Molecular interaction detection and profiling

Abstract

The present invention provides a method for detecting two target molecules in a sample that are independent molecules and that are adjacent to or interact with each other. The method involves performing three separate assay reactions to detect a first target molecule and a second target molecule and their interactions using a proximity probe common among the three assay reactions and a nucleic acid reagent that interacts with the probe. In particular, the methods use a unique nucleic acid substrate molecule, such as a padlock probe, to detect the probes bound to their targets and determine when the probes for the two targets are in close proximity indicative of the interaction between the targets.

Description

Technical Field

[0001] The present invention generally relates to a method for detecting two adjacent or interacting target molecules in a sample. The method allows the detection of two target molecules independently, and also allows the detection of proximity between molecules (which can indicate an interaction between molecules), and the determination of the amount of each molecule that is adjacent to or interacts with each other. The method uses probes including proximity probes that bind to target molecules and nucleic acid reagents that interact with the probes, particularly unique nucleic acid substrate molecules (e.g., padlock probes), to detect probes that bind to their targets and determine when the probes for two targets are in close proximity, which indicates an interaction between the targets. Background Art

[0002] Many methods have been developed in the art to detect target molecules, such as proteins and nucleic acids. Generally, it is convenient to be able to detect proteins in a multiplexed manner, but the multiplexed protein detection methods currently on the market focus on detecting the expression level of independent proteins, and in these methods, functional aspects of these proteins, such as their protein-protein interactions (PPIs) or post-translational modifications (PTMs) are ignored.

[0003] Methods for studying protein interactions have been developed, including proximity assays, which rely on the interaction between proximity probe pairs, when both probes of the pair are adjacently bound to their respective targets (occurring when the respective targets interact), the pair can generate a signal indicating the interaction; when the targets are not adjacent (non-interacting), no signal is generated. A typical representative of this type of proximity assay is the in situ proximity ligation assay (isPLA), which is a powerful and widely used tool for detecting PPIs or PTMs. The limitation of this assay is that it only detects proximity events (i.e., interactions), and cannot detect the expression level of proteins that undergo PPIs or PTMs. In addition, in situ PLA has been rarely used in multiplex environments so far.

[0004] The proximity probe used in the proximity assay generally comprises a binding domain and a nucleic acid structure domain that can be directly (primary binding partner) or indirectly (secondary binding partner) bonded to the target analyte. The nucleic acid structure domain of the proximity probe pair is for example directly interacted with each other by hybridization and extension and / or by connection, or indirectly interacted with each other by another nucleic acid molecule, to produce a nucleic acid product that can be detected, so as to detect the target of proximity assay (as mentioned above, it can be the complex of interacting proteins, but it is not necessarily so; proximity assay is also used to detect independent target molecules, wherein the two proximity probes in the proximity probe pair are all bonded to the same target molecule). The connection product of proximity assay can be produced by connecting the nucleic acid structure domain to each other or being connected to another molecule or by being bonded to the connection of one or more other nucleic acid molecules in the nucleic acid structure domain. Therefore, when two proximity probes are all combined with their respective targets in a proximity mode, one or more nucleic acid molecules can be combined with two nucleic acid structure domains of two proximity probes, and the connection of one or more nucleic acid molecules can be templated by one or two nucleic acid structure domains of proximity probes. One or more nucleic acid molecules connected in this way can be padlock probes, which are formed into rings by connection. This configuration represents the classical form of original position PLA.

[0005] Padlock probes are circularizable probes comprising one or more linear oligonucleotides that can be connected together to form a ring. Padlock probes are well known in the art and widely used. Padlock probes are generally linear oligonucleotides, and the sequences at their 3' and 5' ends are complementary to the sequences in the target nucleic acid molecule. The target-complementary binding sequence is connected by an intermediate "skeleton" region that is not bound to the target molecule. The hybridization of the probe end with the target nucleic acid molecule places the probe end in a position where it can be connected to each other: at this position, the probe ends can be directly connected to each other (wherein the probe ends have been hybridized adjacent to each other), or indirectly connected to each other (wherein there is a gap between the hybridization ends). The gap can be filled by a gap oligonucleotide (which can be regarded as a part of a two-part padlock probe), or by extending the hybridization 3' end of the padlock probe to produce a 3' end that can be connected to the hybridization 5' end of the adjacent padlock probe. The resulting circular nucleic acid molecule can be detected by sequencing or qPCR, etc., or by using a circular padlock as an RCA template for rolling circle amplification (RCA). The RCA product can then be detected, for example, by using a probe to detect the target sequence or by digesting the RCA product into a monomer followed by sequencing.

[0006] RCA utilizes a strand displacement polymerase to extend a primer hybridized to a circular template (the strand displacement activity displaces the primer and effectively causes the ring to "roll"). Adding polymerase and nucleotides to start the synthesis reaction, i.e., polymerization. Since the rolling circle template is circular, the resulting RCA product is a long single-stranded nucleic acid molecule (i.e., concatemer) consisting of linked tandem repeats complementary to the rolling circle template. Such concatemers typically form balls or "spots" that can be easily visualized and counted. For this reason, RCA has been widely used as an amplification and / or detection technique in various nucleic acid-based assays, usually in combination with padlock probes.

[0007] Other methods for detecting the interaction between two analytes (e.g., protein-protein interaction) include the MolBoolean method described in WO2018 / 147794. The MolBoolean method utilizes a single-stranded substrate nucleic acid, which forms a double-stranded fragment when interacting with the nucleic acid domain of the proximity probe, and the double-stranded fragment comprises a cleavage site, in which a label corresponding to the analyte of interest is inserted. Inserting a single label into the substrate nucleic acid can identify the presence of non-interactive analytes, while inserting two labels (corresponding to two analytes) into the substrate nucleic acid can identify the interaction between analytes. In this way, the amount of each analyte that interacts with each other can be determined. This method is superior to the MolBoolean method because no enzyme cleavage step is required, and further by producing different nucleic acid products, such as by using different padlock probes or other nucleic acid-based reagents that interact with the nucleic acid domain of the proximity probe, the method can be easily reused. Summary of the invention

[0008] Developed novel method of this paper to overcome the limitation of the above method, so that it is possible to detect the interaction between two target molecules in the same assay in a way that can be easily reused, or more specifically to indicate the proximity of the interaction, and the independent expression level of two participating molecules. The method uses the proximity probes combined with two corresponding target molecules in a way that can carry out 3 independent detection reactions, to produce three different signals (for example, detectable reaction products), which can be detected individually to detect two independent proteins and interactions. Proximity probes are used to detect target interactions, and the independent components of proximity probe pairs are also used individually to detect independent target molecules. Conveniently, different padlock probes can be used in combination with proximity probes, and padlock probes can interact with one or two nucleic acid domains of proximity probes to produce 3 different circular RCA templates that can be detected by RCA, to detect the independent components of the interaction and the interaction. However, more generally, any nucleic acid reagent that can hybridize with the nucleic acid domain of proximity probes and produce detectable signals or products can be used, and different reagents can be used in 3 independent detection reactions. Therefore, for example, the padlock probe that can hybridize with two nucleic acid structure domains of the adjacent probe pair can be used to detect interactions, and each can specifically bind to a nucleic acid structure domain in the nucleic acid structure domain of the adjacent probe pair but not be bound to the independent detection probe of another nucleic acid structure domain respectively can be used to detect each of the two adjacent probes adjacent to the probe individually, and therefore detect two independent target molecules that the adjacent probe has been combined.Other nucleic acid reagents include other substrates for producing nucleic acid products, such as HCR reagents, and for constructing a signal amplification system based on nucleic acid hybridization, such as the reagent of a branched DNA amplification system.

[0009] Thus, in a broad first aspect, there is provided a method for detecting two target molecules in a sample and detecting an interaction between the two target molecules, the method comprising:

[0010] (i) contacting a sample with (a) a first probe or a first proximity probe pair for detecting a first target molecule and (b) a second probe or a second proximity probe pair for detecting a second target molecule;

[0011] wherein each of the probes comprises a binding domain and a nucleic acid domain capable of binding directly or indirectly to their target molecules, and the first probe and the second probe or one of the probes of the first proximity probe pair and one of the probes of the second proximity probe pair together form a third proximity probe pair for detecting the interaction between the first target molecule and the second target molecule;

[0012] (ii) performing a first assay reaction to detect the nucleic acid domain of the first probe or the nucleic acid domain of at least one member of the first proximity probe pair, thereby detecting the first target molecule;

[0013] (iii) performing a second assay reaction to detect the nucleic acid domain of a second probe or the nucleic acid domain of at least one member of a second proximity probe pair, thereby detecting a second target molecule;

[0014] (iv) performing a third assay reaction to detect the interaction between the first target molecule and the second target molecule, wherein the third assay reaction is a proximity assay using a third proximity probe pair, wherein when the first target molecule and the second target molecule are present in proximity in an interactive manner, the nucleic acid domains of the third proximity probe pair interact with each other directly or indirectly to produce a nucleic acid product, and detecting the product to detect the interaction between the first target molecule and the second target molecule.

[0015] In one embodiment, the first assay and the second assay involve quantifying the amount of target molecules detected, and the third assay involves quantifying the amount of target molecules present in an interactive manner.

[0016] The first assay reaction, the second assay reaction and the third assay reaction are separate independent reactions performed as part of the same assay method. They can be performed simultaneously or sequentially. Each assay reaction is performed individually. More specifically, each assay reaction produces a separate signal. In one embodiment, each assay reaction results in or produces a separate product to be detected. Therefore, the method as a whole produces three separate reaction products, and each product individually indicates two corresponding independent target molecules and target molecule interactions.

[0017] In one embodiment of the method, use the nucleic acid reagent that hybridizes with the nucleic acid structure domain of the proximity probe, described reagent allows to detect this probe or the proximity probe right that has been combined together in a neighboring mode.Nucleic acid reagent can produce detectable signal, for example, they can be detectably labeled, or they cause to produce a kind of product, for example hybridization, extension, connection or amplification product, it can be detected, so that detect two kinds of independent target molecules and interaction respectively.

[0018] The method can use at least two nucleic acid reagents. In one embodiment, one nucleic acid reagent can participate in two assay reactions. In another embodiment, three nucleic acid reagents can be used, with each assay reaction using a separate reagent.

[0019] In a specific implementation scheme:

[0020] (a) a first assay reaction utilizes a first nucleic acid reagent capable of hybridizing to the nucleic acid domain of a first probe or the nucleic acid domain of at least one member of a first proximity probe pair, and detecting the first reagent or its hybridization;

[0021] (b) a second assay reaction utilizes a second nucleic acid agent capable of hybridizing to the nucleic acid domain of the second probe or the nucleic acid domain of at least one member of a second proximity probe pair, and detecting the second agent or hybridization thereof; and

[0022] (c) The third assay either (i) utilizes a third nucleic acid reagent that is capable of hybridizing to both nucleic acid domains of a third proximity probe pair to produce a proximity probe-interactive nucleic acid product, or (ii) utilizes one of the first nucleic acid reagent or the second nucleic acid reagent that is also capable of hybridizing to the nucleic acid domain of the other member of the third proximity probe pair to produce a third proximity probe pair-interactive nucleic acid product.

[0023] In one embodiment, the nucleic acid domain of the proximity probe comprises a tag sequence to be detected.

[0024] In one embodiment, the nucleic acid structure domain of the first probe or the first nucleic acid structure domain of a member of the probe pair comprises the first label sequence detected to detect the first target molecule; The nucleic acid structure domain of the second probe or the second nucleic acid structure domain of a member of the probe pair comprises the second label sequence detected to detect the second target molecule; And the first probe and the second probe or the first nucleic acid structure domain of the member of the probe pair and the second (constituting the third adjacent to the probe pair) adjacent to the probe pair each also comprises a separate complementary region that can mediate the interaction between the nucleic acid structure domain of the third adjacent to the probe pair. In a specific embodiment, the complementary region can be a binding site or hybridization site for a nucleic acid reagent that can hybridize with the third adjacent to two nucleic acid structure domains.

[0025] In one embodiment, the first tag sequence and the second tag sequence constitute the binding / hybridization site for the first nucleic acid agent and the second nucleic acid agent, respectively. In such an embodiment, the separate complementary region in the nucleic acid domain of the third proximity probe pair comprises or constitutes the binding / hybridization site for the third nucleic acid agent.

[0026] In one embodiment, the first and second assay reactions produce first and second nucleic acid products from or using first and second nucleic acid reagents, respectively, and in the third assay reaction produce a third nucleic acid product.

[0027] In one embodiment, the first nucleic acid product, the second nucleic acid product and the third nucleic acid product are produced in the first assay reaction, the second assay reaction and the third assay reaction, respectively. In a more specific embodiment, the first nucleic acid product, the second nucleic acid product and the third nucleic acid product are produced from the first reagent, the second reagent and the third reagent, respectively, or using the first reagent, the second reagent and the third reagent. These three nucleic acid products can be distinguished from each other by sequence and are detected to detect the first target molecule and the second target molecule and the interaction between them.

[0028] In one embodiment, the nucleic acid product is amplified, and the resulting amplicon is detected.

[0029] Nucleic acid reagent can be a substrate for producing nucleic acid product. In one embodiment, substrate is a padlock probe. In this embodiment, product is a circular nucleic acid molecule, which can be amplified by RCA to produce RCA product (RCP). In another embodiment, substrate is a substrate for HCR reaction or for branched DNA amplification reaction.

