes (see, for example, probe units in Figures 3, 4, 5, and 17); Step F5: Process the sample The process includes: washing; step F6: preparing M HCR amplifiers corresponding to M of the N probe sets (if M ≤ N; each is labeled with a different reporter) (see, for example, reporter-labeled amplifiers in Figures 8 and 18); step F7: washing the sample; step F8: detecting M signals corresponding to the M reporters; step F9: removing the M signals from the sample (see, for example, Figure 23); and step F10: repeating one or more of steps 6-9 until signal detection is performed for all N targets (see Figure 26F).

[0177] In some embodiments, the multiplex analysis method using repeated reporter detection is Method G (e.g., Example 21) (which may be further modified optionally with CARD, enzyme deactivation, and / or repeated CARD): 1. Prepare a sample that may contain one or more targets and other non-target molecules. 2. Fix the above sample if desired. 3. If desired, the above sample may be subjected to permeabilization treatment. 4. Prepare one or more probe sets, each containing either a) one or more HCR initiator-labeled probes, or b) one or more probe units, each containing two or more HCR splitting initiators. 5. Wash the above sample if desired. 6. Prepare one or more HCR amplifiers (each labeled with one or more reporters). 7. Wash the above sample if desired. 8. Detecting one or more signals from one or more reporters. 9. Remove one or more probe sets from the sample, if desired. 10. If desired, remove one or more HCR amplifiers from the sample. 11. If desired, remove one or more reporters from the above sample. 12. Remove one or more signals from the sample, if desired. 13. If desired, repeat any of steps 2 to 12 once or more times in any order.

[0178] In some embodiments, Method G (e.g., Example 21) involves: Step G1: Preparing a sample that may contain one or more targets and other non-target molecules; Step G2: Optionally, immobilizing the sample; Step G3: Optionally, permeabilizing the sample; Step G4: Preparing one or more probe sets, each containing either a) one or more HCR initiator-labeled probes (e.g., probes in Figures 39A-39N, 41A, 42A-42F, and 43A), or b) one or more probe units (e.g., probe sets in Figures 8 and 16) each containing two or more HCR splitting initiator probes (e.g., probe units in Figures 3, 4, 5, and 17); Step G5: Optionally, washing the sample; Step G6: 1 or The process includes: preparing multiple HCR amplifiers (each labeled with one or more reporters) (see, for example, reporter-labeled HCR amplifiers in Figures 8 and 18); step G7: optionally washing the sample; step G8: detecting one or more signals from one or more reporters; step G9: optionally removing one or more probe sets from the sample; step G10: optionally removing one or more HCR amplifiers from the sample; step G11: optionally removing one or more reporters from the sample; step G12: optionally removing one or more signals from the sample (see, for example, Figure 23); and step G13: optionally repeating any of steps 2-12 once or multiple times in any order (see Figure 26G).

[0179] In some embodiments, Method G (e.g., Example 21) involves: Step G1: Preparing a sample that may contain one or more targets and other non-target molecules; Step G2: Immobilizing the sample; Step G3: Permeabilizing the sample; Step G4: Preparing one or more probe sets, each containing either a) one or more HCR initiator-labeled probes (e.g., probes in Figures 39A-39N, 41A, 42A-42F, and 43A), or b) one or more probe units (e.g., probe sets in Figures 8 and 16) each containing two or more HCR splitting initiator probes (e.g., probe units in Figures 3, 4, 5, and 17); Step G5: Washing the sample; Step G6: Prepare one or more HCR amplifiers (each labeled with one or more reporters) (see, for example, reporter-labeled HCR amplifiers in Figures 8 and 18); Step G7: Wash the sample; Step G8: Detect one or more signals from one or more reporters; Step G9: Remove one or more probe sets from the sample; Step G10: Remove one or more HCR amplifiers from the sample; Step G11: Remove one or more reporters from the sample; Step G12: Remove one or more signals from the sample (see, for example, Figure 23); Step G13: Repeat any of steps 2-12 once or more times in any order (see Figure 26G).

[0180] In some embodiments, the multiplex analysis method using repeated reporter detection is Method H (e.g., Example 22) (which may be further modified optionally with CARD, enzyme deactivation, and / or repeated CARD): 1. Prepare a sample that may contain the target and other non-target molecules. 2. Fix the above sample if desired. 3. If desired, the above sample may be subjected to permeabilization treatment. 4. Prepare a probe set that includes either a) one or more HCR initiator-labeled probes, or b) one or more probe units, each containing two or more HCR split initiators. 5. Wash the above sample if desired. 6. Prepare an HCR amplifier labeled with the substrate. 7. Wash the above sample if desired. 8. Prepare a labeled probe (bound to the reporter) corresponding to the above substrate. 9. Wash the above sample if desired. 10. Detect the signal from the above reporter.

[0181] In some embodiments, Method H (e.g., Example 22) involves: Step H1: preparing a sample that may contain the target and other non-target molecules; Step H2: optionally immobilizing the sample; Step H3: optionally permeabilizing the sample; Step H4: a) one or more HCR initiator-labeled probes (see, for example, the probes in Figures 39A-39N, 41A, 42A-42F, and 43A), or b) one or more probe units each containing two or more HCR splitting initiator probes (see, for example, the probe units in Figures 3, 4, 5, and 17). The process includes: preparing a probe set (see, for example, the probe sets in Figures 8 and 16); step H5: washing the sample as desired; step H6: preparing a substrate-labeled HCR amplifier (see, for example, the substrate-labeled HCR amplifier in Figure 18); step H7: washing the sample as desired; step H8: preparing a labeled probe (bound to a reporter) corresponding to the substrate (see, for example, the labeled probe in Figure 20); step H9: washing the sample as desired; and step H10: detecting a signal from the reporter (see Figure 26H).

[0182] In some embodiments, Method H (e.g., Example 22) involves: Step H1: preparing a sample that may contain the target and other non-target molecules; Step H2: immobilizing the sample; Step H3: permeabilizing the sample; Step H4: a) one or more HCR initiator-labeled probes (see, for example, the probes in Figures 39A-39N, 41A, 42A-42F, and 43A), or b) one or more probe units, each containing two or more HCR splitting initiator probes (see, for example, the probe units in Figures 3, 4, 5, and 17). Step H5: Prepare a probe set including one of the probe sets in Figures 8 and 16 (see, for example, the probe sets in Figures 8 and 16); Step H6: Wash the sample; Step H7: Prepare a substrate-labeled HCR amplifier (see, for example, the substrate-labeled HCR amplifier in Figure 18); Step H8: Wash the sample; Step H9: Prepare a labeled probe (bound to a reporter) corresponding to the substrate (see, for example, the labeled probe in Figure 20); Step H10: Detect a signal from the reporter (see Figure 26H).

[0183] In some embodiments, the multiplex analysis method using repeated reporter detection is Method I (e.g., Example 23) (which may be further modified optionally with CARD, enzyme deactivation, and / or repeated CARD): 1. Prepare a sample that may contain up to N targets and other non-target molecules. 2. Fix the above sample if desired. 3. If desired, the above sample may be subjected to permeabilization treatment. 4. Prepare N probe sets, each containing either a) one or more HCR initiator-labeled probes, or b) one or more probe units, each containing two or more HCR splitting initiators. 5. Wash the above sample if desired. 6. Prepare N HCR amplifiers (each labeled with a different substrate) corresponding to the N probe sets mentioned above. 7. Wash the above sample if desired. 8. Prepare N labeled probes (each bound to a different reporter) corresponding to the N separate substrates mentioned above. 9. Detect N signals from the N separate reporters mentioned above.

[0184] In some embodiments, Method I (e.g., Example 23) involves: Step I1: Preparing a sample that may contain up to N targets and other non-target molecules; Step I2: Optionally, immobilizing the sample; Step I3: Optionally, permeabilizing the sample; Step I4: a) One or more HCR initiator-labeled probes (see, for example, the probes in Figures 39A-39N, 41A, 42A-42F, and 43A), or b) One or more probe units each containing two or more HCR splitting initiator probes (see, for example, the probe units in Figures 3, 4, 5, and 17); (e.g., the probes in Figures 8 and 16) Step I5: Prepare N probe sets, each containing one of the following (see Set); Step I6: Wash the sample as desired; Step I7: Prepare N HCR amplifiers corresponding to the N probe sets (each labeled with a different substrate) (see, for example, the substrate-labeled HCR amplifiers in Figure 18); Step I8: Wash the sample as desired; Step I9: Prepare N labeled probes corresponding to the N separate substrates (each bound to a different reporter) (see, for example, the labeled probes in Figure 20); Step I9: Detect N signals from the N separate reporters (see Figure 26I).

[0185] In some embodiments, Method I (e.g., Example 23) involves: Step I1: Preparing a sample that may contain up to N targets and other non-target molecules; Step I2: Immobilizing the sample; Step I3: Permeabilizing the sample; Step I4: a) One or more HCR initiator-labeled probes (see, for example, the probes in Figures 39A-39N, 41A, 42A-42F, and 43A), or b) One or more probe units (e.g., the probe units in Figures 3, 4, 5, and 17), each containing two or more HCR splitting initiator probes. Step I5: Prepare N probe sets, each containing one of the following (see Figure 26I); Step I6: Wash the sample; Step I7: Prepare N HCR amplifiers corresponding to the N probe sets (each labeled with a different substrate) (see, for example, the substrate-labeled HCR amplifiers in Figure 18); Step I8: Prepare N labeled probes corresponding to the N separate substrates (each bound to a different reporter) (see, for example, the labeled probes in Figure 20); Step I9: Detect N signals from the N separate reporters (see Figure 26I).

[0186] In some embodiments, the multiplex analysis method using repeated reporter detection is Method J (e.g., Example 24) (which may be further modified optionally with CARD, enzyme deactivation, and / or repeated CARD): 1. Prepare a sample that may contain up to N targets and other non-target molecules. 2. Fix the above sample if desired. 3. If desired, the above sample may be subjected to permeabilization treatment. 4. Prepare N probe sets, each containing either a) one or more HCR initiator-labeled probes, or b) one or more probe units, each containing two or more HCR splitting initiators. 5. Wash the above sample if desired. 6. Prepare N HCR amplifiers (each labeled with a different substrate) corresponding to the N probe sets mentioned above. 7. Wash the above sample if desired. 8. Prepare M labeled probes (if M ≤ N; each is bound to a different reporter) corresponding to M of the N separate substrates mentioned above. 9. Wash the above sample if desired. 10. Detect M signals corresponding to the M separate reporters mentioned above. 11. Remove the above M signals from the above sample. 12. If desired, repeat one or more of steps 8-11 until signal detection is performed for all N targets.