[0030] Therefore, in a more specific embodiment of the first aspect, a method for detecting two target molecules in a sample and detecting the interaction between the two target molecules is provided herein, the method comprising:

[0031] (i) contacting a sample with (a) a first probe or a first proximity probe pair for detecting a first target molecule and (b) a second probe or a second proximity probe pair for detecting a second target molecule;

[0032] wherein each of the probes comprises a binding domain capable of directly or indirectly binding to a target molecule and a nucleic acid domain, and the first probe and the second probe or one of the probes of the first proximity probe pair and one of the probes of the second proximity probe pair together form a third proximity probe pair for detecting an interaction between the first target molecule and the second target molecule;

[0033] (ii) contacting the probe with at least a first nucleic acid substrate molecule and a second nucleic acid substrate molecule, wherein the first substrate molecule hybridizes to the nucleic acid domain of the first probe or one of the probes of a first proximity probe pair, and the second substrate molecule hybridizes to the nucleic acid domain of the second probe or one of the probes of a second proximity probe pair;

[0034] (iii) performing a first assay reaction using the first probe or at least one member of a first proximity probe pair to detect a first target molecule and produce a first nucleic acid product from the first nucleic acid substrate;

[0035] (iv) performing a second assay reaction using a second probe or at least one member of a second proximity probe pair to detect a second target molecule and produce a second nucleic acid product from the second nucleic acid substrate;

[0036] (v) performing a third assay reaction using a third proximity probe pair to detect an interaction between the first target molecule and the second target molecule and producing a third nucleic acid product when the first target molecule and the second target molecule are in proximity in an interactive manner;

[0037] wherein the first nucleic acid product, the second nucleic acid product, and the third nucleic acid product are distinguishable from each other by sequence;

[0038] (vi) optionally generating an amplification product of or from the first nucleic acid product, the second nucleic acid product, and, if present, a third nucleic acid product; and

[0039] (vii) detecting the nucleic acid products or amplification products, wherein the first nucleic acid or amplification product indicates a first target molecule, the second nucleic acid or amplification product indicates a second target molecule, and the third nucleic acid or amplification product indicates an interaction between the first target molecule and the second target molecule.

[0040] In one embodiment, step (ii) above further comprises contacting the probe with a third substrate molecule which hybridizes to both nucleic acid domains of the third proximity probe pair, and step (v) comprises generating a third nucleic acid product from the third substrate.

[0041] In alternative embodiments, the third nucleic acid product is generated by the interaction of the first substrate molecule or the second substrate molecule with the nucleic acid domain of the third proximity probe pair.

[0042] In one embodiment, the nucleic acid substrate is a padlock probe, which can be provided in one or more parts (e.g., two parts). The padlock probes can be directly or indirectly connected using the nucleic acid domains to which they hybridize as ligation templates. The ligated circular product can be amplified by RCA.

[0043] In all embodiments related to contacting a neighborhood probe with a nucleic acid reagent (e.g., substrate), contact can be carried out before sample contacts the neighborhood probe, during sample contacts the neighborhood probe, or after sample contacts the neighborhood probe. This may depend on accurate method form and method steps. Therefore, in some embodiments, nucleic acid reagent, particularly when it is a substrate such as a padlock probe, can prehybridize with the nucleic acid structure domain of the neighborhood probe before the neighborhood probe contacts the sample. In other embodiments, nucleic acid reagent can contact the neighborhood probe after the neighborhood probe contacts the sample, more specifically after the neighborhood probe is combined with its target molecule, for example, wherein the detection reagent is for detecting the first neighborhood probe or the second neighborhood probe detection probe.

[0044] In a specific embodiment, the method can be defined as a method for detecting two target molecules in a sample and detecting an interaction between the two target molecules, the method comprising:

[0045] (i) contacting the sample with:

[0046] (a) a first proximity probe for the first target molecule, wherein the first proximity probe comprises a binding domain capable of binding directly or indirectly to the first target molecule and a first nucleic acid domain comprising one or more (e.g., two) single-stranded regions, wherein the first nucleic acid domain comprises a first padlock binding site for a first padlock probe and a hybridization sequence capable of hybridizing to a third padlock probe, both the first padlock binding site and the hybridization sequence being located within the one or more single-stranded regions;

[0047] (b) a second proximity probe for a second target molecule, wherein the second proximity probe comprises a binding domain capable of directly or indirectly binding to a second target molecule and a second nucleic acid domain comprising one or more single-stranded regions, wherein the second nucleic acid domain comprises a second padlock binding site for a second padlock probe and a hybridization sequence capable of hybridizing to a third padlock probe, both the second padlock binding site and the hybridization sequence being located within the one or more single-stranded regions;

[0048] (c) a first padlock probe comprising target binding sequences at its 5' and 3' ends capable of hybridizing to the first padlock binding site of the first proximity probe;

[0049] (d) a second padlock probe comprising target binding sequences at its 5' and 3' ends capable of hybridizing to the second padlock binding site of the second proximity probe; and

[0050] (e) a third padlock probe, the third padlock probe comprising:

[0051] (I) a single circularisable oligonucleotide comprising a target binding sequence at its 5' and 3' ends capable of hybridising to the hybridising sequence of the second proximity probe and comprising an anchor sequence in the backbone region between the 5' and 3' ends capable of hybridising to the hybridising sequence of the first proximity probe, and wherein when applied to a sample, the anchor sequence hybridises to its complementary hybridising sequence of the first proximity probe; or

[0052] (II) two circularized oligonucleotides that together form a two-part padlock probe, each circularized oligonucleotide comprising at its 5' and 3' ends a first target binding sequence that is capable of hybridizing to the hybridization sequence of the first proximity probe and a second target binding sequence that is capable of hybridizing to the hybridization sequence of the second proximity probe, such that when the first proximity probe and the second proximity probe are adjacent (i.e., when the first target molecule and the second target molecule interact), each circularized oligonucleotide can hybridize to the nucleic acid domains of the two proximity probes, and the respective 5' and 3' ends of the two circularized oligonucleotides are juxtaposed to directly or indirectly link to each other to form a loop;

[0053] (ii) performing a gap-filling reaction (by hybridizing a gap oligonucleotide and / or by extending the 3' end) where the 5' and 3' ends of the padlock probes have hybridized to their respective binding sites or hybridization sequences with a gap therebetween, and ligating the hybridized padlock probes, thereby generating a first circular nucleic acid product from the first padlock probe, a second circular nucleic acid product from the second padlock probe, and a third circular nucleic acid product from the third padlock probe, wherein the first circular nucleic acid product, the second circular nucleic acid product, and the third circular nucleic acid product are distinguishable from each other by sequence (e.g., they may each comprise a distinguishable tag sequence or unique identifier sequence);

[0054] (iii) optionally amplifying the first circular nucleic acid product, the second circular nucleic acid product and the third circular nucleic acid product by rolling circle amplification (RCA) to produce a first RCA product (RCP), a second RCA product and a third RCA product; and

[0055] (iv) detecting a first circular nucleic acid product, a second circular nucleic acid product and a third circular nucleic acid product or RCP, wherein the first circular nucleic acid product or RCP indicates a first target molecule, the second circular nucleic acid product or RCP indicates a second target molecule, and the third circular nucleic acid product or RCP indicates an interaction between the two target molecules.

[0056] It will be observed that the hybridization sequences in the first nucleic acid domain and the second nucleic acid domain can be target binding sites for the target binding region of the third padlock probe, or they can be anchor binding sequences for the anchor sequence in the backbone region of the third padlock probe. Thus, depending on the probe configuration, the hybridization sequence can be a target binding sequence for the third padlock probe, i.e., it can provide a ligation template for the third padlock probe, or it can be a capture sequence for the third padlock probe, i.e., through which the padlock probe can be attached to the nucleic acid domain of the proximity probe.

[0057] The first and second proximity probes of the above embodiments may be replaced by a pair of proximity probes specific for each of the two target molecules.

[0058] Therefore, another specific embodiment of the first aspect is a method for detecting two target molecules in a sample and detecting the interaction between the two target molecules, the method comprising:

[0059] (i) contacting the sample with:

[0060] (a) a first proximity probe pair for a first target molecule, the first proximity probe pair comprising a first proximity probe and a second proximity probe, each proximity probe comprising a binding domain capable of directly or indirectly binding to the first target molecule and a nucleic acid domain comprising one or more (e.g., two) single-stranded regions, wherein the nucleic acid domain of the first proximity probe comprises a first padlock binding site for a first padlock probe, and the nucleic acid domain of the second proximity probe comprises a hybridization sequence capable of hybridizing to a third padlock probe, both the first padlock binding site and the hybridization sequence being located within the one or more single-stranded regions;

[0061] (b) a second proximity probe pair for a second target molecule, the second proximity probe pair comprising a first proximity probe and a second proximity probe, each proximity probe comprising a binding domain capable of binding directly or indirectly to the second target molecule and a nucleic acid domain comprising one or more single-stranded regions, wherein the nucleic acid domain of the first proximity probe comprises a second padlock binding site for a second padlock probe, and the nucleic acid domain of the second proximity probe comprises a hybridization sequence capable of hybridizing to a third padlock probe, the second padlock binding site and the hybridization sequence being located within the one or more single-stranded regions;

[0062] (c) a first padlock probe comprising a target binding sequence at its 5' and 3' ends capable of hybridizing to a first padlock binding site of a first proximity probe of a first proximity probe pair;

[0063] (d) a second padlock probe comprising target binding sequences at its 5' and 3' ends capable of hybridizing to a second padlock binding site of a first proximity probe of a second proximity probe pair;

[0064] (e) a third padlock probe, the third padlock probe comprising:

[0065] (I) a single circularisable oligonucleotide comprising a target binding sequence at its 5' and 3' ends that is capable of hybridising to a hybridising sequence of a second proximity probe of a second proximity probe pair and comprising an anchor sequence in the backbone region between the 5' and 3' ends that is capable of hybridising to a hybridising sequence of the second proximity probe of a first proximity probe pair, and wherein when applied to a sample, the anchor sequence hybridises to its complementary hybridising sequence of the second proximity probe of the first proximity probe pair; or

[0066] (II) two circularized oligonucleotides that together form a two-part padlock probe, each circularized oligonucleotide comprising at its 5′ and 3′ ends a first target binding sequence that is capable of hybridizing to a hybridization sequence of a second proximity probe of a first proximity probe pair and a second target binding sequence that is capable of hybridizing to a hybridization sequence of a second proximity probe of a second proximity probe pair, such that when the first proximity probe and the second proximity probe are adjacent (i.e., when the first target molecule and the second target molecule interact), each circularized oligonucleotide can hybridize to the nucleic acid domains of the two second proximity probes, and the respective 5′ and 3′ ends of the two circularized oligonucleotides are juxtaposed to directly or indirectly link to each other to form a loop;

[0067] (ii) performing a gap-filling reaction (by hybridizing a gap oligonucleotide and / or by extending the 3' end) where the 5' and 3' ends of the padlock probes have hybridized to their respective binding sites or hybridization sequences with a gap therebetween, and ligating the hybridized padlock probes, thereby generating a first circular nucleic acid product from the first padlock probe, a second circular nucleic acid product from the second padlock probe, and a third circular nucleic acid product from the third padlock probe, wherein the first circular nucleic acid product, the second circular nucleic acid product, and the third circular nucleic acid product are distinguishable from each other by sequence (e.g., they may each comprise a distinguishable tag sequence or unique identifier sequence);

[0068] (iii) amplifying the first circular nucleic acid product, the second circular nucleic acid product and the third circular nucleic acid product by rolling circle amplification (RCA) to produce a first RCA product (RCP), a second RCA product and a third RCA product; and

[0069] (v) detecting a first RCP, a second RCP, and a third RCP, wherein the first RCP indicates the first target molecule, the second RCP indicates the second target molecule, and the third RCP indicates an interaction between the two target molecules.

[0070] In a more specific embodiment, the first adjacent to the right second adjacent to the nucleic acid structure territory of the probe also comprises a hybridization sequence that can hybridize with the complementary anchor sequence in the first padlock probe, and the second adjacent to the right second adjacent to the nucleic acid structure territory of the probe also comprises a hybridization sequence that can hybridize with the complementary anchor sequence in the second padlock probe. In this way, it is also necessary to detect the first and second target molecules independently adjacent to an event.

[0071] In another specific embodiment, a method for detecting two target molecules in a sample and detecting an interaction between the two target molecules is provided, the method comprising:

[0072] (i) contacting the sample with:

[0073] (a) a first proximity probe for a first target molecule, wherein the first proximity probe comprises a nucleic acid domain (e.g. a single-stranded nucleic acid domain) that hybridizes to a first padlock probe (more specifically to a backbone region thereof), wherein the 5' and 3' ends of the first padlock probe are hybridized to a blocking oligonucleotide;

[0074] (b) a second proximity probe for a second target molecule, wherein the second proximity probe comprises a nucleic acid domain comprising one or more single-stranded regions, and wherein the nucleic acid domain comprises a first padlock binding site capable of hybridizing to the 5' and 3' ends of the first padlock probe and a second padlock binding site for the second padlock probe, the padlock binding sites both being located within the one or more single-stranded regions; and

[0075] (c) a second padlock probe comprising target binding sequences at its 5' and 3' ends capable of hybridizing to the second padlock binding site of the second proximity probe;

[0076] such that when the first proximity probe and the second proximity probe are in proximity (i.e. when the first target molecule and the second target molecule interact), the blocking oligonucleotide is displaced from the first padlock probe by the single-stranded region of the second proximity probe comprising the first padlock binding site, and when the first proximity probe and the second proximity probe are not in proximity, the first padlock probe remains bound to the blocking oligonucleotide (and is not bound to the nucleic acid domain of the second proximity probe),

[0077] wherein the first padlock binding site of the blocking oligonucleotide and / or the second proximity probe comprises a gap sequence located between complementary binding sites capable of hybridizing to the 5' and 3' ends of the first padlock probe, such that the hybridized 3' and 5' ends of the first padlock probe are separated by the gap (and cannot ligate to each other);

[0078] (ii) when the 5' and 3' ends of the padlock probes have hybridized to their respective binding sites with a gap therebetween, performing a gap-filling reaction (by hybridizing a gap oligonucleotide and / or by extending the 3' end), and ligating the hybridized padlock probes, thereby generating a first circular nucleic acid product from the first padlock probe hybridized to the blocking oligonucleotide, a second circular nucleic acid product from the second padlock probe, and a third circular nucleic acid product from the first padlock probe hybridized to the first padlock binding site of the second proximity probe, wherein the first circular nucleic acid product, the second circular nucleic acid product, and the third circular nucleic acid product are distinguishable from each other by sequence (e.g., they may each comprise a distinguishable tag sequence or unique identifier sequence);

[0079] (iii) amplifying the first circular nucleic acid product, the second circular nucleic acid product and the third circular nucleic acid product by rolling circle amplification (RCA) to produce a first RCA product (RCP), a second RCA product and a third RCA product; and

[0080] (iv) detecting a first RCP, a second RCP, and a third RCP, wherein the first RCP indicates the first target molecule that does not interact with the second target molecule, the second RCP indicates the second target molecule, and the third RCP indicates an interaction between the two target molecules.