[0187] In some embodiments, Method J (e.g., Example 24) involves: Step J1: preparing a sample that may contain up to N targets and other non-target molecules; Step J2: optionally immobilizing the sample; Step J3: optionally permeabilizing the sample; Step J4: preparing N probe sets, each containing either a) one or more HCR initiator-labeled probes (e.g., probes in Figures 39A-39N, 41A, 42A-42F, and 43A), or b) one or more probe units (e.g., probe sets in Figures 8 and 16) each containing two or more HCR splitting initiator probes (e.g., probe units in Figures 3, 4, 5, and 17); Step J5: optionally washing the sample; Step J6: preparing N probe sets Step J7: Prepare N HCR amplifiers corresponding to the probe set (each labeled with a different substrate) (see, for example, the substrate-labeled HCR amplifier in Figure 18); Step J8: Wash the sample as desired; Step J9: Prepare M labeled probes corresponding to M of the N separate substrates (if M ≤ N; each bound to a different reporter) (see, for example, the labeled probes in Figure 20); Step J10: Detect M signals corresponding to the M separate reporters; Step J11: Remove the M signals from the sample (see, for example, Figure 23); Step J12: Repeat one or more of steps J8-J11 until signal detection is performed for all N targets (see Figure 26J).

[0188] In some embodiments, Method J (e.g., Example 24) involves: Step J1: Preparing a sample that may contain up to N targets and other non-target molecules; Step J2: Immobilizing the sample; Step J3: Permeabilizing the sample; Step J4: Preparing N probe sets, each containing either a) one or more HCR initiator-labeled probes (e.g., probes in Figures 39A-39N, 41A, 42A-42F, and 43A), or b) one or more probe units (e.g., probe sets in Figures 8 and 16) each containing two or more HCR splitting initiator probes (e.g., probe units in Figures 3, 4, 5, and 17); Step J5: Washing the sample; Step J6: Immobilizing the N probe sets Step J7: Prepare N HCR amplifiers corresponding to the lobe set (each labeled with a different substrate) (see, for example, the substrate-labeled HCR amplifier in Figure 18); Step J8: Wash the sample; Step J9: Prepare M labeled probes corresponding to M of the N separate substrates (if M ≤ N; each bound to a different reporter) (see, for example, the labeled probes in Figure 20); Step J10: Detect M signals corresponding to the M separate reporters; Step J11: Remove the M signals from the sample (see, for example, Figure 23); Step J12: Repeat one or more of steps J8-J11 until signal detection is performed for all N targets (see Figure 26J).

[0189] In some embodiments, the multiplex analysis method using repeated reporter detection is Method K (e.g., Example 25) (which may be further modified optionally with CARD, enzyme deactivation, and / or repeated CARD): 1. Prepare a sample that may contain one or more targets and other non-target molecules. 2. Fix the above sample if desired. 3. If desired, the above sample may be subjected to permeabilization treatment. 4. Prepare one or more probe sets, each containing either a) one or more HCR initiator-labeled probes, or b) one or more probe units, each containing two or more HCR splitting initiators. 5. Wash the above sample if desired. 6. To provide one or more HCR amplifiers (each labeled with a substrate) corresponding to one or more probe sets. 7. Wash the above sample if desired. 8. Prepare one or more labeled probes (each bound to a reporter) corresponding to one or more substrates. 9. Wash the above sample if desired. 10. Detecting one or more signals corresponding to one or more reporters. 11. Remove one or more signals from the above sample. 12. If desired, repeat any of steps 4 to 11 once or more times in any order.

[0190] In some embodiments, method K (e.g., Example 25) involves: step K1: preparing a sample that may contain one or more targets and other non-target molecules; step K2: optionally immobilizing the sample; step K3: optionally permeabilizing the sample; step K4: preparing one or more probe sets, each containing either a) one or more HCR initiator-labeled probes (e.g., probes in Figures 39A-39N, 41A, 42A-42F, and 43A), or b) one or more probe units (e.g., probe sets in Figures 8 and 16) each containing two or more HCR splitting initiator probes (e.g., probe units in Figures 3, 4, 5, and 17); step K5: optionally washing the sample. The process includes: step K6: providing one or more HCR amplifiers (each labeled with a substrate) corresponding to one or more probe sets (see, for example, the substrate-labeled HCR amplifier in Figure 18); step K7: optionally washing the sample; step K8: preparing one or more labeled probes (each bound to a reporter) corresponding to one or more substrates (see, for example, the labeled probes in Figure 20); step K9: optionally washing the sample; step K10: detecting one or more signals corresponding to one or more reporters; step K11: removing one or more signals from the sample (see, for example, Figure 23); and step K12: optionally repeating any of steps K4-K11 once or more times in any order (see Figure 26K).

[0191] In some embodiments, method K (e.g., Example 25) involves: step K1: preparing a sample that may contain one or more targets and other non-target molecules; step K2: immobilizing the sample; step K3: permeabilizing the sample; step K4: preparing one or more probe sets, each containing either a) one or more HCR initiator-labeled probes (e.g., probes in Figures 39A-39N, 41A, 42A-42F, and 43A), or b) one or more probe units (e.g., probe sets in Figures 8 and 16) each containing two or more HCR splitting initiator probes (e.g., probe units in Figures 3, 4, 5, and 17); step K5: washing the sample. The process includes: step K6: providing one or more HCR amplifiers (each labeled with a substrate) corresponding to one or more probe sets (see, for example, the substrate-labeled HCR amplifier in Figure 18); step K7: washing the sample; step K8: preparing one or more labeled probes (each bound to a reporter) corresponding to one or more substrates (see, for example, the labeled probes in Figure 20); step K9: washing the sample; step K10: detecting one or more signals corresponding to one or more reporters; step K11: removing one or more signals from the sample (see, for example, Figure 23); and step K12: repeating any of steps K4 to K11 once or more times in any order (see Figure 26K).

[0192] In some embodiments, the multiplex analysis method using repeated reporter detection is Method L (e.g., Example 26) (which may be further modified optionally with CARD, enzyme deactivation, and / or repeated CARD): 1. Prepare a sample that may contain one or more targets and other non-target molecules. 2. Fix the above sample if desired. 3. If desired, the above sample may be subjected to permeabilization treatment. 4. a) One or more HCR initiator-labeled probes, or b) Two or more Prepare one or more HCR probe sets, each containing either one or more probe units, each containing an HCR splitting initiator. 5. Prepare one or more HCR amplifiers (each labeled with one or more reporters and / or one or more substrates) corresponding to one or more probe sets. 6. Prepare, if desired, one or more labeled probes corresponding to one or more substrates (each bound to one or more reporters). 7. Detecting one or more signals 8. Wash the above sample if desired. 9. Remove one or more signals from the sample, if desired. 10. If desired, remove one or more reporters from the above sample. 11. If desired, remove one or more labeled probes from the sample. 12. If desired, remove one or more HCR amplifiers from the sample. 13. Remove one or more probe sets from the sample, if desired. 14. Repeat any of the above steps in any order, if desired.

[0193] In some embodiments, Method L (e.g., Example 26) involves: Step L1: Preparing a sample that may contain one or more targets and other non-target molecules; Step L2: Optionally, immobilizing the sample; Step L3: Optionally, permeabilizing the sample; Step L4: Preparing one or more HCR probe sets, each containing either a) one or more HCR initiator-labeled probes (e.g., probes in Figures 39A-39N, 41A, 42A-42F, and 43A), or b) one or more probe units (e.g., probe sets in Figures 8 and 16) each containing two or more HCR splitting initiator probes (e.g., probe units in Figures 3, 4, 5, and 17); Step L5: Preparing one or more HCR amplifiers corresponding to the one or more probe sets (each labeled with one or more reporters and / or one or more substrates) Step L6: Prepare an HCR amplifier (see, for example, the HCR amplifiers in Figures 8 and 18); optionally prepare one or more labeled probes corresponding to one or more substrates (each bound to one or more reporters) (see, for example, the labeled probes in Figure 20); step L7: Detect one or more signals; step L8: optionally wash the sample; step L9: optionally remove one or more signals from the sample (see, for example, Figure 23); step L10: optionally remove one or more reporters from the sample; step L11: optionally remove one or more labeled probes from the sample; step L12: optionally remove one or more HCR amplifiers from the sample; step L13: optionally remove one or more probe sets from the sample; step L14: optionally repeat any of the above steps in any order (see Figure 26L).

[0194] In some embodiments, method L (e.g., Example 26) involves: step L1: preparing a sample that may contain one or more targets and other non-target molecules; step L2: immobilizing the sample; step L3: permeabilizing the sample; step L4: preparing one or more HCR probe sets, each containing either a) one or more HCR initiator-labeled probes (e.g., probes in Figures 39A-39N, 41A, 42A-42F, and 43A), or b) one or more probe units (e.g., probe sets in Figures 8 and 16) each containing two or more HCR splitting initiator probes (e.g., probe units in Figures 3, 4, 5, and 17); step L5: preparing one or more HCR amplifiers corresponding to one or more probe sets (each containing one or more reporters and / or 1 Step L6: Prepare a sample (labeled with multiple substrates) (see, for example, the HCR amplifiers in Figures 8 and 18); Step L7: Prepare one or more labeled probes corresponding to one or more substrates (each bound to one or more reporters) (see, for example, the labeled probes in Figure 20); Step L7: Detect one or more signals; Step L8: Wash the sample; Step L9: Remove one or more signals from the sample (see, for example, Figure 23); Step L10: Remove one or more reporters from the sample; Step L11: Remove one or more labeled probes from the sample; Step L12: Remove one or more HCR amplifiers from the sample; Step L13: Remove one or more probe sets from the sample; Step L14: Repeat any of the above steps in any order (see Figure 26L).

[0195] In some embodiments, the multiplex analysis method using repeated reporter detection is Method M (e.g., Example 27) (which may be further modified optionally with CARD, enzyme deactivation, and / or repeated CARD): 1. Prepare a sample that may contain one or more targets and other non-target molecules. 2. Fix the above sample if desired. 3. If desired, the above sample may be subjected to permeabilization treatment. 4. Perform any of steps 5-9 once or multiple times in any order: 5. Prepare one or more HCR probe sets, each containing either a) one or more HCR initiator-labeled probes, or b) one or more probe units, each containing two or more HCR split initiators. 6. Prepare one or more HCR amplifiers that directly or indirectly generate one or more signals. 7. Wash the above sample if desired. 8. Detecting one or more signals 9. Remove one or more signals as desired.

[0196] In some embodiments, Method M (e.g., Example 27) involves: Step M1: Preparing a sample that may contain one or more targets and other non-target molecules; Step M2: Optionally, immobilizing the sample; Step M3: Optionally, permeabilizing the sample; Step M4: Performing any of Steps M5-M9 once or more times in any order; Step M5: a) one or more HCR initiator-labeled probes (e.g., see probes in Figures 39A-39N, 41A, 42A-42F, and 43A), or b) two or more HCR splitting initiator probes (e.g., see probes in Figures 3, 4, 5, and Step M6: Prepare one or more HCR probe sets, each containing one or more probe units (see, for example, the probe sets in Figures 8 and 16), each containing one or more probe units (see the probe units in Figure 17); Step M6: Prepare one or more HCR amplifiers (see, for example, Figures 8, 18, and 20) that directly or indirectly produce one or more signals; Step M7: Wash the sample as desired; Step M8: Detect one or more signals; Step M9: Remove one or more signals as desired (see, for example, Figure 23), including (see Figure 26M).

[0197] In some embodiments, Method M (e.g., Example 27) involves: Step M1: Preparing a sample that may contain one or more targets and other non-target molecules; Step M2: Immobilizing the sample; Step M3: Permeabilizing the sample; Step M4: Performing any of Steps M5-M9 once or more times in any order; Step M5: a) One or more HCR initiator-labeled probes (e.g., see probes in Figures 39A-39N, 41A, 42A-42F, and 43A), or b) Two or more HCR splitting initiator probes (e.g., probe units in Figures 3, 4, 5, and 17) Step M6: Prepare one or more HCR probe sets, each containing one or more probe units (see, for example, the probe sets in Figures 8 and 16), each containing one or more (see, for example, the probe sets in Figures 8 and 16); Step M6: Prepare one or more HCR amplifiers (see, for example, Figures 8, 18, and 20) that directly or indirectly produce one or more signals; Step M7: Wash the sample; Step M8: Detect one or more signals; Step M9: Remove one or more signals (see, for example, Figure 23), including (see Figure 26M).

[0198] In some embodiments, the targeted analysis method is Method N (e.g., Example 28) (which may be further modified optionally by multiplexing of CARD, enzyme deactivation, repeated CARD, repeated reporter detection, and / or repeated signal removal): 1. Prepare a sample that may contain the target and other non-target molecules. 2. Fix the above sample if desired. 3. If desired, the above sample may be subjected to permeabilization treatment. 4. Prepare a probe set comprising either a) one or more HCR initiator-labeled probes, or b) one or more probe units, each containing two or more HCR splitting initiators, wherein the target binding region on the probe within each probe unit is configured to bind to the overlap binding site on the target. 5. Wash the above sample if desired. 6. Prepare an HCR amplifier labeled with a reporter and / or substrate. 7. Wash the above sample if desired. 8. Prepare a labeled probe (bound to a reporter) corresponding to the above substrate, if desired. 9. Wash the above sample if desired. 10. Detect the signal from the above reporter.

[0199] In some embodiments, Method N (e.g., Example 28) involves: Step N1: preparing a sample that may contain a target and other non-target molecules; Step N2: optionally immobilizing the sample; Step N3: optionally permeabilizing the sample; Step N4: a) one or more HCR initiator-labeled probes (see probes in Figures 39A-39N, 41A, 42A-42F, and 43A), or b) two or more HCR splitting initiator probes, each configured such that the target binding region on the probe within each probe unit binds to the overlapping binding site on the target (see probe unit in Figure 22) The process includes: preparing a probe set containing one of several probe units (see, for example, the probe sets in Figures 8 and 16); step N5: optionally washing the sample; step N6: preparing an HCR amplifier labeled with a reporter and / or substrate (see, for example, the HCR amplifiers in Figures 8 and 18); step N7: optionally washing the sample; step N8: optionally preparing a labeled probe (bound to the reporter) corresponding to the substrate (see, for example, the labeled probe in Figure 20); step N9: optionally washing the sample; and step N10: detecting a signal from the reporter (see Figure 26N).

[0200] In some embodiments, Method N (e.g., Example 28) involves: Step N1: preparing a sample that may contain the target and other non-target molecules; Step N2: immobilizing the sample; Step N3: permeabilizing the sample; Step N4: a) one or more HCR initiator-labeled probes (see probes in Figures 39A-39N, 41A, 42A-42F, and 43A, e.g.,) or b) two or more HCR splitting initiator probes, wherein the target binding region on the probe within each probe unit is the same as above. Step N5: Prepare a probe set comprising one or more probe units (e.g., see probe sets in Figures 8 and 16) configured to bind to overlap binding sites on a target (e.g., see probe unit in Figure 22); Step N6: Wash the sample; Step N7: Prepare an HCR amplifier labeled with a reporter and / or substrate (e.g., see HCR amplifier in Figures 8 and 18); Step N8: Wash the sample; Step N9: Prepare a labeled probe (bound to the reporter) corresponding to the substrate (e.g., see labeled probe in Figure 20); Step N10: Detect a signal from the reporter (see Figure 26N).

[0201] In some embodiments, the targeted analysis method is Method O (e.g., Example 29) (which may be further modified optionally by multiplexing of CARD, enzyme deactivation, repeated CARD, repeated reporter detection, and / or repeated signal removal): 1. Prepare a sample that may contain the target and other non-target molecules. 2. Fix the above sample if desired. 3. If desired, the above sample may be subjected to permeabilization treatment. 4. Prepare a probe set comprising either a) one or more HCR initiator-labeled probes, or b) one or more probe units, each containing two or more HCR split initiators, configured such that the split initiators on the probes within each probe unit are coupled to overlap junction sites on HCR hairpins. 5. Wash the above sample if desired. 6. Prepare an HCR amplifier labeled with a reporter and / or substrate. 7. Wash the above sample if desired. 8. Prepare a labeled probe (bound to a reporter) corresponding to the above substrate, if desired. 9. Wash the above sample if desired. 10. Detect the signal from the above reporter.

[0202] In some embodiments, Method O (e.g., Example 29) comprises: Step O1: preparing a sample that may contain the target and other non-target molecules; Step O2: optionally immobilizing the sample; Step O3: optionally permeabilizing the sample; Step O4: a) one or more HCR initiator-labeled probes (see, for example, the probes in Figures 39A-39N, 41A, 42A-42F, and 43A), or b) two or more HCR splitting initiator probes, each configured such that the splitting initiator on the probe within each probe unit binds to the overlapping binding site on the HCR hairpin (see, for example, the probe unit in Figure 21). The process includes: preparing a probe set containing either multiple probe units (see, for example, the probe sets in Figures 8 and 16); step O5: optionally washing the sample; step O6: preparing an HCR amplifier labeled with a reporter and / or substrate (see, for example, the HCR amplifiers in Figures 8 and 18); step O7: optionally washing the sample; step O8: optionally preparing a labeled probe (bound to the reporter) corresponding to the substrate (see, for example, the labeled probe in Figure 20); step O9: optionally washing the sample; and step O10: detecting a signal from the reporter (see Figure 26O).

[0203] In some embodiments, method O (e.g., Example 29) is: step O1: prepare a sample which may contain the target and other non-target molecules; step O2: fix the sample; step O3: permeate the sample; step O4: a) 1 or more HC A probe set comprising either a) an R initiator-labeled probe (see, for example, the probes in Figures 39A-39N, 41A, 42A-42F, and 43A), or b) one or more probe units (see, for example, the probe sets in Figures 8 and 16), each containing two or more HCR split initiator probes, configured such that the split initiators on the probes within each probe unit are coupled to overlap junctions on the HCR hairpins (see, for example, the probe unit in Figure 21). Step O5: Wash the above sample; Step O6: Prepare an HCR amplifier labeled with a reporter and / or substrate (see, for example, the HCR amplifiers in Figures 8 and 18); Step O7: Wash the above sample; Step O8: Prepare a labeled probe corresponding to the above substrate (bound to the reporter) (see, for example, the labeled probe in Figure 20); Step O9: Wash the above sample; Step O10: Detect the signal from the reporter (see Figure 26O).

[0204] In some embodiments, the targeted analysis method is Method P (e.g., Example 30) (which may be further modified optionally by multiplexing of CARD, enzyme deactivation, repeated CARD, repeated reporter detection, and / or repeated signal removal): 1. Prepare a sample that may contain the target and other non-target molecules. 2. Fix the above sample if desired. 3. If desired, the above sample may be subjected to permeabilization treatment. 4. Prepare a probe set comprising either a) one or more HCR initiator-labeled probes, or b) one or more probe units, each containing two or more HCR split initiators, wherein the target binding region on the probe within each probe unit is configured to bind to the overlap binding site on the target, and the split initiator on the probe within each probe unit is configured to bind to the overlap binding site on the HCR hairpin. 5. Wash the above sample if desired. 6. Prepare an HCR amplifier labeled with a reporter and / or substrate. 7. Wash the above sample if desired. 8. Prepare a labeled probe (bound to a reporter) corresponding to the above substrate, if desired. 9. Wash the above sample if desired. 10. Detect the signal from the above reporter.