[0081] In a more specific embodiment of this version of the method, the blocking oligonucleotide comprises complementary binding sites for the 5' and 3' ends of the first padlock probe separated by an intervening gap sequence such that the hybridized 3' and 5' ends are separated by the gap.

[0082] Another specific embodiment of the first aspect is a method for detecting two target molecules in a sample and detecting an interaction between the two target molecules, the method comprising:

[0083] (i) contacting the sample with:

[0084] (a) a first proximity probe pair for a first target molecule, the first proximity probe pair comprising a first proximity probe and a second proximity probe, wherein the first proximity probe comprises a nucleic acid domain (e.g. a single stranded nucleic acid domain) comprising a first padlock binding site, and the second proximity probe comprises a nucleic acid domain (e.g. a single stranded nucleic acid domain) that hybridizes to the first padlock probe (more specifically to its backbone region), the first padlock probe comprising binding sequences at its 5' and 3' ends that are capable of hybridizing to the first padlock binding site (i.e. the first padlock binding site of the first proximity probe of the first pair); and

[0085] (b) a second proximity probe pair for a second target molecule, the second proximity probe pair comprising a first proximity probe and a second proximity probe, wherein the first proximity probe comprises a nucleic acid domain (e.g. a single stranded nucleic acid domain) comprising a second padlock binding site, and the second proximity probe comprises a nucleic acid domain (e.g. a single stranded nucleic acid domain) that hybridizes to the second padlock probe (more specifically to its backbone region), the second padlock probe comprising binding sequences at its 5' and 3' ends that are capable of hybridizing to the second padlock binding site (i.e. the second padlock binding site of the first proximity probe of the second pair);

[0086] wherein the binding sequence of the first padlock probe is also capable of hybridizing to the second padlock binding site, and / or the binding sequence of the second padlock probe is also capable of hybridizing to the first padlock binding site;

[0087] wherein the first padlock probe and the second padlock probe each comprise an identifier sequence, and the first padlock binding site and / or the second padlock binding site comprises a gap sequence (which may be or comprise, for example, a tag or barcode sequence) located between complementary binding sites capable of hybridizing to the 5' and 3' ends of the respective padlock probes;

[0088] (ii) where the 5' and 3' ends of the padlock probes have hybridized to their respective binding sites with a gap between them, performing a gap-filling reaction (by hybridizing a gap oligonucleotide and / or by extending the 3' end), and ligating the hybridized padlock probes, thereby generating a first circular nucleic acid product from the first padlock probe hybridized to the first padlock binding site, a second circular nucleic acid product from the second padlock probe hybridized to the second padlock binding site, and a third circular nucleic acid product and optionally a fourth circular nucleic acid product from the first padlock probe hybridized to the second padlock binding site and / or the second padlock probe hybridized to the first padlock binding site, wherein the first circular nucleic acid product, the second circular nucleic acid product, the third circular nucleic acid product and optionally the fourth circular nucleic acid product are distinguishable from each other by sequence (e.g., they may each comprise a unique combination of an identifier sequence and an optional gap-filling sequence);

[0089] (iii) amplifying the first circular nucleic acid product, the second circular nucleic acid product, the third circular nucleic acid product and optionally the fourth circular nucleic acid product by rolling circle amplification (RCA) to produce a first RCA product (RCP), a second RCA product, a third RCA product and optionally a fourth RCA product; and

[0090] (iv) detecting a first RCP, a second RCP, a third RCP and an optional fourth RCP (e.g., by detecting a unique combination of their identifiers and an optional gap-filling sequence), wherein the first RCP indicates a first target molecule, the second RCP indicates a second target molecule, and the third RCP and the optional fourth RCP indicate an interaction between the two target molecules.

[0091] In these various embodiments, the RCP can be detected by detecting its tag sequence (eg, unique identifier sequence) or a combination thereof.

[0092] In one embodiment, the relative levels of the first RCP, the second RCP, and the third RCP, and optionally the fourth RCP, respectively indicate the relative levels of the two target molecules and the proportion of each of the two target molecules that interact.

[0093] In one embodiment, the target molecule is a protein. In another embodiment, the first target molecule is a protein and the second target molecule is a modification group on the protein (eg, a post-translational modification (PTM)).

[0094] In a second aspect, the present invention provides a kit for detecting two target molecules in a sample and detecting the interaction between the two target molecules, the kit comprising:

[0095] (i) a first probe or a first proximity probe pair for detecting a first target molecule, and a second probe or a second proximity probe pair for detecting a second target molecule, wherein the probes each comprise a binding domain capable of binding directly or indirectly to their target molecules and a nucleic acid domain, and wherein the first probe and the second probe or one of the probes of the first proximity probe pair and one of the probes of the second proximity probe pair together form a third proximity probe pair for detecting an interaction between the first target molecule and the second target molecule; and

[0096] (ii) a first nucleic acid agent and a second nucleic acid agent, wherein the first agent is capable of hybridizing to the nucleic acid domain of the first probe or one of the probes of a first proximity probe pair, and the second agent is capable of hybridizing to the nucleic acid domain of the second probe or one of the probes of a second proximity probe pair, optionally wherein the first agent and / or the second agent is also capable of hybridizing to the nucleic acid domain of the other member of a third proximity probe pair; and

[0097] (iii) an optional third nucleic acid agent capable of hybridizing to the nucleic acid domain of a third proximity probe pair.

[0098] In one embodiment, the nucleic acid agent is a substrate molecule capable of producing a distinguishable nucleic acid product.

[0099] Specific description

[0100] The present method provides a method for detecting two target molecules in a sample and detecting the interaction between the two target molecules. As further defined and described below, more precisely, the method detects the proximity between the two target molecules, and the proximity can indicate that the molecules are interacting. The method can independently detect the two target molecules and detect their proximal positions (e.g., the complex of the two target molecules) simultaneously. Therefore, the method can be regarded as a method for analyzing the interaction.

[0101] In order to detect at least the interaction between two target molecules, the method relies on the principle of "proximity probing", in which each target molecule is bound by a probe. The interaction of the two target molecules brings the probes into proximity, thereby allowing an interaction between the two probes, which results in the generation of a signal. Proximity probing can also be used to detect independent target molecules.

[0102] Effectively, proximity probe or proximity probe pair (each specific to one of two target molecules that may interact) is used in combination with additional auxiliary reagent, and the auxiliary reagent interacts with the nucleic acid structure domain of proximity probe to produce signal, and the signal not only indicates interaction, but also indicates each member of interaction.By using additional reagent, identical or common proximity probe or proximity right member can be used for producing different signals for two target molecules and their interaction.This allows to carry out three separate determinations (that is, three separate determination reactions as described above) in the case of the same determination, and also allows to disclose these determinations simultaneously in a manner that is easy to reuse.In a specific embodiment, the same probe independently used for detecting independent target molecules individually is also used together as the proximity probe pair for detecting target interaction.Therefore, a kind of specific probe can be used for both detecting target molecules independently, and can also be used in combination with another kind of probe for detecting other target molecules independently, to detect target interaction.In other words, in such an embodiment, a kind of probe is used for the first determination reaction and the third determination reaction, and another kind of probe is used for the second determination reaction and the third determination reaction.Therefore, proximity probe pairs can be provided, and its independent probe member is shared or shared between the determination reaction and the interaction for detecting target molecules independently.

[0103] Proximity assays can be performed in homogeneous (ie, in solution) or in solid phase format, and the present methods can be performed with any format of proximity assays using padlock probes, including in solution and solid phase or hybrid formats.

[0104] The two target molecules in the sample are different molecules (i.e., molecules of different types). That is, the method is not suitable for detecting homologous interactions. The target molecule can be any type of biomolecule, and the two target molecules can be different types of biomolecules. Therefore, the target molecule can be, for example, a protein or a nucleic acid molecule, such as a DNA molecule or an RNA molecule.

[0105] In the case of nucleic acid molecules, particularly DNA molecules, the target molecule can be a specific nucleotide sequence in a much larger molecule, for example it can be a specific DNA sequence in a genome or chromosome. When one of the target molecules is a DNA molecule, it can be a natural or synthetic DNA molecule. The target DNA molecule (or target DNA sequence) can be coding or non-coding DNA, for example it can be genomic DNA, or can be derived from genomic DNA, for example it can be a copy or amplicon thereof, or it can be cDNA or an amplicon or copy thereof, etc.

[0106] When one of the target molecules is an RNA molecule, it can be an RNA molecule in a pool of RNA or other nucleic acid molecules (e.g., genomic nucleic acid), whether human or from any other source, from a transcriptome, or any other nucleic acid (e.g., organelle nucleic acid, i.e., mitochondrial or plastid nucleic acid or viral nucleic acid), whether naturally occurring or synthetic. Therefore, the target RNA molecule can be or can be from a coding RNA sequence (i.e., precursor mRNA or mRNA) or a non-coding RNA sequence (such as tRNA, rRNA, snoRNA, miRNA, siRNA, snRNA, exRNA, piRNA, and long ncRNA). Alternatively, the target RNA molecule can be a genomic RNA, such as ssRNA or dsRNA of a virus with RNA as its genetic material. It is noteworthy that such viruses include Ebola, HIV, SARS, SARS-CoV2, influenza, hepatitis C, West Nile fever, poliomyelitis, and measles. Therefore, the target RNA molecule can be a sense RNA, an antisense RNA, or a double-stranded RNA from a viral genome, or a sense RNA from a retroviral RNA genome. When the target molecule is an RNA molecule, the method may include a preliminary step of generating a cDNA copy of the target RNA molecule.

[0107] Most commonly, the target molecules are proteins. "Protein" herein refers to any amino acid-based biomolecule, and thus the target protein can be any polypeptide, oligopeptide or peptide comprising amino acids sufficient to be recognized by the probe used according to the present invention. The protein can be a natural (wild-type) protein from humans or other organisms, or a synthetic protein (e.g., a fusion protein), a protein fragment, or a modified protein or a mutant protein.

[0108] Therefore, the method is most commonly used to detect protein-protein interactions between two proteins and between them. However, in other embodiments, the method can be used to detect interactions between a protein and another type of molecule (e.g., nucleic acids), such as protein-DNA or protein-RNA interactions.

[0109] In another embodiment, the two target molecules are a protein and a post-translational modification (PTM) of the protein. In this case, the protein itself (modified and unmodified) is detected, and the post-translational modified version of the protein is detected separately, so that the proportion of the protein modified post-translationally in the manner of interest can be quantified. Any post-translational protein modification, such as alkylation (such as methylation), acetylation, glycosylation, phosphorylation, lipidation, ubiquitination, etc. can be detected according to the method of the present invention.

[0110] Therefore, more generally, the two target molecules can be a protein and a modifying group on a protein, or in other words, between a protein and a non-protein (non-amino acid) chemical group. Such a modifying group can be any group that can be recognized by a binding molecule (i.e., a molecule that is used as or forms the binding domain of a proximity probe).

[0111] Similarly, the two target molecules can be nucleic acids (eg, RNA or DNA) or any biomolecules, and modification groups on the nucleic acids or other biomolecules.

[0112] For example, the first target molecule may be a protein, DNA or RNA, and the second target molecule may be a lipid, carbohydrate, phosphate, alkyl, acetyl, etc., which may be recognized by the binding agent without limitation.

[0113] The interaction between two target molecules is the interaction of two target molecules coming together. Most generally, this includes the proximal position of two molecules together. The interaction includes the combination or physical connection between the two molecules. Generally, the interaction is a direct interaction, whereby the two molecules bind to each other, but the interaction can also be an indirect interaction, whereby the two target molecules are linked together by a connecting molecule. In any case, in order to detect the interaction, the two target molecules must be sufficiently close to each other so that the proximity probes that are bonded to them can interact. The interaction can be a covalent interaction, but is generally a non-covalent interaction.

[0114] Although traditionally interaction represents direct or indirect physical interaction between two molecules, it will be understood that the close proximity of two molecules can be simply detected using the proximity assay of proximity probes, even if they do not compound or physically interact with each other. Therefore, the term "interaction" used herein includes the physical proximity between two target molecules, so that when two molecules are adjacent, proximity probes can be used to detect. It is not required that there is a combination or physical connection or connection between the two molecules. It is enough for two target molecules to be positioned adjacent to each other. Alternatively, in all cases of mentioning "interaction", it can be replaced by mentioning the interaction or proximity between two target molecules. As mentioned above, proximity herein refers to two target molecules close enough to each other so that they can be detected by proximity assay. For example, this may actually mean that they are located or positioned to be no more than 100nm, 90nm, 80nm or more particularly 70nm away from each other, such as 10nm to 80nm, 20nm to 80nm, 20nm to 70nm, 30nm to 70nm, 40nm to 70nm, etc.

[0115] The sample for carrying out the method of the present invention can be any sample containing the target molecule. It can be a biological sample, such as a research sample or a clinical sample. Therefore, the method of the present invention can be used as a research tool or a diagnostic tool. The sample can be any type of biological sample, such as a cell or tissue sample, a fluid sample, a cell lysate, etc. The sample can contain any virus or cell material, including all prokaryotic or eukaryotic cells, viruses, bacteriophages, mycoplasmas, protoplasts, and organelles. Therefore, this biological material can include all types of mammalian and non-mammalian cells, plant cells, algae (including blue-green algae), fungi, bacteria, protozoa, etc., or viruses. The cell can be, for example, a human cell, a bird cell, a reptile cell, etc., without limitation.

[0116] Thus, representative samples include whole blood and blood-derived products such as plasma, serum and buffy coat, blood cells, urine, stool, cerebrospinal fluid or any other body fluid (e.g., respiratory secretions, saliva, breast milk, etc.), tissue, biopsy, cell culture, cell suspension, conditioned medium or other samples of cell culture components, etc. The method is particularly suitable for the analysis of histological and tissue and cell samples.

[0117] The sample can be freshly prepared, or can be pre-treated in any convenient or desired manner to prepare for use in the method, such as by cell lysis or purification, nucleic acid separation, etc. Therefore, the sample can be processed as required, for example, cells or tissues can be fixed or permeabilized as required according to the target molecule and other relevant factors. Therefore, fresh, frozen or fixed cells or tissues can be used, such as FFPE tissues (formalin fixed paraffin embedded). This method is particularly advantageous because it can detect target molecules and interactions therebetween at very low concentrations.