[0205] In some embodiments, method P (e.g., Example 30) comprises: step P1: preparing a sample which may contain a target and other non-target molecules; step P2: optionally immobilizing the sample; step P3: optionally permeabilizing the sample; step P4: a) one or more HCR initiator-labeled probes (see probes in Figures 39A-39N, 41A, 42A-42F, and 43A), or b) two or more HCR split initiator probes, each comprising a target binding region on the probe within each probe unit configured to bind to overlap binding sites on the target, and the split initiators on the probe within each probe unit configured to bind to overlap binding sites on the HCR hairpins. Step P5: Prepare a probe set comprising one or more probe units (see, for example, probe sets in Figures 8 and 16) configured as follows (see, for example, probe unit in Figure 27); Step P6: Wash the sample as desired; Step P7: Prepare an HCR amplifier labeled with a reporter and / or substrate (see, for example, HCR amplifier in Figures 8 and 18); Step P7: Wash the sample as desired; Step P8: Prepare a labeled probe (bound to the reporter) corresponding to the substrate (see, for example, labeled probe in Figure 20); Step P9: Wash the sample as desired; Step P10: Detect a signal from the reporter (see Figure 26P).

[0206] In some embodiments, Method P (e.g., Example 30) involves: Step P1: preparing a sample that may contain a target and other non-target molecules; Step P2: immobilizing the sample; Step P3: permeabilizing the sample; Step P4: a) comprising one or more HCR initiator-labeled probes (see, for example, the probes in Figures 39A-39N, 41A, 42A-42F, and 43A), or b) comprising two or more HCR split initiator probes, each configured such that the target binding region on the probe within each probe unit binds to the overlap binding site on the target, and the split initiator on the probe within each probe unit binds to the overlap binding site on the HCR hairpin. Step P5: Prepare a probe set including one or more probe units (see, for example, probe sets in Figures 8 and 16) configured to perform the following actions (see, for example, probe unit in Figure 27); Step P6: Wash the sample; Step P7: Prepare an HCR amplifier labeled with a reporter and / or substrate (see, for example, HCR amplifier in Figures 8 and 18); Step P8: Wash the sample; Step P9: Prepare a labeled probe (bound to the reporter) corresponding to the substrate (see, for example, labeled probe in Figure 20); Step P10: Detect a signal from the reporter (see Figure 26P).

[0207] In some embodiments, the targeted analysis method is Method Q (e.g., Example 31) (which may be further modified optionally by multiplexing of CARD, enzyme deactivation, repeated CARD, repeated reporter detection, and / or repeated signal removal): 1. Prepare a sample that may contain the target and other non-target molecules. 2. Fix the above sample if desired. 3. If desired, the above sample may be subjected to permeabilization treatment. 4. Prepare a probe set comprising either a) one or more HCR initiator-labeled probes, or b) one or more probe units, each containing two or more HCR split initiators, wherein the target binding region on the probe within each probe unit is configured to bind to a non-overlapping binding site on the target, and the split initiators on the probe within each probe unit are configured to bind to an overlapping binding site on an HCR hairpin. 5. Wash the above sample if desired. 6. Prepare an HCR amplifier labeled with a reporter and / or substrate. 7. Wash the above sample if desired. 8. Prepare a labeled probe (bound to a reporter) corresponding to the above substrate, if desired. 9. Wash the above sample if desired. 10. Detect the signal from the above reporter.

[0208] In some embodiments, Method Q (e.g., Example 31) comprises: Step Q1: preparing a sample which may contain a target and other non-target molecules; Step Q2: optionally immobilizing the sample; Step Q3: optionally permeabilizing the sample; Step Q4: a) one or more HCR initiator-labeled probes (see probes in Figures 39A-39N, 41A, 42A-42F, and 43A), or b) two or more HCR split initiators, each comprising a target binding region on the probe within each probe unit configured to bind to a non-overlapping binding site on the target, and the split initiators on the probe within each probe unit binding to overlapping binding on the HCR hairpin. Step Q5: Prepare a probe set comprising one or more probe units configured to bind to a site (see, for example, the probe unit in Figure 21); Step Q6: Wash the sample as desired; Step Q7: Prepare an HCR amplifier labeled with a reporter and / or substrate (see, for example, the HCR amplifiers in Figures 8 and 18); Step Q8: Wash the sample as desired; Step Q9: Prepare a labeled probe (bound to the reporter) corresponding to the substrate (see, for example, the labeled probe in Figure 20); Step Q10: Detect a signal from the reporter (see Figure 26Q).

[0209] In some embodiments, Method Q (e.g., Example 31) involves: Step Q1: preparing a sample that may contain a target and other non-target molecules; Step Q2: immobilizing the sample; Step Q3: permeabilizing the sample; Step Q4: a) one or more HCR initiator-labeled probes (see probes in Figures 39A-39N, 41A, 42A-42F, and 43A, e.g., probes in Figures 39A-39N, 41A, 42A-42F, and 43A), or b) two or more HCR split initiators, each comprising a target binding region on the probe within each probe unit configured to bind to a non-overlapping binding site on the target, and the split initiators on the probe within each probe unit binding to an overlapping HCR hairpin. Step Q5: Prepare a probe set including one or more probe units configured to bind to wrap binding sites (see, for example, the probe unit in Figure 21); Step Q6: Wash the sample; Step Q7: Prepare an HCR amplifier labeled with a reporter and / or substrate (see, for example, the HCR amplifiers in Figures 8 and 18); Step Q8: Wash the sample; Step Q9: Prepare a labeled probe corresponding to the substrate (bound to the reporter) (see, for example, the labeled probe in Figure 20); Step Q10: Detect a signal from the reporter (see Figure 26Q).

[0210] In some embodiments, the targeted analysis method is Method R (e.g., Example 32) (which may be further modified optionally by multiplexing CARD, enzyme deactivation, repeated CARD, repeated reporter detection, and / or repeated signal removal): 1. Prepare a sample that may contain the target and other non-target molecules. 2. Fix the above sample if desired. 3. If desired, the above sample may be subjected to permeabilization treatment. 4. Prepare a probe set comprising either a) one or more HCR initiator-labeled probes, or b) one or more probe units, each containing two or more HCR split initiators, wherein the target binding region on the probe within each probe unit is configured to bind to the overlap binding site on the target, and the split initiators on the probe within each probe unit are configured to bind to the non-overlap binding site on the HCR hairpin. 5. Wash the above sample if desired. 6. Prepare an HCR amplifier labeled with a reporter and / or substrate. 7. Wash the above sample if desired. 8. Prepare a labeled probe (bound to a reporter) corresponding to the above substrate, if desired. 9. Wash the above sample if desired. 10. Detect the signal from the above reporter.

[0211] In some embodiments, method R (e.g., Example 32) involves: step R1: preparing a sample which may contain the target and other non-target molecules; step R2: optionally immobilizing the sample; step R3: optionally permeabilizing the sample; step R4 a) One or more HCR initiator-labeled probes (see, for example, the probes in Figures 39A-39N, 41A, 42A-42F, and 43A), or b) One or more probe units, each containing two or more HCR split initiators, wherein the target binding region on the probe within each probe unit is configured to bind to the overlapping binding site on the target, and the split initiator on the probe within each probe unit is configured to bind to the non-overlapping binding site on the HCR hairpin (see, for example, the probe unit in Figure 22). Step R5: Prepare a probe set including the reporter and / or substrate; optionally wash the sample; Step R6: Prepare an HCR amplifier labeled with the reporter and / or substrate (see, for example, the HCR amplifiers in Figures 8 and 18); Step R7: optionally wash the sample; Step R8: optionally prepare a labeled probe (bound to the reporter) corresponding to the substrate (see, for example, the labeled probe in Figure 20); Step R9: optionally wash the sample; Step R10: Detect the signal from the reporter (see Figure 26R).

[0212] In some embodiments, method R (e.g., Example 32) involves: step R1: preparing a sample that may contain a target and other non-target molecules; step R2: immobilizing the sample; step R3: permeabilizing the sample; step R4: a) one or more HCR initiator-labeled probes (e.g., see probes in Figures 39A-39N, 41A, 42A-42F, and 43A), or b) two or more HCR splitting initiators, each comprising a target binding region on the probe within each probe unit configured to bind to overlapping binding sites on the target, and the splitting initiators on the probe within each probe unit binding to non-overlapping HCR hairpins. Step R5: Prepare a probe set including one or more probe units configured to bind to wrap binding sites (see, for example, the probe unit in Figure 22); Step R6: Wash the sample; Step R7: Prepare an HCR amplifier labeled with a reporter and / or substrate (see, for example, the HCR amplifiers in Figures 8 and 18); Step R8: Wash the sample; Step R9: Prepare a labeled probe corresponding to the substrate (bound to the reporter) (see, for example, the labeled probe in Figure 20); Step R10: Detect a signal from the reporter (see Figure 26R).

[0213] In some embodiments, the targeted analysis method is Method S (e.g., Example 33) (which may be further modified optionally by multiplexing of CARD, enzyme deactivation, repeated CARD, repeated reporter detection, and / or repeated signal removal): 1. Prepare a sample that may contain the target and other non-target molecules. 2. Fix the above sample if desired. 3. If desired, the above sample may be subjected to permeabilization treatment. 4. Prepare a probe set comprising either a) one or more HCR initiator-labeled probes, or b) one or more probe units, each containing two or more HCR split initiators, wherein the target binding region on the probe within each probe unit is configured to bind to a non-overlapping binding site on the target, and the split initiator on the probe within each probe unit is configured to bind to a non-overlapping binding site on an HCR hairpin. 5. Wash the above sample if desired. 6. Prepare an HCR amplifier labeled with a reporter and / or substrate. 7. Wash the above sample if desired. 8. Prepare a labeled probe (bound to a reporter) corresponding to the above substrate, if desired. 9. Wash the above sample if desired. 10. Detect the signal from the above reporter.

[0214] In some embodiments, Method S (e.g., Example 33) comprises: Step S1: preparing a sample which may contain a target and other non-target molecules; Step 2: optionally immobilizing the sample; Step S3: optionally permeabilizing the sample; Step S4: a) one or more HCR initiator-labeled probes (see, for example, the probes in Figures 39A-39N, 41A, 42A-42F, and 43A), or b) two or more HCR split initiator probes, each comprising a target binding region on the probe within each probe unit configured to bind to a non-overlapping binding site on the target, and a split initiator on the probe within each probe unit configured to bind to a non-overlapping binding site on an HCR hairpin. Step S5: Prepare a probe set including either a probe unit (see, for example, the probe units in Figures 3, 4, 5, and 17), one or more probe units (see, for example, the probe sets in Figures 8 and 16); Step S6: Wash the sample as desired; Step S7: Prepare an HCR amplifier labeled with a reporter and / or substrate (see, for example, the HCR amplifiers in Figures 8 and 18); Step S8: Wash the sample as desired; Step S9: Prepare a labeled probe (bound to the reporter) corresponding to the substrate (see, for example, the labeled probe in Figure 20); Step S9: Wash the sample as desired; Step S10: Detect a signal from the reporter (see Figure 26S).

[0215] In some embodiments, Method S (e.g., Example 33) involves: Step S1: preparing a sample that may contain a target and other non-target molecules; Step 2: immobilizing the sample; Step S3: permeabilizing the sample; Step S4: a) one or more HCR initiator-labeled probes (see, for example, the probes in Figures 39A-39N, 41A, 42A-42F, and 43A), or b) two or more HCR split initiator probes, each comprising a target binding region on the probe within each probe unit configured to bind to a non-overlapping binding site on the target, and the split initiators on the probe within each probe unit configured to bind to a non-overlapping binding site on the HCR hairpin. Step S5: Prepare a probe set comprising one or more probe units (see, for example, probe units in Figures 3, 4, 5, and 17) (see, for example, probe sets in Figures 8 and 16); Step S6: Wash the sample; Step S7: Prepare an HCR amplifier labeled with a reporter and / or substrate (see, for example, HCR amplifiers in Figures 8 and 18); Step S8: Wash the sample; Step S9: Prepare a labeled probe (bound to the reporter) corresponding to the substrate (see, for example, labeled probe in Figure 20); Step S9: Wash the sample; Step S10: Detect a signal from the reporter (see Figure 26S).

[0216] In some embodiments, the multiplex analysis method using repeated reporter detection is Method T (e.g., Example 34) (which may be further modified optionally with CARD, enzyme deactivation, and / or repeated CARD): 1. Prepare a sample that may contain one or more targets and other non-target molecules. 2. Fix the above sample if desired. 3. If desired, the above sample may be subjected to permeabilization treatment. 4. Each probe unit comprises either a) one or more HCR initiator-labeled probes, or b) two or more HCR-splitting initiators, wherein the target binding region on the probe within each probe unit binds to either overlapping or non-overlapping binding sites on the target. Prepare one or more probe sets, each containing one or more probe units, wherein the split initiator on the probe within each probe unit is configured to connect to either overlapping or non-overlapping connection points on the HCR hairpin. 5. Wash the above sample if desired. 6. Prepare one or more HCR amplifiers, each labeled with one or more reporters and / or substrates. 7. Wash the above sample if desired. 8. Prepare, if desired, one or more labeled probes corresponding to one or more substrates (each bound to one or more reporters). 9. Wash the above sample if desired. 10. Detecting signals from one or more reporters. 11. Remove one or more signals from the sample, if desired. 12. If desired, remove one or more reporters from the above sample. 13. If desired, remove one or more labeled probes from the sample. 14. If desired, remove one or more amplifiers from the sample. 15. Remove one or more probe sets from the sample, if desired. 16. Repeat any of the above steps in any order, if desired.

[0217] In some embodiments, method T (e.g., Example 34) involves: step T1: preparing a sample which may contain one or more targets and other non-target molecules; step T2: optionally immobilizing the sample; step T3: optionally permeabilizing the sample; step T4: a) one or more HCR initiator-labeled probes (see probes in Figures 39A-39N, 41A, 42A-42F, and 43A, e.g., probes in Figures 39A-39N, 41A, 42A-42F, and 43A), or b) two or more HCR splitting initiators, each containing a target binding region on the probe within each probe unit. Step T5: Prepare one or more probe sets, each containing one or more probe units, wherein the region is configured to bind to overlapping or non-overlapping binding sites on the target, and the split initiator on the probe within each probe unit is configured to bind to overlapping or non-overlapping binding sites on the HCR hairpin (see, for example, the probe units in Figures 3, 4, 5, 17, 21, 22, and 27); Step T6: Wash the sample as desired; Step T7: Wash one or more probe sets Step T7: Prepare one or more HCR amplifiers labeled with several reporters and / or substrates, respectively (see, for example, the HCR amplifiers in Figures 8 and 18); Step T8: Optionally, wash the above sample; Step T9: Optionally, prepare one or more labeled probes corresponding to one or more substrates (each bound to one or more reporters) (see, for example, the labeled probes in Figure 20); Step T9: Optionally, wash the above sample; Step T10: Detect signals from one or more reporters; Step T Step 11: optionally remove one or more signals from the sample (see, for example, Figure 23); step T12: optionally remove one or more reporters from the sample; step T13: optionally remove one or more labeled probes from the sample; step T14: optionally remove one or more amplifiers from the sample; step T15: optionally remove one or more probe sets from the sample; step T16: optionally repeat any of the above steps in any order (see Figure 26T).

[0218] In some embodiments, method T (e.g., Example 34) involves: step T1: preparing a sample which may contain one or more targets and other non-target molecules; step T2: immobilizing the sample; step T3: permeabilizing the sample; step T4: a) one or more HCR initiator-labeled probes (e.g., Figures 39A-39N, Figure 41A, Step T5: Prepare one or more probe sets, each containing one or more probe units, each containing one or more probe units: b) each containing two or more HCR splitting initiators, wherein the target binding region on the probe within each probe unit is configured to bind to an overlapping or non-overlapping binding site on the target, and the splitting initiator on the probe within each probe unit is configured to bind to an overlapping or non-overlapping binding site on the HCR hairpin (see, for example, the probe units in Figures 3, 4, 5, 17, 21, 22, and 27); Step T5: Wash the above sample; Step T6: Attach one or more HCR amplifiers labeled with one or more reporters and / or substrates (see, for example, the HCR amplifiers in Figures 8 and 18) Step T7: Prepare the Fire (see Fire); Step T8: Wash the sample; Step T9: Prepare one or more labeled probes corresponding to one or more substrates (each bound to one or more reporters) (see, for example, the labeled probes in Figure 20); Step T10: Detect signals from one or more reporters; Step T11: Remove one or more signals from the sample (see, for example, Figure 23); Step T12: Remove one or more reporters from the sample; Step T13: Remove one or more labeled probes from the sample; Step T14: Remove one or more amplifiers from the sample; Step T15: Remove one or more probe sets from the sample; Step T16: Repeat any of the above steps in any order, if desired (see Figure 26T).

[0219] Multiplexing using single-molecule barcoding In some embodiments, the number of analyzable targets in a sample can be increased by analyzing each target molecule in multiple analysis rounds, such that the labeling used for different target species changes in different analysis rounds, so that different barcodes are formed for each target molecule species. The barcode for a given target molecule is then read as a barcode for signal measurement. For example, consider three rounds of imaging using single-molecule imaging to read the signal for each target molecule as a diffraction-limited dot. For each given target species, consider assigning a probe set containing one or more probe units such that each probe unit in the probe set colocalizes a complete HCR initiator corresponding to an HCR amplifier containing an HCR hairpin labeled with either a red reporter or a green reporter, depending on the target species and the imaging round. Next, for example, a type 1 target molecule can be read by a barcode (red, red, green; the red dot represents the first round using an HCR amplifier labeled with a red reporter, the red dot represents the second round using an HCR amplifier labeled with a red reporter, and the green dot represents the third round using an HCR amplifier labeled with a green reporter), a type 2 target molecule can have a barcode (red, green, red), a type 3 target molecule can have a barcode (red, red, red), a type 4 target molecule can have a barcode (green, red, green), and so on.

[0220] In some embodiments, the number of analyzable targets in a sample can be increased by detecting each target molecule in only a portion of the barcoding rounds. For example, in a 4-round experiment, type 1 target molecules can be read by the barcode (red, ---, ---, red; representing a red dot for round 1, no dot for round 2, no dot for round 3, and a red dot for round 4), and type 2 target molecules can be read by the barcode (green, red, ---, ---), and so on.

[0221] Additional Embodiments Any embodiment and / or method provided herein may be used in conjunction with any of the following: It can be used, or in an alternative form thereof. That is, for example, the above method can use any of the following compositions or methods. Similarly, the above method should be understood to also provide a method employing the following methods, or to be part of the following methods.

[0222] Similarly, embodiments and / or methods provided herein should be understood to also provide embodiments relating to such methods, such as compositions, components of such methods, kits, etc. In some embodiments, any of the components of one or more methods and / or processes provided herein may be provided as a kit comprising one or more of the above components (and optionally, a target or target sequence or sample).

[0223] composition Several embodiments of the composition are outlined in Figures 12 and 13, and other figures provided herein. In some embodiments, a composition is provided comprising a first split initiator probe (1190) containing a first split initiator (1151), and a second split initiator probe (1290) containing a second split initiator (1251). In some embodiments, the first and second split initiators (1151, 1251) combine to form a complete initiator (1050), from which HCR can proceed via first and second hairpin monomers (1510, 1610). In some embodiments, the first split initiator probe (1190) further comprises a first target binding section (1141), and the second split initiator probe (1290) further comprises a second target binding section (1241), wherein the first target binding section (1141) is configured to bind to the first target section (1100), and the second target binding section (1241) is configured to bind to the second target section (1200). When both split initiator probes are bound to both targets, the first and second split initiators (within the split initiator probes) are close enough to form a complete initiator (1050), and these target binding sections are effectively adjacent on the target molecule so that HCR can be initiated from that complete initiator. In some embodiments, these split initiator probes can be supplied as a kit together with hairpin monomers for HCR polymerization.

[0224] In some embodiments, a composition is provided comprising a first hairpin monomer (1510), a second hairpin monomer (1610), a first split initiator probe (1190) including a first split initiator (1151), and a second split initiator probe (1290) including a second split initiator (1251). In some embodiments, the first split initiator and the second split initiator (1151, 1251) combine to form a complete initiator (1050), from which HCR can proceed via the first hairpin monomer and the second hairpin monomer (1510, 1610).

[0225] In some embodiments, the segmented initiator probe and the hairpin monomer can be introduced into the same sample simultaneously. In some embodiments, the segmented initiator probe can be introduced into the sample, washed, and then the hairpin monomer can be introduced into the sample and washed. In some embodiments, the segmented initiator probe and the hairpin monomer can be introduced into the sample at different times.