[0118] In one embodiment, sample and the first neighboring probe for detecting the first target molecule and the second neighboring probe for detecting the second target molecule are contacted.In another embodiment, sample and the first neighboring probe for detecting the first target molecule and the second neighboring probe for detecting the second target molecule are contacted.Single neighboring probe for detecting the first target molecule can be used in combination with the neighboring probe for detecting the second target molecule, but usually two kinds of target molecules will be contacted with single neighboring probe or neighboring probe.

[0119] Probes each comprise a binding domain and a nucleic acid domain. The binding domain can be directly or indirectly bound to a target molecule. In one embodiment, the binding domain of each probe specifically binds to the target molecule that it is designed to detect. In other words, it is a primary binding partner for the target molecule. In another embodiment, it indirectly binds to an intermediate binding partner that is bound to the target molecule itself, such as Figure 6In this case, the binding domain is a secondary binding partner for the target molecule which is able to bind specifically to a primary binding partner which is itself bound to the target molecule.

[0120] The nature of the binding domain depends on the type of target molecule it is designed to detect. The binding domain can be any affinity binding partner for the target molecule, i.e., any entity capable of specifically binding to the target molecule. When the target molecule is a protein, the binding domain is generally an antibody or antigen binding fragment or derivative thereof specific for the protein of interest. Examples of suitable antibody fragments and derivatives include Fab, Fab', F(ab'), 2 and scFv molecules.

[0121] The Fab fragment consists of the antigen-binding domain of the antibody. It can be observed that a single antibody contains two Fab fragments, each of which consists of a light chain and the N-terminal portion of the heavy chain to which it is attached. Therefore, the Fab fragment contains the entire light chain and the V-terminal portion of the heavy chain to which it is attached. H and C H 1 domain. Fab fragments can be obtained by digesting antibodies with papain.

[0122] F(ab') 2 The fragment consists of two Fab fragments of an antibody plus the hinge region of the heavy chain domain (including the disulfide bonds that link the two heavy chains together). In other words, F(ab') 2 The fragment can be viewed as two covalently linked Fab fragments. F(ab') 2 Fragments can be obtained by digesting antibodies with pepsin. F(ab') 2 Reduction of the fragments produces two Fab' fragments, which can be considered as Fab fragments containing additional sulfhydryl groups that can be used for conjugation of the fragments to other molecules. ScFv molecules are synthetic constructs produced by fusing together the variable domains of the antibody light and heavy chains. Typically, this fusion is achieved recombinantly by engineering antibody genes to produce a fusion protein comprising a heavy chain variable domain and a light chain variable domain.

[0123] While binding domains based on the antigen binding site of antibodies are typically used in proximity probes, the use of other binding domains is not excluded, including, for example, those based on receptor-ligand pairs or other binding pairs or aptamers and the like.

[0124] As is known in the art, when proximity probe pairs are used to detect a single protein, the two binding domains bind at different sites, for example the two antibodies (or antibody derivatives) in the pair bind to the protein at different epitopes.

[0125] When the target molecule is a nucleic acid molecule, the binding domain is typically also a nucleic acid molecule. In this case, the binding domain nucleic acid molecule is at least partially single-stranded, and the single-stranded region of the binding domain is or comprises a region complementary to the target molecule, such that the single-stranded region of the binding domain specifically hybridizes with the target molecule. When the binding domain is a nucleic acid molecule, it can be any nucleic acid, including a DNA or RNA molecule or any derivative thereof, but is typically DNA.

[0126] The nucleic acid structure domain of the vicinity probe can be similarly any nucleic acid molecule, but it will generally be a DNA domain. The nucleic acid structure domain is at least partially single-stranded. That is, the nucleic acid structure domain is either single-stranded or comprises at least one single-stranded region. The single-stranded region includes a binding site or hybridization site for nucleic acid reagents, etc., or in other words, the single-stranded region includes a region complementary to another nucleic acid molecule, and they are designed to hybridize or combine with this another nucleic acid molecule. When the binding domain of the probe is an antibody or other non-nucleic acid molecule, the binding domain is conjugated to the nucleic acid structure domain. When the binding domain is a nucleic acid, the probe can be a single nucleic acid comprising a binding domain at one end and a nucleic acid structure domain at the other end, which is optionally linked by a joint nucleic acid sequence. Alternatively, both binding domain nucleic acid and nucleic acid structure domain can be conjugated to a core group that is not a nucleic acid molecule.

[0127] The nucleic acid structure domain of each neighboring probe is or comprises a unique nucleotide sequence.Usually, each nucleic acid structure domain comprises at least one unique nucleotide sequence, or a nucleic acid sequence, by which detection or differentiation can be performed. "Unique" refers to that other neighboring probes used in the method do not carry the same nucleotide sequence in their nucleic acid structure domain.In an alternative, the nucleic acid structure domain can include a tag sequence, by which it can be distinguished from the nucleic acid structure domain of another neighboring probe.The tag sequence can be used to detect the nucleic acid structure domain of the neighboring probe, and therefore detects the target molecule that the neighboring probe combines.In addition, the tag sequence can provide a binding site for the nucleic acid reagent used in the assay reaction.

[0128] The nucleic acid in the proximity probe can be arranged in any orientation. For example, when the binding domain is conjugated to a single-stranded nucleic acid, the binding domain can be conjugated to the 3' or 5' end of the nucleic acid. Similarly, in other probe designs, the nucleic acid domain can be oriented in any direction in appropriate circumstances.

[0129] The first neighboring probe and the second neighboring probe, or the first neighboring probe in the case of using neighboring probe right neighboring probe and the second neighboring probe in the case of detecting another target molecule, a neighboring probe formed additional neighboring probe pairs.If a single neighboring probe is used to detect a kind of target molecule in the target molecule, and use neighboring probe to detect another kind of target molecule, then a neighboring probe formed additional neighboring probe pairs by a single neighboring probe and a neighboring probe pair.When using a single neighboring probe to detect every kind of independent target molecule, this additional probe is to the only probe pair used in the method.When using a single neighboring probe to detect a target molecule and using neighboring probe to detect another target molecule, additional probe is to the second probe pair used in the method.When using the first neighboring probe and the second neighboring probe to detect the first target molecule and the second target molecule, additional probe is to the 3rd probe pair used in the method.

[0130] Nevertheless, for simplicity, for all reaction settings, including those embodiments in which a single proximity probe is used to independently detect a first target molecule and a second target molecule, the probe pair formed between the probes that recognize the first target molecule and the second target molecule is referred to herein as a third probe pair. In fact, the single proximity probes in the above discussion are single probes because they are not used as proximity probes when used individually.

[0131] In the most general sense, the method includes carrying out the first determination reaction and the second determination reaction, to detect the first target molecule and the second target molecule by using the nucleic acid structure domain of any known or convenient method detection probe. The third determination reaction detects the interaction between two target molecules. In the third determination reaction, the interaction of the nucleic acid structure domain of the third neighboring probe pair produces a nucleic acid product, and detects the nucleic acid product to detect the interaction. The nucleic acid product is conveniently referred to as the third nucleic acid product (because the nucleic acid product occurs due to the third neighboring probe pair). In some embodiments, the first determination reaction and the second determination reaction can also cause the generation of the first nucleic acid product and the second nucleic acid product respectively.

[0132] Since the first determination reaction and the second determination reaction can include a simple detection of the nucleic acid domain of the probe, this can be carried out using any convenient or desired method for detecting nucleic acid. In this regard, as described above, the first probe and the second probe for detecting the first target molecule and the second target molecule include a unique sequence or a label sequence, which can be detected in order to detect the domain. Therefore, a detection probe specifically combined with a unique / label sequence can be used, and various such probes for detecting specific nucleic acid sequences are known in the art. These probes include simple hybridization probes that can be directly labeled with a detectable marker, or molecular beacons, etc. Alternatively, the unique or label sequence of a probe can be detected by various sequencing methods.

[0133] More complex labeling systems based on hybridization probes are also known and widely used in the art, such as "sandwich-type" systems, in which secondary or additional detection probes are used that hybridize to multiple binding sites on a primary detection probe to amplify the number of labeling sites, etc. In addition, nucleic acid detection assays are known, which involve nucleic acid reactions, such as extension, connection or cutting to produce a nucleic acid product produced. Such products can be produced by the nucleic acid domain itself, for example, they can be cut to produce a product that is detected or connected (to each other or to another nucleic acid) or extended by a polymerase-catalyzed extension reaction, or they can be used to produce a product from another nucleic acid molecule that interacts with one or more nucleic acid domains, such as a primer hybridized to a nucleic acid domain, which can be extended by a polymerase-catalyzed reaction, or one or more oligonucleotides connected using a nucleic acid domain as a connection template. In addition, oligonucleotides can hybridize with nucleic acid domains to produce a cleavage site (e.g., for a restriction enzyme, etc.).

[0134] As mentioned above, proximity assays are well known and used in the art to detect molecular interactions as well as independent target molecules; in the latter case, the identification of independent target molecules relies on the dual identification of the target by each of the two components of the proximity probe pair. The nucleic acid domains of the proximity probe pair can interact directly with each other (e.g., they can directly hybridize with each other and can, for example, use another domain as an extension template for extension), or they can be connected together when both are hybridized with a common connection template. They can also interact indirectly, such as when both interact with one or more other common nucleic acid molecules, such as two nucleic acid domains can template-connect added oligonucleotides, such as, for example, padlock probes (e.g., see Figure 5 , Figure 6 , Figure 7 and Figure 8 ), or one nucleic acid domain may carry a padlock probe that targets another nucleic acid domain (which serves as a ligation template), such that a proximity event is required to detect a target molecule or interaction via the padlock probe (see e.g. Figure 1 The padlock C in Figure 2 , Figure 3 , Figure 4 or Fig.13 ).

[0135] Therefore, in general, the detection reaction of the interaction between the nucleic acid structure territory of the detection probe or the nucleic acid structure territory of the detection proximity probe relates to another nucleic acid molecule (for example, oligonucleotide) that uses and one or more nucleic acid structure territory interacts or hybridizes.Therefore, the detection reaction, or more generally the first determination reaction, the second determination reaction and the third determination reaction, can relate to the use of the nucleic acid reagent that hybridizes with one or more nucleic acid structure territory of the probe.

[0136] As mentioned above, nucleic acid reagent can be a simple detection probe, and the hybridization of the nucleic acid structure domain with the first probe or the second probe can be detected, so as to detect the first target molecule or the second target molecule independently. However, more complicated signal generation systems based on nucleic acid known in the art are used to enhance signal and assist detection, which can improve the sensitivity of the method. In such a system, other nucleic acid reagents (for example, oligonucleotides) are used to build or produce a nucleic acid product that can be detected. Any such signal amplification system based on DNA can be used. These methods include methods based on amplification or polymerase chain extension reaction and / or ligation reaction and methods wherein using the hybridization of oligonucleotides to build large nucleic acid products. These include hybridization chain reaction (HCR) and branched DNA amplification reaction, in the case of hybridization chain reaction, short single-stranded oligonucleotides (HCR monomers) are hybridized together to build long double-stranded nucleic acid chains with gaps, in the case of branched DNA amplification reaction, the sequence of intermediate hybridization probes is used, and intermediate hybridization probes are hybridized to provide multiple hybridization sites for other probes, so that branched nucleic acid structures are built, thereby multiple binding sites for multiple labeled detection probes are finally provided.

[0137] Thus, a template or scaffold molecule can be provided that hybridizes with its target nucleic acid sequence in the nucleic acid domain of the probe (e.g., hybridizes with a tag or unique sequence in the domain) and contains multiple binding sites for additional hybridization probes. These templates or scaffold molecules can include detection sequences to which labeled detection probes can bind, or they can include binding sites for additional hybridization probes, etc. Thus, a "layered" or "branched" structure composed of multiple hybridization probes can be formed. A hybridization probe or a subset thereof (e.g., the last or final hybridization probe added to the structure) can include a detection sequence. In such an embodiment, the nucleic acid product can be referred to as a hybridization assembly. This is, for example, described in WO2011 / 094669 RNAscope TM Although RNAscope TM It was developed for in situ hybridization for RNA detection, but it illustrates the principle of using sandwich or intermediate hybridization probes that each provide multiple binding sites for labeled detection probes to produce a detectable nucleic acid product containing multiple labels.

[0138] This amplification method for producing nucleic acid products can be applicable to the assay reaction based on proximity.For example, in WO2015 / 118029, a contiguous HCR method is described, which describes a system, wherein the interaction between the nucleic acid structure domain of the contiguous probe opens the secondary structure in the nucleic acid structure domain to reveal the initiator of HCR reaction. A separate external activator molecule can be used, which is combined with the nucleic acid structure domain to open the secondary structure (for example, hairpin), and the secondary structure opens the nucleic acid structure domain of another contiguous probe to reveal the HCR initiator. Then, the HCR initiator opens the hairpin of the first HCR monomer, to trigger the HCR reaction between two groups of HCR monomers. HCR monomers can be marked, or the detection site for the detection probe for binding labeling can be provided for HCR monomers, to allow detection of HCR products.

[0139] Similarly, as is known in the context of branched DNA amplification techniques, proximity binding can be used for binding of a primary scaffold molecule upon which a branched DNA structure is constructed. Thus, when the nucleic acid domains of the proximity probes are brought together and adjacent by binding of the proximity probe pair to the first and second target molecules, the nucleic acid domains of the proximity probes allow for binding of a scaffold oligonucleotide for a branched DNA amplification reaction.

[0140] Thus, as can be seen above, in one embodiment, a nucleic acid agent may be a substrate from which or using which a nucleic acid product is produced.

[0141] As mentioned above, separate substrate can be used for each assay reaction in three assay reactions, but this is not necessary, and one or two substrates used for the first assay reaction and the second assay reaction can also be used for the third assay reaction. In this respect, it should be understood that in this method, substrates will be used in different ways to produce different nucleic acid products.

[0142] In the method, probe can contact with at least the first nucleic acid substrate molecule and the second nucleic acid substrate molecule. The contact step can be carried out before, during or after the probe is applied to the sample as required or preferably depending on the specific setting for the method. The first substrate molecule hybridizes with the first nucleic acid structure domain of the probe or the first nucleic acid structure domain of one of the probes adjacent to the right probe, and the second substrate molecule hybridizes with the second nucleic acid structure domain of the probe or the second nucleic acid structure domain of one of the probes adjacent to the right probe.

[0143] More specifically, the first nucleic acid substrate and the first nucleic acid structure domain of the first neighboring probe or the first neighboring probe in the nucleic acid structure domain of one of the probes are specifically hybridized.As described above, each nucleic acid structure domain includes at least one sequence that can be distinguished by it in the nucleic acid structure domain, such as at least one unique nucleotide sequence or at least one label sequence.The first nucleic acid substrate includes one or more homologous sequences, which are complementary or complementary together with the first neighboring probe in the nucleic acid structure domain or the first neighboring probe in the nucleic acid structure domain of one of the distinguishable or unique sequences.Equivalently, one or more distinguishable or unique sequences in the second nucleic acid substrate are complementary and specifically hybridized with the second neighboring probe in the nucleic acid structure domain or the second neighboring probe in the nucleic acid structure domain of one of the distinguishable or unique sequences.In other words, substrate includes binding site, which is complementary to the homologous substrate binding site in the nucleic acid structure domain and can hybridize with it.

[0144] In a specific embodiment, the first nucleic acid substrate and the second nucleic acid substrate are padlock probes (i.e., the first padlock probe and the second padlock probe, respectively). The operation of the method using the padlock probe will be described in more detail below. However, it will be appreciated by the skilled person that the padlock probe can be replaced by other types of substrates, such as components of HCR reactions or branched DNA amplification reactions as described above.

[0145] The selection of substrate or detection mode can depend on the properties of target molecule and sample, etc. For example, for the in situ detection method that needs to detect the target molecule locally, it may be necessary to produce nucleic acid products, such as RCP. Therefore, padlock probes and RCA-based detection are convenient under this setting. However, other detection methods can also be used according to selection. These methods can include, for example, PCR-based methods. For non-in situ detection, it may be convenient to detect the target molecule (or more specifically the nucleic acid domain and / or one or more nucleic acid products of the detection probe) by sequencing or PCR-based methods (e.g., qPCR). However, the properties of this detection and the nucleic acid products thus produced, or the detection probes used, etc. are not limited. For example, nucleotide detection methods can be used, such as STROM (Rust et al., Nature Methods 2006, 3 (10), 793-795) or DNAPaint (Schnitzbauer et al., 2017, Nature Protocols, 12, 1198-1228).

[0146] As mentioned above, padlock probes can be defined as circularizable probes. The use of padlocks or circularizable probes is well known in the art, including in the case of RCA reactions. Circularizable probes include one or more linear oligonucleotides that can be connected together to form a ring. Therefore, the principle of padlock detection is well known, and the design and use of padlock probes are known and described in the art. Padlock probes are typically linear circularizable oligonucleotides, which are connected together with their target nucleic acid sequence or molecular hybridization by juxtaposing the 5' and 3' connectable ends of the probe to directly or indirectly (in this case, when the probe is hybridized with its target sequence, the gap is located between the connectable ends of the probe). By connecting the 5' and 3' ends of the probe hybridization, the probe is circular. It should be understood that in order to carry out circularization (connection), the connectable 5' end of the padlock probe has a free 5' phosphate group. In the case of the presence of a gap, it is filled by a gap oligonucleotide (which can be regarded as a part of a padlock probe) or by an extension of the hybridization 3' end of the padlock probe.

[0147] In order to allow the ends of the padlock probe to be juxtaposed for ligation, the padlock probe is designed to have target binding sites at its 5' and 3' ends. That is, the complementary regions that allow the padlock probe to bind to its target are located at the ends of the padlock probe. The region of the padlock probe that connects to the target complementary ends or is located between the target complementary ends and cannot hybridize to its target molecule is generally referred to as the backbone region.

[0148] To allow ligation, the 3' and 5' ends to be ligated (the "ligatable" 3' and 5' ends) are hybridized to a target sequence that serves as a template for ligation. Herein, the target sequence for a padlock probe (i.e., the region of the target molecule that is complementary to the target binding region of the padlock probe) is generally referred to as a padlock binding site. The term "hybridization sequence" in relation to a padlock probe more generally includes sequences that are capable of hybridizing to the padlock probe at any site in the padlock probe (and this includes hybridizing to complementary sequences in the backbone region as well as the target binding region of the padlock probe).

[0149] The combination of the padlock probe and its target sequence places its end in the juxtaposition position. When the complementary binding sites in the target molecule or sequence are directly adjacent to each other (or adjacent), the ends of the padlock probe will be directly adjacent to each other (i.e., there is no gap) and can be directly connected to each other. Therefore, in this case, the connectable end of the probe is provided by the actual end of the probe. In the case of gap filling padlock probes, the target binding region at the end of the padlock probe does not hybridize with the adjacent binding site, but hybridizes with the non-adjacent (non-adjacent) binding site in the target molecule. In this arrangement, the 5' connectable end of the probe is provided by the actual 5' end of the probe. However, the connectable 3' end of the probe can be generated by extending the hybridization 3' end of the probe using the target sequence as an extension template to fill the gap between the hybridization ends of the probe. The extension reaction juxtaposes the extended 3' ends of the probe for connection. In this case, the connectable 3' end of the probe is therefore the extended 3' end of the probe. In another embodiment, the juxtaposed connectable 5' and 3' ends are provided by gap oligonucleotides, which hybridize between the two ends of the padlock probe and connect to form a ring. In this case, there are two ligation junctions. This can be viewed as a two-part padlock probe format. In another configuration, a two-part padlock probe can have two target sequences, each of which serves as a ligation template for the probe, wherein the 5' end of one part (the first circularizable oligonucleotide) is juxtaposedly hybridized to ligate to the 3' end of the other part (the second circularizable oligonucleotide), and the two parts are ligated together to form a ring (see, for example, Figure 1 B. Figure 1 C. Figure 5 , Figure 6 , Figure 7 and Figure 8 ).

[0150] The term "hybridization" as used herein generally refers to the formation of a duplex between nucleotide sequences that are sufficiently complementary to form a duplex by Watson-Crick base pairing or any similar base pair interaction. When the molecules of two nucleotide sequences have the same base pair structural homology, the two nucleotide sequences are "complementary". Therefore, the complementary region in a molecule or probe or sequence refers to the portion of the molecule or probe or sequence that can form a duplex. Hybridization does not require 100% complementarity between sequences, so the regions that are complementary to each other do not require complete sequence complementarity, but this is not excluded. Therefore, the complementary region may contain one or more mismatches. Therefore, "complementary" as used herein refers to "functional complementarity", that is, a level of complementarity sufficient to mediate productive hybridization, which covers a degree of complementarity less than 100%. The degree of mismatch tolerated can be controlled by appropriately adjusting the hybridization conditions. Following the guidance provided in the art, a technician in the field of nucleic acid technology can determine the stability of the duplex based on experience considering many variables, including, for example, the length and base pair composition of the corresponding molecule or probe oligonucleotide, ionic strength, and the incidence of mismatched base pairs. Therefore, the design of suitable probes and their binding regions, as well as the conditions under which they hybridize to their respective targets are well within the routine skills of those skilled in the art.

[0151] The region complementary to the target sequence in the padlock probe binding region, for example, or to the target sequence between the detection sequence and the detection oligonucleotide, or to the RCA primer of the circular padlock probe, etc. can be at least 6 nucleotides long to ensure the specificity of binding, or more particularly at least 7, 8, 9 or 10 nucleotides long. The upper limit of the length of this region is not critical, but can be, for example, up to 50, 40, 35, 30, 25, 20 or 15 nucleotides. Therefore, the length of the complementary region can be in the range between any one of the above-mentioned lower length limit and the upper length limit. In the case of padlock probes, the length of a single target binding region can be in a lower range, so when hybridizing with their targets, the total length of the two binding regions is in a higher range. For example, a single target binding region can be 8 to 15, for example 10 to 12 nucleotides, so that the total hybridization length is 16 to 30 nucleotides, for example 20-24 nucleotides long. Under the constraints of probe conformation, spacing of domains and desired or preferred hybridisation, it may be desirable to minimise the overall length of the padlock probe to minimise the size of the loop subject to RCA and therefore minimise the length of the complementary region where possible.

[0152] In the embodiment that the first nucleic acid substrate and the second nucleic acid substrate are padlock probes, the nucleic acid domain (" first nucleic acid domain ") of one of the first adjacent probe or the first adjacent probe pair includes a padlock binding site complementary to the target binding sequence at 5' and 3' ends of the first padlock probe. Equivalently, the nucleic acid domain (" second nucleic acid domain ") of one of the second adjacent probe or the second adjacent probe pair includes a padlock binding site complementary to the target binding sequence at 5' and 3' ends of the second padlock probe. The term " padlock binding site " used herein simply refers to the sequence in the nucleic acid of the padlock probe hybridization in the method. In particular, the term refers to the sequence that the padlock probe that constitutes the first nucleic acid substrate molecule or the second nucleic acid substrate molecule combines.

[0153] Once the probe has been contacted with the first and second nucleic acid substrate molecules and the sample (in any order in which these steps are performed), two assay reactions are performed to detect the first and second target molecules. The first target molecule is detected by producing a first nucleic acid product from the first nucleic acid substrate, and the second target molecule is detected by producing a second nucleic acid product from the second nucleic acid substrate. The first and second nucleic acid products are detected (as further discussed below) to detect the first and second target molecules. The first and second assays for detecting the first and second target molecules are typically performed simultaneously, but may also be performed sequentially depending on the specific configuration of the method used.

[0154] When the first nucleic acid substrate and the second nucleic acid substrate are padlock probes, the assay comprises circularization and (generally but not necessarily) amplification of the padlock probes. As described above, when a padlock probe hybridizes to a padlock binding site in its target nucleic acid domain, its target binding region (i.e., its 3' and 5' ends that hybridize to the nucleic acid domain) may bind to the padlock binding site either directly adjacent to each other or with a gap therebetween, which is filled prior to ligation into a circular shape.

[0155] As noted above, other types of nucleic acid substrates (other than padlock probes) may be used if desired, in which case the first and second assay reactions selected are appropriate to the type of nucleic acid substrate used.

[0156] Also use the 3rd neighbouring probe to carry out the 3rd determination reaction to detect the interaction between the first target molecule and the second target molecule.When the first target molecule and the second target molecule interact, the 3rd neighbouring probe right member directly or indirectly interacts.The 3rd determination reaction produces the 3rd nucleic acid product indicating the interaction between the third neighbouring right probe.The detection of the 3rd nucleic acid product corresponds to the interaction detection of the third neighbouring right probe, and therefore corresponds to the interaction detection between the first target molecule and the second target molecule.

[0157] As mentioned above, the interaction between the third neighboring probe right member can be direct.That is to say, the third neighboring probe right member can directly hybridize each other.In this embodiment, the third neighboring probe right probe interaction formation adjacent to the basis of extension assay (PEA).PEA method is well known in the art, and described in, for example, WO 03 / 044231 and WO2004 / 094456.In this case, the end (that is, the end away from the binding domain of the reporter nucleic acid) of the third neighboring probe right is complementary, to allow them to hybridize each other.

[0158] In another embodiment, the 3rd nucleic acid substrate molecule is a probe that is adjacent to the probe of the probe pair. However, generally, the 3rd probe pair interacts indirectly, that is, by one or more intermediate nucleic acid molecules or oligonucleotides with two probe hybridizations. Especially, the 3rd nucleic acid substrate molecule that can contact with two probe hybridizations in the pair is adjacent to the probe. The 3rd nucleic acid product is then produced by the 3rd nucleic acid substrate molecule. In specific embodiments, the 3rd nucleic acid substrate molecule is a padlock probe.

[0159] As mentioned above, the padlock probe used herein can be provided in one or two parts. That is, generally, the padlock probe is provided in a single part, i.e., as a single linear nucleic acid molecule with target binding regions at both ends, so that the two ends of the probe bind to the target sequence adjacent to each other, thereby enabling the probe to cyclize. The first nucleic acid substrate molecule and the second nucleic acid substrate molecule are conveniently one-part padlock probes in many cases, but it will be clear from the more detailed description below that it is not excluded that they can be two-part probes with a gapped oligonucleotide configuration (see, for example, Figure 3 B). However, the padlock probe used in certain embodiments, in particular the padlock probe as the third nucleic acid substrate molecule, can be provided in two parts in other configurations. When the padlock probe is to bind to two separate target nucleic acid molecules, such as two separate nucleic acid domains, the padlock probe can be provided in two parts (see, for example, Figure 1 B and Figure 1 C). As described above, in one such embodiment, two linear nucleic acid molecules (circularizable oligonucleotides) are provided, each of which binds to two target nucleic acid molecules (i.e., both portions of the padlock probe have one end that binds to one target nucleic acid molecule and one end that binds to the other target nucleic acid molecule). The two portions of the padlock probe bind to the two target nucleic acid molecules such that on the two target molecules, the ends of the respective padlock probe portions are adjacent to each other, such that the two portions together have a circular conformation and can be linked to form a circular nucleic acid (directly or indirectly, as described above).

[0160] Therefore, the third nucleic acid substrate can be provided as a padlock probe with two parts.Alternatively, the third nucleic acid substrate can be provided in the form of a traditional part of a padlock probe.However, in this case, a probe pre-hybridized with one of the probes in the third probe pair must be provided so that its end is freed from another probe in the pair for hybridization, thereby linking the probe of the third probe pair in an indirect interactive manner.Therefore, in such an embodiment, the third padlock probe comprises the anchor sequence in the skeleton region between the target binding ends of the padlock.The anchor sequence is a binding site for the complementary binding site in the nucleic acid structure domain of the neighboring probe in the third neighboring probe pair (for example, the first neighboring probe can include a hybridization sequence that can hybridize with the anchor sequence in its nucleic acid structure domain).Therefore, a hybridization sequence that can hybridize with the anchor sequence (or more generally the skeleton sequence) in the padlock probe can be provided in the nucleic acid structure domain of the neighboring probe.Therefore, it will be understood that the anchor sequence is only a sequence that allows the third padlock probe to hybridize with the nucleic acid structure domain of the neighboring probe in a manner that its end is freed for target binding.It can be alternatively referred to as a capture sequence. The third padlock probe may be pre-hybridized with one of the proximity probes of the third proximity probe pair (e.g., the first proximity probe) prior to performing the method (e.g., it may be provided in this way), or it may be pre-hybridized during use, for example by contacting the third padlock probe with the proximity probe prior to contacting it with the sample.