[0226] In some embodiments, one or more of the hairpin monomers (first and second, and optionally more hairpin monomers) may contain a reporter molecule so that polymerization of the hairpin monomers results in a detectable signal transduction phenomenon. In some embodiments, the reporter molecule is covalently bonded to the hairpin monomer. This is possible. In some embodiments, the reporter molecule can be subsequently attached to the HCR polymer after polymerization (e.g., in a post-hybridization event). In some embodiments, the first hairpin monomer (1510) includes a labeling site (not shown) configured to hybridize to a complementary chain to the labeling site (not shown). In some embodiments, the complementary chain to the labeling site further includes the reporter molecule.

[0227] In some embodiments, a composition is provided comprising a first hairpin monomer (1510) comprising a first input domain (1852) including a first toehold (1851) and a first stem portion (1755), a first output domain (1854) including a first hairpin loop (1853) and a complementary chain (1756) to the first stem portion, and a first reporter molecule (1850). The composition may further comprise a second hairpin monomer (1610) comprising a second input domain (1952) including a second toehold (1951) and a second stem portion (1855), a second output domain (1954) including a second hairpin loop (1953) and a complementary chain to the second stem portion (1856), and a second reporter molecule (1950). The above composition may further include a) a first split initiator probe (1190) containing a first split initiator (1151), and b) a second split initiator probe (1290) containing a second split initiator (1251). As mentioned above, the first hairpin monomer and the second hairpin monomer can be introduced into the sample together with the first split initiator probe and the second split initiator probe, or separately from the first split initiator probe and the second split initiator probe. In some embodiments, each monomer can be supplied together but separated from the first split initiator probe and the second split initiator probe. In some embodiments, the hairpin monomer and the split initiator probe can be introduced into the same sample simultaneously. In some embodiments, the hairpin monomer and the split initiator probe can be introduced into the same sample at different, non-overlapping time intervals (including washing to remove unbound molecules).

[0228] In some embodiments, the first stem portion has the same arrangement as the second stem portion. In some embodiments, the complementary chain to the first stem portion has the same arrangement as the complementary chain to the second stem portion. In some embodiments, the complementary chain to the first stem portion has the same arrangement as the complementary chain to the second stem portion, and the first stem portion has the same arrangement as the second stem portion. In some embodiments, the toehold arrangements of the two hairpin monomers are the same, and the loop arrangements of the two hairpin monomers are the same (however, the two hairpin monomers are not identical because their orientations are opposite).

[0229] In some embodiments, the first toehold (1851) is complementary to the second hairpin loop. In some embodiments, the second toehold is complementary to the first hairpin loop. In some embodiments, this cyclicality enables the generation of a hybridization chain reaction. In some embodiments, the first toehold is not 100% complementary to the second hairpin loop, but is sufficient to enable hybridization.

[0230] In some embodiments, three or more different input domains can be used, for example, 3, 4, 5, 6, 7, 8, 9, 10, 100, 1000, 2000, 4000, or more input domains can be used. In some embodiments, a number of subparts corresponding to each input domain can be used.

[0231] In some embodiments, any of the compositions described herein includes a target molecule (1020) comprising a first target section (1100) and a second target section (1200) (e.g., illustrated in Figure 12). In some embodiments, the target molecule includes additional target sections, e.g., 3, 4, 5, 6, 7, or more (e.g., 10, 50, 100, etc.) target sections. In some embodiments, the number of target sections means that there is a corresponding number of target-binding sections (e.g., 1141, 1241). In some embodiments, this allows for greater specificity / selectivity of the initial complete initiator formation (because more target-binding sections are required to bind to the above target sections). In some embodiments, this allows for assaying two or more target sequences in parallel at once (allowing for a single assay of multiple targets, each containing two or more target-binding sections / divided initiator probes).

[0232] In some embodiments, any of the first split initiator probes (1190) described herein further include a first target binding section (1141), and any of the second split initiator probes (1290) described herein further include a second target binding section (1241). In some embodiments, the first target binding section (1141) is configured to bind to a first target section (1100). In some embodiments, the second target binding section (1241) is configured to bind to a second target section (1200). In some embodiments, the first and second split initiator probes include additional target binding sections, for example, two, three, four, five, ten, twenty, thirty, forty, fifty, one hundred, one thousand, one hundred, or more target binding sections.

[0233] In some embodiments, the first target-binding section is configured to bind to the first target section via selective protein-protein interactions (e.g., via an antibody as the first target-binding section). In some embodiments, the first target-binding section is configured to bind to the first target section via selective nucleic acid-protein interactions (e.g., via an aptamer as the first target-binding section). In some embodiments, the second target-binding section is configured to bind to the second target section via selective protein-protein interactions (e.g., via an antibody as the second target-binding section). In some embodiments, the second target-binding section is configured to bind to the second target section via selective nucleic acid-protein interactions (e.g., via an aptamer as the second target-binding section). In some embodiments, the first target-binding section is configured to bind to the first target section via hybridization. In some embodiments, the second target-binding section is configured to bind to the second target section via hybridization. In some embodiments, the first target-binding section is configured to bind to the first target section via covalent or ionic bonding. In some embodiments, the second target-binding section is configured to bind to the second target section via covalent or ionic bonding.

[0234] In some embodiments, any reporter molecule capable of monitoring the presence or absence of a substance can be used. In some embodiments, the reporter molecule includes a fluorescent molecule, such as a fluorophore, i.e., a colorimetric compound, which enables visualization of the resulting polymer. In some embodiments, the reporter molecule is directly observable. In some embodiments, the reporter molecule is indirectly observable. In some embodiments, the reporter molecule contains or is enzymatic and / or can mediate enzymatic signaling after HCR polymerization. In some embodiments, the reporter molecule The terminating action is achieved by catalytic reporter deposition ("CARD"). In some embodiments, a labeling site on each hairpin monomer can be used to bind a complementary chain to the labeling site, and the complementary chain to the labeling site contains a reporter molecule. In some embodiments, one reporter molecule supported on the hairpin monomer or the complementary chain to the labeling site can mediate enzymatic signal amplification (CARD) after HCR polymerization so that a second reporter molecule deposited near the HCR polymer / target molecule is subsequently detected. In some embodiments, the reporter molecule is at least one of a luminescent molecule, a FRET molecule, a fluorophore / quencher molecule pair, or other detectable marker. In some embodiments, the reporter molecule can make a second molecule (such as a secondary antibody) available for detection of the polymerization event. In some embodiments, the hairpin monomer can be labeled with a reporter molecule (e.g., a fluorophore and a quencher) so that the hairpin monomer is quenched, but the higher-order structural changes that occur during HCR polymerization result in a fluorescent HCR-amplified polymer.

[0235] In some embodiments, any component in one or more methods and / or processes provided herein can be provided as a composition comprising one or more of the above components (and optionally, a target or target sequence or sample). In some embodiments, Figures 1A, 1B, 2A, 2B, 3A, 3B, 4A, 4B, 4C, 4D, 5A, 5B, 5C, 5D, 5E, 8A, 8B, 12, 13, 14, 15, 16A, 16B, 16C, 16D, 17A, 17B, 17C, 18A, 18B, 18C, 18D, 18E, 18F, 19A, 19 B, Figure 20A, Figure 20B, Figure 20C, Figure 20D, Figure 20E, Figure 20F, Figure 21A, Figure 21B, Figure 22, Figure 23A, Figure 23B, Figure 23C, Figure 23D, Figure 23E, Figure 23F, Figure 23G, Figure 23H, Figure 23I, Figure 23J, Figure 23K, Figure 23L, Figure 23M, Figure 23N, Figure 23O, Figure 27, Figure 28A, Figure 29A, Figure 29B, Figure 30A, Figure 30B, Figure 31A, Figure 32A, Figure 33A, Figure 33B, Figures 33C, 33D, 33E, 34A, 34B, 34C, 35-1, 35-2, 36A, 36B, 37A, 38-1, 38-2, 39A, 39B, 39C, 39D, 39E, 39F, 39G, 39H, 39I, 39J, 39K, 39L, 39M, 39N, 40A, 40B, 40C, 40D, 40E, 40F, Figure Figures 40G, 40H, 40I, 40J, 40K, 40L, 40M, 40N, 41A, 42A, 42B, 42C, 42D, 42E, 42F, 43A, 44A, 44B, 44C, 44D, 44E, 44F, 44G, 44H, 44I, 44J, 44K, 44L, 44M, 44N, 44O, 44P, 44Q, 44R, In any one or more of Figures 44S, 44T, 44U, 44V, 44W, 44X, 44Y, 44Z, 46A, 46B, 46C, 46D, 46E, 46F, 46G, 46H, 46I, 46J, 46K, 46L, and / or 46M, one or more molecules or combinations of molecules are intended to be a composition of their indicated structural components.

[0236] In some embodiments, the composition or component is added to the sample. In some embodiments, the composition or component is added in each step of the method shown in the figure. In some embodiments, the composition or component is added first in the reaction and / or in any reaction step shown in the figure. In some embodiments, the composition is as shown in Figures 1A, 1B, 2A, 2B, 3A, 3B, 4A, 4B, 4C, 4D, 5A, 5B, 5C, 5D, 5E, 8A, 8B, 12, 13, 14, 15, 16A, 16B, 16C, 16D, 17A, 17B, 17C, 18A, 18B, 18C, 18D, 18E, 18F, 19A, 19B, 20A, 20B, 20C, 20D, 20E, 20F, 21A, 21B, 22, 23A, 23B, 23C, 23D, 23E, 23F Figures 23G, 23H, 23I, 23J, 23K, 23L, 23M, 23N, 23O, 27, 28A, 29A, 29B, 30A, 30B, 31A, 32A, 33A, 33B, 33C, 33D, 33E, 34A, 34B, 34C, 35-1, 35-2, 36A, 36B, 37A, 38-1, 38-2, 39A, 39B, 39C, 39D, 39E, 39F, 39G, 39H, 39I, 39J, 39 K, Figure 39L, Figure 39M, Figure 39N, Figure 40A, Figure 40B, Figure 40C, Figure 40D, Figure 40E, Figure 40F, Figure 40G, Figure 40H, Figure 40I, Figure 40J, Figure 40K, Figure 40L, Figure 40M, Figure 40N, Figure 41A, Figure 42A, Figure 42B, Figure 42C, Figure 42D, Figure 42E, Figure 42F, Figure 43A, Figure 44A, Figure 44B, Figure 44C, Figure 44D, Figure 44E, Figure 44F, Figure 44G, Figure 44H, Figure 44I, Figure 44J, Figure 44K, Figure 44L, Figure 44M, Figure 44N, Figure 44O, Figure 44P, Figure 44Q, Figure 44R, Figure This includes the hybridization of a molecule to a target, as shown in any one of the following: 44S, 44T, 44U, 44V, 44W, 44X, 44Y, 44Z, 46A, 46B, 46C, 46D, 46E, 46F, 46G, 46H, 46I, 46J, 46K, 46L, and / or 46M.