[0161] In another embodiment, the third nucleic acid substrate molecule is the first substrate molecule or the second substrate molecule, that is, a single substrate molecule is used alone to detect the first target molecule or the second target molecule and the interaction between the target molecules. This arrangement can be achieved in a variety of ways.

[0162] For example, a padlock probe pre-hybridized with the first neighboring probe can be provided, and the padlock probe is compounded with the blocking oligonucleotide hybridized with the target binding region of the padlock probe. The target binding region of the padlock probe is hybridized with the nucleic acid structure domain of the second neighboring probe, and its affinity is higher than the affinity of the hybridization of the blocking oligonucleotide. Therefore, when two target molecules interact, the nucleic acid structure domain of the second neighboring probe surpasses and replaces the blocking oligonucleotide, and hybridizes with the padlock probe. The padlock probe hybridizes with one or both of the blocking oligonucleotide and the second nucleic acid structure domain and has a gap between its 3' and 5' ends. Therefore, the gap sequence is present in the blocking oligonucleotide and / or the second nucleic acid structure domain, and it is located between the binding site / hybridization sequence (that is, the complementary binding site on the blocking oligonucleotide or the second nucleic acid structure domain) hybridized with the target binding end of the padlock probe. This gap sequence provides a method by which the obtained cyclization product of the padlock probe can be distinguished. In other words, the gap sequence can provide or include an identifier sequence. For example, it can provide or include a barcode sequence or a tag sequence (or be composed of a barcode sequence or a tag sequence). As described above, the gap can be filled by a gap oligonucleotide complementary to the gap sequence or by gap-filling extension of the hybridized 3' end. After circularization of the padlock probe, the associated gap sequence is bound, allowing the padlock probe bound to the blocking oligonucleotide (corresponding to the non-interacting target molecule) to be distinguished from the padlock probe bound to the second nucleic acid domain (corresponding to the interacting target molecule). This arrangement of the method is as follows Figure 2 shown.

[0163] More generally, blocking oligonucleotides can be used in other forms and configurations of the method to control hybridization and ligation of padlock probes. This can allow for better control of unwanted non-specific background reactions. Thus, the target binding end of the padlock probe used in any embodiment of the method described herein can be protected by a blocking oligonucleotide, which prevents the padlock probe from hybridizing to its intended target until the blocking oligonucleotide is removed. Thus, the padlock probe can be provided or used in the form of a complex with a blocking oligonucleotide. As an example of this form, Figure 1 A or Figure 3 The configuration shown in A can be modified to provide each of padlocks A, B and C with a blocking oligonucleotide hybridized to the target binding end of the padlock. Fig.13 The three padlocks described in can be provided with a blocking oligonucleotide. Removal can be by displacement of the blocking oligonucleotide by the intended target, or by a separately added key oligonucleotide that is able to invade and displace the blocking oligonucleotide.

[0164] In another embodiment, two target molecules are detected using proximity probe pairs. In each probe pair, one probe has a nucleic acid domain with a pre-hybridized padlock probe, and the other probe has a nucleic acid domain comprising a padlock binding site with a gap sequence (the gap sequence may comprise a barcode / tag sequence). Each pair of padlock probes is capable of binding to the nucleic acid domains of both its paired proximity probe and another pair of proximity probes with a gap sequence. When the target molecules do not interact, each pair of padlock probes hybridizes with the nucleic acid domain of the paired proximity probes; when the target molecules interact, the padlock probes interact with the free (i.e., non-padlock-carrying) nucleic acid domains of the proximity probes of another probe pair. As described above, after the padlock probes are cyclized, they are combined with the relevant gap sequences so that the padlock probes bound to the proximity probes (corresponding to the non-interactive target molecules) and the padlock probes bound to the proximity probes (corresponding to the interactive target molecules) of the other pair can be distinguished. This arrangement of the method of the present invention is Figure 3 Shown in B is a diagram illustrating a gap oligonucleotide hybridized to a gap sequence.

[0165] Other configurations using proximity probe pairs to detect independent target molecules are also possible. For example, Figure 3 A shows Figure 1 A variant, wherein the first adjacent probe pair and the second adjacent probe pair are used to detect the first target molecule and the second target molecule in the first determination reaction and the second determination reaction respectively. In this case, one of the adjacent probes of the pair carries a pre-hybridization padlock probe, which has a target binding site (end) specific for the nucleic acid structure domain of another member of the adjacent probe pair. When the first pair or the second pair of two probes have been attached to their target molecule, the padlock can hybridize with its target binding site in the adjacent probe of its pairing, and detects the circular padlock in the first determination reaction and the second determination reaction. In order to detect the interaction between two molecules, the third padlock probe is used in the third determination reaction. This can also be pre-hybridized with the nucleic acid structure domain of the adjacent probe of one of the first pair and the second pair carrying the first or second padlock. The third padlock probe has a target binding site (end) that can be hybridized with the single binding site (hybridization sequence) in the nucleic acid structure domain of another pair of adjacent probes. When the two target molecules are in proximity (due to interaction with each other) and both the first proximity probe and the second proximity probe pair are bound to their respective target molecules, the third padlock probe in one of the first or second pairs is able to hybridize to its target sequence in the other pair and detect the circular padlock in a third assay reaction. This will be described in more detail below.

[0166] Fig.13 The function is similar to Figure 3Configuration of A. This is based on the first proximity probe pair, the second proximity probe pair and the third proximity probe pair for detecting the first target molecule and the second target molecule and the interaction therebetween respectively in the first determination reaction, the second determination reaction and the third determination reaction. However, in this case, the nucleic acid structure domain is designed differently; each nucleic acid structure domain is single-stranded, and in the first pair and the second pair for constituting the third pair of proximity probes, the nucleic acid structure domain is conjugated with the binding domain at an internal site (rather than at the end), so that it can provide two separate binding sites, one binding site for combining the first padlock or the second padlock, and another binding site for combining the third padlock. A padlock pre-hybridized at its skeleton / anchor sequence with the nucleic acid structure domain of the nucleic acid structure domain can be provided, so that its target binding end is free hybridized with the nucleic acid structure domain of another member of the proximity probe pair. In addition, as described above, three padlocks can each be provided with the complex of the blocking oligonucleotides of the free end hybridization with its target. By allowing the hybridization of the corresponding binding sites of the control padlock and their nucleic acid structure domains on the proximity probe, the performance of this configuration can be improved using blocking oligonucleotides. As described above, the blocking oligonucleotide can be removed by displacement of the target sequence of the padlock probe, or by a separately added key oligonucleotide capable of invading and displacing the blocking oligonucleotide.

[0167] Figure 6 Another configuration of using adjacent probe to detect single target molecule is shown.Here, the first adjacent probe and the second adjacent probe are for detecting the secondary reagent of independent target molecule, which is combined with the primary binding partner that itself is specifically and directly combined with target molecule.One of the adjacent probes in a pair of adjacent probes is used to detect and report molecule; The adjacent probe has a nucleic acid structure domain, and the nucleic acid structure domain is detected by a padlock probe specific for the domain (that is, the first padlock probe and the second padlock probe are each respectively specific for the probe from the first adjacent probe to the second adjacent probe pair). Therefore, each of these adjacent probes is used as an independent probe, to use the first padlock probe and the second padlock probe to detect the first target molecule and the second target molecule in the first determination reaction and the second determination reaction respectively.The third determination reaction uses the first adjacent to the second adjacent to another member, and they together constitute the third adjacent probe pair for the third determination reaction. In the configuration shown herein, the third padlock probe is provided as a two-part probe, and one end of each part of the two-part probe is hybridized with one of the nucleic acid domains of the third adjacent probe pair, so that the corresponding ends of the two parts are juxtaposed to connect using the nucleic acid domains of the third adjacent probe pair as two separate connection templates. In this way, three separate products, i.e., circular padlock probes, are produced respectively by the first padlock probe, the second padlock probe, and the third padlock probe. The circular padlock can be amplified by RCA, and as Figure 6As shown, the RCA reaction can be conveniently initiated by the nucleic acid domain of the proximity probe.

[0168] Figure 7 It is shown in Figure 6 Here, the proximity probe pair used to independently detect each of the two target molecules is replaced by a single probe, but in this case, the probe carries two separate nucleic acid domains (i.e., each of which is individually conjugated to a binding domain of the probe (the binding domain is in Figure 7 As shown, each of the two separate nucleic acid domains is single-stranded, but all that is required is that the nucleic acid domain is at least partially single-stranded in the region containing the binding site for the padlock probe. Thus, in this scheme, there are two probes, each carrying two separate nucleic acid domains (a total of four oligonucleotides, corresponding to the number of oligonucleotides that make up Figure 6 4 oligonucleotides of the nucleic acid structure domain of two neighboring probe pairs shown). The first nucleic acid structure domain of the first probe is detected by the first padlock probe to detect the first target molecule (depicted as A). The first nucleic acid structure domain of the second probe is detected by the second padlock probe to detect the second target molecule (depicted as B). Probe (the first probe and the second probe) is also (and individually) used together as a neighboring probe pair (according to the terminology herein as "the third" neighboring probe pair), and in this case, the third padlock probe is used to detect the neighboring interaction between the second nucleic acid structure domain of the first probe and the second nucleic acid structure domain of the second probe, to detect the interaction between the first target molecule and the second target molecule (depicted as AB). The third padlock probe is provided as a two-part probe, and one end of each of its parts is hybridized with one of the second nucleic acid structure domains of the third neighboring probe pair, and the other end of each of its parts is hybridized with another in the second domain, so as to use the third neighboring probe pair second nucleic acid structure domain as two independent connection templates and make the corresponding ends of the two parts of the padlock probe juxtaposed to be connected. In this way, three independent products are produced respectively in the first padlock probe, the second padlock probe and the third padlock probe, that is, circular padlock probe. The ring padlock can be amplified by RCA and Figure 7 As shown, the RCA reaction can be conveniently initiated by the nucleic acid domain of the proximity probe. The use of a single probe with two different nucleic acid domains allows the probe to be used as a primary binding reagent for a target molecule (rather than as Figure 6 secondary reagent as shown).

[0169] In a specific embodiment, the padlock probe used herein does not have a secondary structure, more specifically does not include an intramolecular double-stranded region or a stem-loop structure. However, the dumbbell-type probe that does have a secondary structure is a special subtype of a padlock probe that can be used. The dumbbell-type probe includes two stem-loop structures connected by a stem-stem, in which one "loop" is not closed, but open, with free 5' and 3' ends that can be used to connect to each other. The "open loop" is used as the target binding domain of the probe. The closed loop is only used as a spacer to connect the ends of the duplex (stem). In other words, it can be regarded as a padlock probe, wherein a duplex region is formed between the complementary sequences (regions) of the padlock. The duplex region is used as a signal domain, and an intercalator can be combined with the signal domain. Therefore, the "open loop" of the dumbbell-type probe can include a complementary target binding region.

[0170] In order to perform the padlock probe binding step, the padlock probe is usually incubated with the neighboring probe. As mentioned above, according to the specific setting of the method, this can be performed before the neighboring probe is applied to (i.e., contacted) the sample, so that the padlock probe is pre-hybridized with the neighboring probe, or this can be performed after the neighboring probe is applied to the sample, in which case the padlock probe is also applied to the sample. For gap filling padlock probes, dNTPs and polymerases can also be included. Conveniently, a ligase can also be included in the padlock probe binding step. Reagents can be added in a single reaction mixture, or added separately before the probe binding step or during the probe binding step. In order to allow the probe to bind, there can be an initial heating step, for example, to denature the double-stranded nucleic acid molecules. According to principles and procedures known in the art, reagents are usually provided in a buffer. For example, a buffer suitable for the selected ligase can be selected.

[0171] The reaction mixture can be incubated under conditions suitable for promoting or achieving padlock probe binding (so-called "annealing" step). If there is a previous denaturation step, this may involve a reduction in temperature. The conditions of these steps are known in the art and are selected or designed within the routine skills of those skilled in the art. For example, room temperature or an annealing temperature in the range of 20°C to 40°C, such as 25°C to 40°C or 25°C to 37°C can be used. In one embodiment, a higher temperature, such as 50°C to 65°C, such as 53°C to 60°C, or 55°C to 60°C, can be used. If a higher annealing temperature is selected, the annealing temperature of the extension step can be reduced in the case of using a gap-filling padlock. Similarly, suitable conditions can be selected according to what is known in the art and the specific reagents used, such as enzymes. For example, after the initial annealing step, the temperature can be reduced to 28°C to 40°C, such as 28°C to 35°C, 30°C to 35°C, 28°C to 33°C, 30°C to 33°C or 30°C to 32°C, etc.

[0172] To ligate the padlock probes, any convenient ligase may be used, representative ligases of interest include, but are not limited to, temperature-sensitive ligases such as SplintR ligase (also known as PBCV-1 DNA ligase or Chlorella virus DNA ligase), bacteriophage T4 DNA ligase, bacteriophage T7 ligase, and E. coli ligase, as well as thermostable ligases such as Taq ligase, Tth ligase, Pfu ligase and 9°N TM DNA ligase.

[0173] Suitable ligation conditions are known in the art, and any necessary and / or desired reagents may be combined with the reaction mixture and maintained under conditions sufficient for ligation. It will be apparent that the ligation conditions may depend on the ligase used in the method of the invention. Thus, for example, Ampligase may be used, and the temperature of the ligation step may be increased. Alternatively, SplintR ligase may be used at room temperature.

[0174] Where temperature changes or temperature control steps are required, the method can be performed in a thermal cycler. This allows temperature changes to be controlled at any time. However, one advantage of the method is that no extreme temperature changes are required, for example, the method can be performed at room temperature or, for example, at 20°C to 37°C. The probe binding and ligation steps can, for example, be performed at room temperature.

[0175] The conditions for the probe binding and ligation reactions can be optimized by routine experiments according to principles known in the art. Thus, temperature, buffer, incubation time, heating rate, etc. can be adjusted to find the optimal conditions.

[0176] Therefore, the first nucleic acid product, the second nucleic acid product and the third nucleic acid product can be produced, which correspond to the interaction between the independent first target molecule, the independent second target molecule and the two target molecules respectively. These three nucleic acid products can be distinguished from each other by sequence. That is to say, each of the three products has or comprises different sequences, that is, detection sequences, so that the three nucleic acid products can be distinguished based on their sequences. The term "detection sequence" used herein includes both the detection sequence that appears in the product (for example, in the circular padlock probe) and the complementary copy that appears in its amplified product. The three nucleic acid products can each have completely different sequences, from which a unique detection sequence can be selected. Alternatively, each nucleic acid product can have a shared skeleton, and the difference is only in the identification detection sequence for detecting each product. Therefore, it will be understood that the detection sequence in the product corresponds to the identifier sequence as described above.

[0177] Therefore, the padlock probe can carry an identifier sequence by which it can be detected and identified. This is a sequence that distinguishes it from other padlock probes, i.e., a sequence that is different in different padlock probes. As described above, this can be referred to as a unique identifier sequence. This can be, for example, a tag or barcode sequence. The tag sequence is essentially a recognition sequence, and can therefore be considered to be equivalent to or synonymous with the identifier sequence. The barcode sequence is also a recognition sequence, which can be conveniently detected by decoding the sequence, such as by sequencing, and sequencing can include hybridization sequencing (sequencing by hybridisation) and ligation reaction sequencing (sequencing by ligation reactions), and conventional sequencing reactions, such as sequencing by synthesis (sequencing by synthesis). Therefore, more generally, the barcode sequence can be simply considered to be a sequence that identifies a nucleic acid molecule (e.g., a padlock probe) and can be detected by detecting and identifying its sequence. The padlock probe can carry such an identifier sequence in its backbone region, or the identifier sequence can be located in one or more target binding regions of the padlock probe. In the latter case, the identifier sequence will reflect the sequence in the target of the padlock probe (for example, complementary to the sequence in the target of the padlock probe), that is, the identifier (or label) sequence in the nucleic acid structure domain of the neighboring probe. In addition, the identifier sequence in the probe skeleton can be located in a region / sequence designed to hybridize with the nucleic acid structure domain, for example, hybridized with the complementary padlock hybridization sequence in the nucleic acid structure domain, such as the anchor sequence of the padlock probe. In this way, the identifier sequence can also reflect the identifier sequence present in the nucleic acid structure domain of the neighboring probe. Alternatively or additionally, as described above, the identifier sequence can be incorporated into the circular padlock probe by gap filling reaction. The gap sequence between the hybridization ends of the padlock in the connection template (for example, nucleic acid structure domain or blocking oligonucleotide) that the padlock probe has hybridized with can include or constitute the identifier sequence, and its complementary sequence is incorporated into the circular padlock probe. Therefore, in general, the identifier sequence includes a complementary sequence. In certain embodiments, the padlock probe can include more than one identifier sequence.

[0178] Similarly, the identifier sequence may be incorporated or provided into other nucleic acid substrates used to generate nucleic acid products, for example into primers, or HCR monomers, or oligonucleotides used in the above-mentioned hybridization-based branched DNA methods.

[0179] Furthermore, in the case of a simple detection assay using a detection probe to directly detect the nucleic acid domains of a first probe and a second probe for independently detecting a first target molecule and a second target molecule, the identifier sequence may be present in the nucleic acid domains of the probes and may be targeted by the detection probe.

[0180] The first, second, and third assays are generally performed simultaneously, but sequential performance may be feasible in some embodiments.

[0181] In the case where determination has been performed to produce nucleic acid products, an amplification reaction can be performed to produce an amplification product of each nucleic acid product or to produce an amplification product by each nucleic acid product. Amplification reaction can be performed in any suitable manner, which may depend on the property of nucleic acid products. In some cases, nucleic acid products themselves are the result of signal amplification reaction, such as HCR or branched chain amplification, and therefore may not need or may not be suitable for further amplification. However, further amplification is not necessarily excluded. For example, the breach in the HCR product can be connected, and the HCR product connected can be further amplified, such as by PCR reaction, the HCR product connected is further amplified. In the case where nucleic acid products are annular, they are advantageously amplified by rolling circle amplification (RCA). Any suitable amplification reaction known in the art can be used.

[0182] RCA utilizes a strand displacement polymerase and requires a circular amplification template, which can be provided by a circular padlock probe. Amplification of the circular template provides a tandem RCA product (RCP) containing multiple copies of a sequence complementary to that of the amplification template (thus the RCP from the padlock probe contains repeating units corresponding to the padlock probe). Such concatemers typically form balls or "spots" that can be easily visualized and detected.

[0183] Prior to the RCA reaction, there may be an optional washing step. The RCA reaction is then typically initiated by adding one or more reagents for RCA (referred to as "RCA reagents"). This is typically a polymerase and nucleotides (particularly dNTPs) for RCA, but primers for RCA may also be optionally added. One or more RCA reagents may be added in advance.

[0184] Primers for RCA reaction can be added to reaction mixture, or can be pre-hybridized with padlock probe. Binding sites for RCA primers can be provided in padlock probes in regions different from target binding regions (i.e., in the backbone region of padlock). In some cases, the target nucleic acid molecule of padlock probes (i.e., molecules used as connection templates for padlock probes, such as nucleic acid domains or blocking oligonucleotides) can be used as or provide primers. Each padlock probe used in the present method can include a single common RCA primer binding site, so the same primer can be used to trigger RCA for all nucleic acid products. Alternatively, different primers can be used. The strand displacement polymerase for RCA is typically Phi29 or a derivative thereof.

[0185] Alternatively, PCR or other amplification reactions can be used to amplify partial nucleic acid products. In particular, PCR reactions can be used to amplify identifier (e.g., barcode) sequences in each nucleic acid product. Nucleic acid products can each include a shared primer binding site located next to their barcode sequence so that a single primer pair can be used to amplify the identifier sequence of each nucleic acid product.

[0186] The amplification products are then detected, or if there is no subsequent amplification step, the nucleic acid products are detected. Any suitable detection means may be used for this purpose. Detection of the first amplification / nucleic acid product indicates the first target molecule (i.e., detection of the first amplification / nucleic acid product is a substitute for detecting the first target molecule), detection of the second amplification / nucleic acid product indicates the second target molecule (i.e., detection of the second amplification / nucleic acid product is a substitute for detecting the second target molecule), and detection of the third amplification / nucleic acid product indicates the interaction between the first target molecule and the second target molecule (i.e., detection of the second amplification / nucleic acid product is a substitute for detecting the interaction between the first target molecule and the second target molecule).

[0187] Detection may include detecting a label incorporated into the product, for example by synthesizing the product using labeled nucleotides, or by attaching the label to the product in a subsequent step, for example by binding to the product a labeled detection oligonucleotide (or detection probe) that is capable of specifically hybridizing to the product, or detection may include or involve sequencing of the product.

[0188] In the case of amplification by RCA, the amplification template (for example, circular padlock probe) comprises a specific detection sequence (that is, identifier sequence), and therefore RCA product (RCP) (as mentioned above, it is a concatemer of circular nucleic acid substrates such as padlock probes) comprises a repeated identifier sequence. Each of the first nucleic acid product, the second nucleic acid product and the third nucleic acid product comprises a unique identifier sequence. These products can be detected in any suitable manner. Typically, the identifier sequence provides a binding site for the detection oligonucleotide hybridized with the identifier sequence. Detection oligonucleotide can be similarly used for combining and detecting other products, such as HCR products or branched chain amplification products.

[0189] Detection oligonucleotide can carry detectable marker, also referred to as detection part. Detection part is any part that can be detected, i.e., can directly or indirectly produce the part of the signal that can be detected. Therefore, the detection part can be regarded as any detectable marker, which can directly or indirectly send a signal. For example, the detection part can be detectable on the spectrum or under the microscope, for example, it can be a fluorescent or colorimetric marker, a particle (such as a bead) or an enzyme marker. Any marker used in the immunohistochemical technique can be used. The hybridization of detection oligonucleotide and its multiple binding sites is concentrated in RCP, thereby allowing detection with high sensitivity.

[0190] However, the detection oligonucleotide does not need to be directly labeled. For example, the detection oligonucleotide can be an unlabeled probe used as a sandwich probe. The concept of sandwich probes is well known in the art and can be applied according to any convenient scheme. The sandwich probes can bind to the RCP (or other products), but are not directly labeled themselves; alternatively, they contain sequences to which the labeled secondary oligonucleotide can bind, thus forming a "sandwich" between the RCP and the labeled secondary oligonucleotide. Alternatively, the RCP can be detected indirectly, for example, by amplifying the product by PCR and detecting the amplified product.

[0191] Detection oligonucleotide or any secondary label probe can be labeled with a directly detectable or indirectly detectable marker. A directly detectable marker is a marker that can be directly detected without using additional reagents, while an indirectly detectable marker is a marker that can be detected by using one or more additional reagents, for example, wherein the marker is a member of a signal generation system consisting of two or more components. In many embodiments, the marker is a directly detectable marker, wherein the directly detectable marker of interest includes but is not limited to: fluorescent markers, colored markers, radioisotope labels, chemiluminescent markers, etc. In many embodiments, the marker is a fluorescent marker, wherein the labeling agent used in these embodiments is one or more fluorescently labeled nucleotides, such as fluorescently labeled CTP (such as Cy3-CTP, Cy5-CTP), etc. The fluorescent moiety that can be used to label nucleotides to produce labeled probe nucleic acids (i.e., detection probes) includes but is not limited to: fluorescein, cyanine dyes, such as Cy3, Cy5, Alexa 555, Bodipy 630 / 650, etc. Other markers known in the art, such as those described above, can also be used.

[0192] Conveniently, the detection moiety may be a colored bead. Colored beads can be easily visualized. Such beads, for example colored polystyrene beads, are widely available.

[0193] Although various detection modes can be adopted, RCP and other larger polymer products can be conveniently detected by visualization (including microscopy) or flow cytometry. In both cases, directly or indirectly labeled detection oligonucleotides can be used, for example, together with fluorescent or colored markers that are easy to detect. In this regard, the marker can include beads or other detectable particles. In the method based on the microscope, RCP or other polymer products can be detected by imaging.

[0194] In other embodiments, the identifier sequence can be detected by quantitative PCR (qPCR) or sequencing. For example, qPCR can be performed using a "TaqMan" probe. In this case, a probe complementary to each identifier sequence is used, wherein each different probe is conjugated to a different, distinguishable fluorophore. Therefore, each identifier sequence (and therefore each nucleic acid product) can be detected and quantified independently. For example, when qPCR is used for detection, the circular padlock probe can be used directly as a PCR template, or, as described above, RCA can be performed and RCP is used as a template.

[0195] Alternatively, when the identifier sequence is detected by sequencing, a form of high throughput DNA sequencing may be used. Synthesis sequencing is a commonly used DNA sequencing method. Examples of sequencing by synthesis techniques include pyrophosphate sequencing, reversible dye terminator sequencing, and ion torrent sequencing, any of which may be used in the present method. Conveniently, the identifier sequence is sequenced using massively parallel DNA sequencing. Massively parallel DNA sequencing is particularly applicable to synthesis sequencing (e.g., reversible dye terminator sequencing, pyrophosphate sequencing, or ion torrent sequencing as described above). Reversible dye terminator methods, such as using NovaSeq TM The system performs massively parallel DNA sequencing.

[0196] As known in the art, massively parallel DNA sequencing is a technique for sequencing multiple (e.g., thousands or millions or more) DNA chains in parallel (i.e., simultaneously). Massively parallel DNA sequencing requires that the target DNA molecule be fixed to a solid surface, such as to a flow cell surface or beads. Each fixed DNA molecule is then sequenced independently. Generally, massively parallel DNA sequencing using reversible dye terminator sequencing utilizes flow cells as a fixed surface, and massively parallel DNA sequencing using pyrophosphate sequencing or ion torrent sequencing utilizes beads as a fixed surface.

[0197] As known to the technician, in the case of large-scale parallel sequencing, DNA molecules are fixed to the surface and usually one or more sequencing adapters are attached to the molecular end to achieve.Therefore, in the method herein, when the identifier sequence is amplified by PCR, PCR primers can include the adapter (sequencing adapter) for sequencing, for adding to the identifier sequence, so that the product can be sequenced.Generally, the sequencing adapter is a nucleic acid molecule (particularly DNA molecule).In this case, the short oligonucleotide complementary to the adapter sequence is conjugated to a fixed surface (for example, the surface of a bead or a flow cell) so that the target DNA molecule can be annealed to the surface by the adapter sequence.Alternatively, any other binding partner can be used to conjugate the target DNA molecule to a fixed surface, such as biotin and avidin / streptavidin.In this case, biotin can be used as a sequencing adapter, and avidin or streptavidin are conjugated to a fixed surface to combine biotin sequencing adapters, and vice versa.

[0198] Thus, the sequencing adapter can be a short oligonucleotide (preferably DNA), generally 10 to 30 nucleotides long (e.g., 15 to 25 or 20 to 25 nucleotides long). As described above, the purpose of the sequencing adapter is to anneal the target DNA molecule to the fixed surface, so the nucleotide sequence of the nucleic acid adapter is determined by the sequence of its binding partner conjugated to the fixed surface. In addition, there is no particular limitation on the nucleotide sequence of the nucleic acid sequencing adapter.

[0199] Generally, in the case of nucleic acid product amplification by RCA, a specific labeled detection oligonucleotide is used to detect RCP. Such methods are particularly suitable for in situ detection. In the case of amplification by PCR, detection is generally performed by qPCR or sequencing. Such methods are particularly suitable for non-in situ detection.

[0200] Therefore, a nucleic acid / amplification product comprising a specific identifier sequence is detected. As described above, when the first amplification / nucleic acid product is detected, this indicates the presence of the first target molecule; when the second amplification / nucleic acid product is detected, this indicates the presence of the second target molecule; when the third amplification / nucleic acid product is detected, this indicates the presence of a complex formed by the interaction of the first target molecule and the second target molecule. In embodiments where a single padlock probe is used to detect a target molecule individually (when the target molecule does not interact with another target molecule) or an interaction between two molecules, the presence of the third amplification product (rather than the first amplification product or the second amplification product) indicates that the entirety of the target molecule in question is complexed with the entirety of another target molecule.