[0237] In some embodiments, the composition or components are as shown in Figures 1A, 1B, 2A, 2B, 3A, 3B, 4A, 4B, 4C, 4D, 5A, 5B, 5C, 5D, 5E, 8A, 8B, 12, 13, 14, 15, 16A, 16B, 16C, 16D, 17A, 17B, 17C, 18A, 18B, 18C, 18D, 18E, 18F, and Figure 19A, Figure 19B, Figure 20A, Figure 20B, Figure 20C, Figure 20D, Figure 20E, Figure 20F, Figure 21A, Figure 21B, Figure 22, Figure 23A, Figure 23B, Figure 23C, Figure 23D, Figure 23E, Figure 23F, Figure 23G, Figure 23H, Figure 23I, Figure 23J, Figure 23K, Figure 23L, Figure 23M, Figure 23N, Figure 23O, Figure 27, Figure 28A, Figure 29A, Figure 29B, Figure 30A, Figure 30B, Figure 31A, Figure 32A, Figure 33A, Figures 33B, 33C, 33D, 33E, 34A, 34B, 34C, 35-1, 35-2, 36A, 36B, 37A, 38-1, 38-2, 39A, 39B, 39C, 39D, 39E, 39F, 39G, 39H, 39I, 39J, 39K, 39L, 39M, 39N, 40A, 40B, 40C, 40D, 40E, 40 F, Figure 40G, Figure 40H, Figure 40I, Figure 40J, Figure 40K, Figure 40L, Figure 40M, Figure 40N, Figure 41A, Figure 42A, Figure 42B, Figure 42C, Figure 42D, Figure 42E, Figure 42F, Figure 43A, Figure 44A, Figure 44B, Figure 44C, Figure 44D, Figure 44E, Figure 44F, Figure 44G, Figure 44H, Figure 44I, Figure 44J, Figure 44K, Figure 44L, Figure 44M, Figure 44N, Figure 44O, Figure 44P, Figure 44Q, Figure 44R, Figure This is during the final step of a method or protocol, as shown in any one of the following: 44S, Figure 44T, Figure 44U, Figure 44V, Figure 44W, Figure 44X, Figure 44Y, Figure 44Z, Figure 46A, Figure 46B, Figure 46C, Figure 46D, Figure 46E, Figure 46F, Figure 46G, Figure 46H, Figure 46I, Figure 46J, Figure 46K, Figure 46L, and / or Figure 46M.

[0238] In some embodiments, the composition or component is appropriate (having a label attached to or bound to the target) Figures 1A, 1B, 2A, 2B, 3A, 3B, 4A, 4B, 4C, 4D, 5A, 5B, 5C, 5D, 5E, 8A, 8B, 12, 13, 14, 15, 16A, 16B, 16C, 16D, 17A, 17B, 17C, 18A, 18B, 18C, 18D, 18E, 18F, 19A, 19B, 20A, 20B, 20C, 20D, 20E, 20F, 21 A, Figure 21B, Figure 22, Figure 23A, Figure 23B, Figure 23C, Figure 23D, Figure 23E, Figure 23F, Figure 23G, Figure 23H, Figure 23I, Figure 23J, Figure 23K, Figure 23L, Figure 23M, Figure 23N, Figure 23O, Figure 27, Figure 28A, Figure 29A, Figure 29B, Figure 30A, Figure 30B, Figure 31A, Figure 32A, Figure 33A, Figure 33B, Figure 33C, Figure 33D, Figure 33E, Figure 34A, Figure 34B, Figure 34C, Figure 35-1, Figure 35-2, Figure 36A, Figure 36B, Figure 37A, Figure 38-1, Figure 38-2, Figure 39A, Figure 39B, Figure 39C, Figure 39D, Figure 39E, Figure 39F, Figure 39G, Figure 39H, Figure 39I, Figure 39J, Figure 39K, Figure 39L, Figure 39M, Figure 39N, Figure 40A, Figure 40B, Figure 40C, Figure 40D, Figure 40E, Figure 40F, Figure 40G, Figure 40H, Figure 40I, Figure 40J, Figure 40K, Figure 40L, Figure 40M, Figure 40N, Figure 41A, Figure 42A, Figure 42B, Figure 42C, Figure 42D, Figure 42E, Figure 42F, Figure 43A, Figure 44A, Figure 44B, Figure 44C, Figure 44D, Figure 44E, Figure 44F, Figure 44G, Figure 44H, Figure 44I, Figure 44 J, having a label attached to or bound to the target, as shown in any one of Figures 44K, 44L, 44M, 44N, 44O, 44P, 44Q, 44R, 44S, 44T, 44U, 44V, 44W, 44X, 44Y, 44Z, 46A, 46B, 46C, 46D, 46E, 46F, 46G, 46H, 46I, 46J, 46K, 46L, and / or Figure 46M.

[0239] method In some embodiments, a method is provided. The above method comprises (i) preparing a first split initiator probe, (1190), a second split initiator probe (1290), a first hairpin monomer (1510), a second hairpin monomer (1610), and a target molecule (1020), (ii) incubating to enable binding, and (iii) detecting a signal.

[0240] In some embodiments, a method for performing HCR is provided. The method comprises (i) adding a first split initiator (1151) and a second split initiator (1251) to a sample to combine the first split initiator (1151) and the second split initiator (1251) to form a complete HCR initiator, and (ii) adding a series of HCR hairpin monomers to the sample in the presence of the complete HCR initiator to generate HCR. The series of HCR hairpin monomers are configured to polymerize via HCR.

[0241] In some embodiments, methods are provided. The above methods include (a) preparing a first split initiator probe (1190) containing a first split initiator (1151), and a second split initiator probe (1290) containing a second split initiator (1251). The above methods further include preparing a first hairpin monomer (1510) containing a first input domain (1852) containing a first toehold (1851) and a first stem portion (1755), a first output domain (1854) containing a first hairpin loop (1853) and a complementary chain to the first stem portion (1756), and a first reporter molecule (1850). Furthermore, a second hairpin monomer (1610) is prepared, comprising IV.a. a second input domain (1952) including a second toehold (1951) and a second stem portion (1855), b. a second output domain (1954) including a second hairpin loop (1953) and a complementary chain to the second stem portion (1856), and c. a second reporter molecule (1950). Furthermore, V. a target molecule (1020) is prepared. The above method further comprises (b) incubation the prepared first split initiator probe and second split initiator probe together with the target. As described herein, the split initiator probe may further include a target binding section to form a complete initiator by colocalizing the two split initiators.

[0242] In some embodiments, by first adding a segmented initiator probe to a sample that may contain a target, washing away the bulk solution, retaining the bound segmented initiator probe, and then adding a hairpin monomer, the segmented initiator probe specifically bound to the target can colocalize the segmented initiator and induce HCR, while a nonspecifically bound segmented initiator probe alone can not colocalize the complete initiator and thus not induce HCR.

[0243] In any of the methods described herein, one or more of the following can be detected and / or quantified: molecules, DNA molecules, RNA molecules, protein molecules, small molecules, synthetic molecules, or molecular complexes. In some embodiments, the target is two or more targets, such as a protein complex or a protein-nucleic acid complex. That is, protein binding can be quantified by the splitting initiator method of the present invention. In some embodiments, inorganic or non-organic substances can also be quantified. In some embodiments, any target can be detected insofar as there is a corresponding target-binding section that can bind to the target and can form part of the splitting initiator probe. In some embodiments, the target is any nucleic acid molecule. In some embodiments, the target is a protein. In some embodiments, the target consists of at least one of mRNA, miRNA, lncRNA, rRNA, non-coding RNA, or genomic DNA. In some embodiments, the target consists of an amino acid sequence. In some embodiments, the target consists of a molecular complex. In some embodiments, the target is at least one of DNA, RNA, protein, or small molecule target molecules or complexes, in vitro, in sights, or in vivo. In some embodiments, the target is a molecular complex comprising at least one of DNA target molecules, RNA target molecules, protein target molecules, or small molecule target molecules. In some embodiments, the target includes molecules or complexes, in vitro, in sights, or in vivo.

[0244] In some embodiments, the target molecule can be complexed such that when the target binding sites within the split initiator (also known as split initiator) probe pair specifically bind to those target sites in the complex, two halves of the HCR initiator are in close proximity, allowing the complete initiator to initiate HCR signal amplification. Figure 5 shows the detection of the target complex using the split initiator (also known as split initiator) probe (panel a), the detection of the target nucleic acid complex using the split initiator (also known as split initiator) nucleic acid probe (panel b), the detection of the target protein complex using the split initiator (also known as split initiator) antibody probe (panel c), the detection of the target protein complex using the primary antibody probe and the split initiator (also known as split initiator) secondary antibody probe (panel d), and the detection of the target protein / nucleic acid complex using the split initiator antibody and nucleic acid probe (panel e).

[0245] In some embodiments, any of the methods described herein can be used as part of an insights method for imaging a DNA target, RNA target, protein target, or small molecule target, including DNA insights hybridization (ISH), RNA insights hybridization (ISH), or protein immunohistochemistry (IHC).

[0246] In some embodiments, any of the methods described herein further include applying one or more of the above components to a target. In some embodiments, the target is hydrated. In some embodiments, the target is present in a solution but can be immobilized on a solid phase. In some embodiments, the target is present in a solution. In some embodiments, the target is immobilized on beads or other carriers. In some embodiments, the carrier is a mesh, gel, or rigid surface. In some embodiments, the target is not immobilized. In some embodiments, the detection occurs in vivo, in vitro, or in insights. In some embodiments, an HCR method is provided, comprising an in vitro method in which the target is immobilized on beads or a microarray. In some embodiments, the target is immobilized on beads. In some embodiments, the target is immobilized on a microarray. In some embodiments, an HCR method is provided, comprising an in vivo or in vitro method in which the target is not immobilized. In some embodiments, an HCR method is provided, comprising an in vivo or in vitro method in which the target is immobilized.

[0247] In some embodiments, incubation causes a first segmented initiator probe (1190) to bind to the target molecule, and a second segmented initiator probe (1290) to bind to the target molecule. The target molecule can be a single molecule or multiple molecules linked together. In some embodiments, incubation is carried out at room temperature. In some embodiments, incubation is carried out at 4°C. In some embodiments, incubation is carried out at 37°C. In some embodiments, incubation is carried out at 45°C. In some embodiments, incubation is carried out at 50°C. In some embodiments, incubation is carried out at 55°C. In some embodiments, incubation is carried out at 60°C. In some embodiments, incubation includes an incubation time of at least 1 minute, for example, 5 minutes, 15 minutes, 30 minutes, or 1 hour. In some embodiments, the incubation time is greater than 1 hour, for example, 2 hours, 4 hours, 12 hours, 16 hours, or 24 hours. In some embodiments, incubation is carried out in a hybridization buffer containing 0% formamide. In some embodiments, incubation is carried out in a hybridization buffer containing formamide. In some embodiments, the percentage concentration of formamide is 1% to 80%, for example, 10% to 70%, or 30% to 60%. In some embodiments, the hybridization buffer contains citric acid. In some embodiments, the molar concentration of citric acid is 1 nM to 30 nM, for example, 5 nM to 15 nM, or 8 nM to 12 nM. In some embodiments, the hybridization buffer contains Tween®. In some embodiments, the percentage concentration of Tween® is 0% to 1.0%, for example, 0.05% to 0.5%. In some embodiments, the hybridization buffer contains heparin. In some embodiments, the heparin concentration is 20 μg / mL to 80 μg / mL, for example, 30 μg / mL to 70 μg / mL, for example, 40 μg / mL to 60 μg / mL.In some embodiments, the hybridization buffer contains Denhart's solution. In some embodiments, the hybridization buffer contains dextran sulfate. In some embodiments, the percentage concentration of dextran sulfate is 1% to 60%, for example, 40% to 60%.

[0248] In some embodiments, a first split initiator (1151) of any of the methods described herein is part of a first split initiator probe (1190), and a second split initiator (1251) is part of a second split initiator probe (1290). In some embodiments, the first split initiator probe (1190) further includes a first target binding section (1141), and the second split initiator probe (1290) further includes a second target binding section (1241). In some embodiments, the first target binding section (1141) is configured to bind adjacent to the second target binding section (1241) on the target when both the first split initiator probe (1190) and the second split initiator probe (1290) specifically bind to the target (1020).

[0249] The term “proximity” is used in reference to the binding of the first and second split initiator probes (and / or the binding of the first and second target binding sections), but the binding does not need to be directly adjacent, as long as the first and second initiator probes are close enough to each other to form a complete initiator capable of inducing an HCR. In some embodiments, the first target binding section binds to the second target binding section within at least 10 amino acids, e.g., within 5 amino acids or within 1 amino acid. In some embodiments, the first target binding section binds to the second target binding section within at least 10 nucleotides, e.g., within 5 nucleotides or within 1 nucleotide. In some embodiments, the first initiator probe and the second split initiator probe (and / or the first and second target binding sections) are within 2 Å, 5 Å, 10 Å, 50 Å, 100 Å, or 1000 Å of each other. In some embodiments, the first and second split initiator probes may include spacers to increase the space between the two target sections. Including additional nucleic acid sequences in such spacers can increase the degrees of freedom in the position of the first and second (or additional) target sections. In some embodiments, each target section on the target is sufficiently proximal to one another to colocalize a complete initiator with two or more split initiators.

[0250] In some embodiments, the first split initiator probe (1190) includes two or more target-binding sections, for example, two target-binding sections, three target-binding sections, four target-binding sections, or five target-binding sections. This allows assaying multiple potential targets at once. In some embodiments, the first split initiator probe (1190) includes more than five target-binding sections. In some embodiments, the second split initiator probe (1290) includes two or more target-binding sections, for example, two target-binding sections, three target-binding sections, four target-binding sections, or five target-binding sections. In some embodiments, the second split initiator probe (1290) includes more than five target-binding sections.

[0251] In some embodiments, the target-binding section (e.g., region) within each of the split initiator (also known as split initiator) probe pairs can consist of DNA, RNA, 2'OMe-RNA, PNA, amino acids, or any synthetic nucleic acid analog, or any synthetic amino acid analog. Figure 4 illustrates the detection of target molecules using split initiator (also known as split initiator) probes (panel a), the detection of target mRNA using split initiator (also known as split initiator) nucleic acid probes (panel b), the detection of target proteins using split initiator (also known as split initiator) antibody probes (panel c), and the detection of target proteins using primary antibody probes and split initiator secondary antibody probes (panel d). In each case, the selective binding of the probe pair to the cognitive target molecule results in the colocalization of two halves of the complete HCR initiator, inducing the growth of an anchored HCR amplification polymer. The target molecule can be a protein or small molecule such that when the target binding sites within a split initiator probe pair specifically bind to those target sites on a protein or small molecule, two halves of the HCR initiator are in close proximity, allowing the complete initiator to initiate HCR signal amplification.

[0252] In some embodiments, the HCR hairpin monomer may be labeled with a non-fluorescent reporter molecule (e.g., an isotopically pure rare earth element, a chromophore, etc.).

[0253] In Scheme E, each probe within a split initiator probe pair contains a target binding site and half of the HCR initiator such that when the split initiator probe specifically base pairs with its target section (e.g., a cognitive proximal target site), two halves of the HCR initiator colocalize; this functionality can be achieved by arranging the target binding site and initiator fragments within the split initiator probe pair in various stereochemistrys (Figure 3).

[0254] In some embodiments, the HCR initiator within a split initiator (also known as a split initiator) probe pair can be split between the two probes (it does not have to be half and half) so that HCR amplification is initiated only when both probes are proximal to each other.

[0255] In some embodiments, the HCR initiator can be split between two or more split initiator (also known as split initiator) probes.

[0256] In some embodiments, a split initiator probe set (or probe set) containing one or more split initiator (also known as split initiator) probe pairs can be used to detect target mRNA; each probe pair contains a target binding site for a different subsequence of the target mRNA. Within the split initiator probe set, each split initiator (also known as split initiator) probe pair colocalizes to form the same HCR initiator sequence, making it possible to simultaneously grow HCR amplification polymers from multiple probe pairs bound to the same target mRNA.

[0257] In some embodiments, the target molecule can be mRNA, miRNA, lncRNA, rRNA, genomic DNA, or any nucleic acid molecule.

[0258] In some embodiments, the split initiator (also known as a split initiator) between the two probes in a pair may be composed of DNA, RNA, 2'OMe-RNA, PNA, or any synthetic polymer capable of initiating HCR amplification.

[0259] In some embodiments, any of the methods described herein include adding a plurality of first split initiators (1151) and second split initiators (1251), for example, 10, 20, 50, 100, 1,000, 10,000, and so on.

[0260] In some embodiments, any of the methods described herein may consist of a two-step approach with washing after each step. In some embodiments, the target is immobilized and / or the sample is immobilized. In some embodiments, the first step is a detection step including binding a split initiator probe to the target and washing away any unbound split initiator probes, and the second step is an amplification step performing HCR amplification. After this, washing away any unpolymerized HCR monomers (such as hairpin monomers) may be performed. In some embodiments, there are two or more washes after each step, for example, two washes, three washes, four washes, or five washes. In some embodiments, the HCR method consists of a single step without washing.

[0261] In some embodiments, rather than directly inserting the reporter molecule into the hairpin monomer itself, the HCR hairpin monomer includes a labeling site. In some embodiments, any of the methods described herein further include washing the sample to remove unpolymerized HCR hairpin monomers, adding a labeled probe containing a complementary chain to the labeling site and a reporter molecule; washing away the unbound labeled probe; and detecting the presence or absence of the reporter molecule. In some embodiments, the labeled probe is a hairpin molecule further comprising a fluorophore / quencher pair such that the fluorophore is quenched when the hairpin monomer is closed, but the fluorophore is not quenched when the labeled probe binds to the labeling site on the HCR polymer. In some embodiments, the labeled probe is a double chain in which one chain carries a fluorophore and the other chain carries a quencher such that when the labeled probe binds to the labeling site on the HCR polymer, the quencher-labeled chain moves and the fluorophore-labeled chain binds to the labeling site on the HCR polymer. In some embodiments, the hairpin monomer carries a reporter, which is one portion of a FRET pair, ...

Claims

[Claim 1] A method for detecting repetitive signals using reporter-marked hairpins, a) Prepare a sample that may contain up to N targets and other non-target molecules; b) Prepare N probe sets (each targeting one of N target species), each containing i) one or more HCR initiator-labeled probes, or ii) one or more probe units, each containing two or more HCR splitting initiators; c) Wash the sample as desired; d) Prepare M HCR amplifiers corresponding to M of the N probe sets (if M ≤ N; each is labeled with a separate reporter); e) Wash the sample as desired; f) Detecting M signals corresponding to the M reporters; g) Removing the M signals from the sample; and h) optionally repeating one or more of steps b to g until signal detection is performed for all N targets; The probe set includes one of the following: a) One or more HCR initiator-labeled probes, or b) One or more probe units, The HCR initiator labeled probe includes: One or more target binding regions, and One or more initiators, The probe unit includes two or more HCR splitting initiator probes. The HCR splitting initiator probe includes: Target binding region, and Split initiator, The HCR amplifier contains two or more HCR hairpins. The HCR hairpin includes the following input domains: Single-chain toehold; and Stem section, The HCR hairpin further includes the following output domains: Single-chain loops; and Complementary chain to the aforementioned stem portion, HCR Hairpin includes reporters, Repeated signal detection method.

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