[0201] The method of the present invention allows detection of a pair of target molecules and their interactions, and in some embodiments, the method can also allow their quantification. Quantification can be relative, that is, the relative amount of the two target molecules in the sample and the ratio of each target molecule that interacts can be quantified. Alternatively, quantification can be absolute, thereby calculating the concentration of the two target molecules in the sample and the concentration of the interaction complex containing the two target molecules.

[0202] Relative quantification can be achieved by comparing the detection level or amount between two or more different target molecules to provide relative quantification of each of two or more different nucleic acid molecules or sequences, i.e., relative quantification relative to each other. Thus, the ratio of target molecules present in a sample can be determined. Absolute quantification generally requires the use of target molecules of known concentration to generate a standard curve to compare the detection values ​​against the standard curve.

[0203] In a specific embodiment, the nucleic acid product (cyclic substrate) is amplified by RCA. Detect RCP, and the amount of the corresponding target is quantitatively counted by RCA signal. Because RCP can be detected independently, RCA and RCP detection provide a method that can be easily quantitative, by which, by counting or enumerating the number or quantity of RCP, the detected molecule or interaction can be quantitatively measured. The quantification of this RCP is well known in the art and has been widely described in the literature.

[0204] Alternatively, quantification of the target detected by qPCR is a routine operation in the art, including using a standard curve for absolute quantitative qPCR. If sequencing is used to detect amplified products, the level of the target molecule can also be quantified during sequencing. The level of the amplified product produced should be proportional to the amount of the target present in the sample. Using common primer binding sites for amplification helps to ensure that the levels of different amplified products are comparable and proportional to their concentrations. During sequencing, the level of each amplified product sequenced is detected, thereby allowing the relative concentration to be calculated. Similarly, using the known concentration of each target molecule to generate a standard curve (which can be compared to the standard curve comparison experimental value) allows absolute quantification of the target molecule concentration.

[0205] The present method can be carried out in a multiplexed manner, to detect many pairs of different target molecules and interactions thereof. To reuse the method, multiple options are arranged. Obviously, the neighboring probes of each independent reaction in the multiplexed reaction will comprise different binding domains specific for the target molecule interested. In one embodiment, the nucleic acid structure domains in all neighboring probe pairs are identical. In this embodiment, before substrate nucleic acid molecules (particularly padlock probes) are applied to sample, they are pre-hybridized with neighboring probes. Different padlock probes (for example, carrying different identifier sequences so that they can be distinguished) are to pre-hybridized with each neighboring probe. Then perform the method as described above. Figure 4 A multiplexing embodiment is illustrated, where different padlock probes are used to detect different target molecules and their interactions. Here, padlocks A, B, and C are used to detect target molecules X and Y and their interactions, and padlocks D, E, and F are used to detect target molecules X1 and Y1 and their interactions. The same nucleic acid domain is conjugated to different binding domains (represented here by antibodies) for detecting different targets X and X1 and Y and Y1, respectively.

[0206] In another embodiment, different nucleic acid structure domains are used in each neighboring probe pair. These probes can be used together with different padlock probes specific for each pair of nucleic acid structure domains. However, in another embodiment, a single padlock probe group is used for all multiplexed reactions. In this embodiment, the padlock probe used is a gap filling padlock probe. Each nucleic acid structure domain comprises a padlock binding site, and the padlock binding site comprises a unique identifier sequence, and the side of the unique identifier sequence is a sequence complementary to the target binding sequence of the relevant padlock probe. After the padlock probe is combined with the nucleic acid structure domain, a gap filling reaction is carried out, and the padlock probe is subsequently connected, and the identifier sequence from the nucleic acid structure domain is incorporated into the padlock probe for subsequent identification. Gap filling can be carried out by extension or by a gap oligonucleotide complementary to a gap sequence (which comprises or constitutes an identifier sequence). Figure 5 Such an embodiment is depicted.

[0207] In this way, the method can be used to detect multiple pairs of target molecules and their interactions in a sample. As used herein, the term "plurality" refers to two or more, for example, 3, 4, 5, 6, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90 or 100 or more.

[0208] When the method is operated in a multiplexed manner, the detection of the amplified product can be carried out by sequencing as described above. In this way, an essentially unlimited number of different sequences can be detected in any one detection. This may be conducive to a very high multiplexing method. Alternatively, different or distinguishable markers can be used, but the multiplexing level may be limited by the number of available markers. In order to improve the multiplexing level, according to principles well known in the art, a combination or sequential labeling method can be used. For example, it is known to use a method for decoding barcode sequences by hybridization sequencing, which method includes sequentially using labeled hybridization probes.

[0209] Figure 1 A specific embodiment of the method is shown in FIG. This embodiment provides a method for detecting two target molecules (X and Y) in a sample and detecting the interaction between the two target molecules, the method comprising:

[0210] (i) contacting the sample with:

[0211] (a) a first proximity probe (A probe) for a first target molecule, wherein the first proximity probe comprises a first nucleic acid domain comprising one or more single-stranded regions (shown as single stranded), wherein the first nucleic acid domain comprises a padlock binding site and a second sequence for hybridizing to the padlock (padlock hybridization sequence), both of which are located within the one or more single-stranded regions;

[0212] (b) a second proximity probe (B probe) for a second target molecule, wherein the second proximity probe comprises a second nucleic acid domain comprising one or more single-stranded regions (shown as single-stranded), wherein the second nucleic acid domain comprises a padlock binding site and a further padlock hybridization sequence, both located within the one or more single-stranded regions;

[0213] (c) a first padlock probe (A) comprising binding sequences at its 5' and 3' ends capable of hybridizing to the padlock binding site of the first proximity probe;

[0214] (d) a second padlock probe (B) comprising binding sequences at its 5' and 3' ends capable of hybridizing to the padlock binding site of the second proximity probe; and

[0215] (e) a third padlock probe comprising one or two circularized oligonucleotides (C, or C1 and C2), wherein:

[0216] (I) the one circularizing oligonucleotide (C) comprises a target binding sequence capable of hybridizing to the hybridizing sequence of the second proximity probe at its 5' and 3' ends, and comprises an anchor sequence capable of hybridizing to the hybridizing sequence of the first proximity probe between the 5' and 3' ends, and wherein when applied to a sample, the circularizing oligonucleotide pre-hybridizes with the hybridizing sequence of the first proximity probe at the anchor sequence; or

[0217] (II) the two circularized oligonucleotides (C1 and C2) together form a two-part padlock probe, each circularized oligonucleotide comprising at its 5' and 3' ends a first target binding sequence capable of hybridizing to a hybridization sequence of a first proximity probe and a second target binding sequence capable of hybridizing to a hybridization sequence of a second proximity probe, such that when the first proximity probe and the second proximity probe are in proximity, the two circularized oligonucleotides hybridize to the two proximity probes and juxtapose their respective ends to directly or indirectly link to each other to form a loop,

[0218] (ii) performing a gap-filling reaction in which the 5′ and 3′ ends of the padlock probes have hybridized to their respective binding sites or hybridization sequences with a gap therebetween, and ligating the hybridized padlock probes and one or two circularized oligonucleotides, thereby generating a first circular nucleic acid product from the first padlock probe, a second circular nucleic acid product from the second padlock probe, and a third circular nucleic acid product from the third padlock probe, wherein the first circular nucleic acid product, the second circular nucleic acid product, and the third circular nucleic acid product are distinguishable from each other by sequence (e.g., each comprises a distinguishable identifier sequence);

[0219] (iii) amplifying the first circular nucleic acid product, the second circular nucleic acid product, and the third circular nucleic acid product to produce a first amplification product, a second amplification product, and a third amplification product; and

[0220] (iv) detecting a first amplification product, a second amplification product, and a third amplification product, wherein the first amplification product indicates the first target molecule, the second amplification product indicates the second target molecule, and the third amplification product indicates an interaction between the two target molecules.

[0221] The figure illustrates a particular design of proximity probes, but any suitable design may be used (in this and all embodiments). For example, the figure shows that the padlock binding site is located at the end of the nucleic acid domain distal to the binding domain, and the hybridization sequence is located proximal to the binding domain. However, this arrangement is not required: any functional arrangement of binding sites / hybridization sequences may be used. Similarly, any design with respect to single-stranded and double-stranded regions of the probes may be used, as long as the binding site and hybridization sequence on each probe are located within the single-stranded region. The figure shows two probe designs in this regard: the A probe is completely single-stranded, while the B probe has a double-stranded central region with both chains having single-stranded overhangs containing binding sites and hybridization sequences (see Figure 1 A or Figure 1 B). This setup may be used, but the probes used may alternatively all have the same structure (eg the structure of either the A probe or the B probe). Figure 1 C shows that both nucleic acid domains (one on each probe) are single-stranded. Completely different probe structures can also be used, for example the nucleic acid can have a double-stranded region at one end (distal or proximal to the binding domain) and a single-stranded overhang containing two hybridization sequences at only one end.

[0222] As Figure 1 In a variation of the format shown in A, each of the three padlock probes may be provided with a blocking oligonucleotide as described above.

[0223] Figure 8Another variant is shown in, which shows an embodiment, wherein two nucleic acid domains of the neighboring probe are partially double-stranded. In particular, each nucleic acid domain has a double-stranded central region, wherein two chains have single-stranded protrusions comprising binding sites and hybridization sequences. This configuration works well in the following examples. In such an embodiment, one or two chains of the nucleic acid domain that the third padlock probe binds (and the template that the third padlock probe is connected to) can be further used as a primer for connecting and the RCA of the circular third padlock probe.

[0224] As used herein, the term "hybridization sequence" refers to a sequence to which a padlock probe corresponding to a third nucleic acid substrate molecule binds. As described above, in this embodiment, the third nucleic acid substrate molecule is a one-part or two-part padlock probe.

[0225] Figure 2 Another embodiment is shown in FIG. This embodiment provides a method for detecting two target molecules (X and Y) in a sample and detecting the interaction between the two target molecules, the method comprising:

[0226] (i) contacting the sample with:

[0227] (a) a first proximity probe (A probe) for a first target molecule, wherein the first proximity probe comprises a nucleic acid domain (described herein as a single-stranded nucleic acid domain) that hybridizes to a first padlock probe, wherein the 5' and 3' ends of the first padlock probe are hybridized to a blocking oligonucleotide;

[0228] (b) a second proximity probe (B probe) for a second target molecule, wherein the second proximity probe comprises a nucleic acid domain comprising one or more single-stranded regions, wherein the nucleic acid domain comprises a first padlock binding site capable of hybridizing to the 5' and 3' ends of the first padlock probe and a second padlock binding site for the second padlock probe, the padlock binding sites being both located within the one or more single-stranded regions; and

[0229] (c) a second padlock probe (B) comprising a target binding sequence at its 5' and 3' ends capable of hybridizing to the second padlock binding site of the second proximity probe;

[0230] such that when the first proximity probe and the second proximity probe are in proximity, the blocking oligonucleotide is displaced from the first padlock probe by the first padlock binding site of the second proximity probe, wherein the first padlock binding site of the blocking oligonucleotide and / or the second proximity probe comprises a gap sequence located between sequences capable of hybridizing to the 5' and 3' ends of the first padlock probe, such that

[0231] The hybridized 3' and 5' ends of the first padlock probe are separated by a gap;

[0232] (ii) performing a gap-filling reaction in which the 5' and 3' ends of the padlock probes have hybridized to their respective binding sites with a gap therebetween, and ligating the hybridized padlock probes, thereby generating a first circular nucleic acid product from the first padlock probe hybridized to the blocking oligonucleotide, a second circular nucleic acid product from the second padlock probe, and a third circular nucleic acid product from the first padlock probe hybridized to the first padlock binding site of the second proximity probe, wherein the first circular nucleic acid product, the second circular nucleic acid product, and the third circular nucleic acid product are distinguishable from each other by sequence (e.g., each comprises a distinguishable identifier sequence);

[0233] (iii) amplifying the first circular nucleic acid product, the second circular nucleic acid product, and the third circular nucleic acid product to produce a first amplification product, a second amplification product, and a third amplification product; and

[0234] (iv) detecting a first amplification product, a second amplification product, and a third amplification product (e.g., by detecting their identifier sequences), wherein the first amplification product indicates the first target molecule that has not interacted with the second target molecule, the second amplification product indicates the second target molecule, and the third amplification product indicates an interaction between the two target molecules.

[0235] Figure 3 Another embodiment of the present invention is shown in A. This embodiment provides a method for detecting two target molecules (X and Y) in a sample and detecting the interaction between the two target molecules, the method comprising:

[0236] (i) contacting the sample with:

[0237] (a) a first proximity probe pair for a first target molecule, the first proximity probe pair comprising a first proximity probe and a second proximity probe (x1 and x2 probes), each proximity probe comprising a nucleic acid domain comprising one or more single-stranded regions, wherein the nucleic acid domain of the first proximity probe comprises a first padlock binding site for a first padlock probe, and the nucleic acid domain of the second proximity probe comprises a hybridization sequence capable of hybridizing to a third padlock probe, the first padlock binding site and the hybridization sequence being located within the one or more single-stranded regions;

[0238] (b) a second proximity probe pair for a second target molecule, the second proximity probe pair comprising a first proximity probe and a second proximity probe (y1 and y2 probes), each proximity probe comprising a nucleic acid domain comprising one or more single-stranded regions, wherein the nucleic acid domain of the first proximity probe comprises a second padlock binding site for a second padlock probe, and the nucleic acid domain of the second proximity probe comprises a hybridization sequence capable of hybridizing to a third padlock probe, both the second padlock binding site and the hybridization sequence being located within the one or more single-stranded regions;

[0239] (c) a first padlock probe (A) comprising a target binding sequence at its 5' and 3' ends capable of hybridizing to a first padlock binding site of a first proximity probe of a first proximity probe pair;

[0240] (d) a second padlock probe (B) comprising a target binding sequence at its 5' and 3' ends capable of hybridizing to a second padlock binding site of a first proximity probe of a second proximity probe pair;

[0241] (e) a third padlock probe comprising one or two circularized oligonucleotides, wherein:

[0242] (I) the one circularized oligonucleotide (C) comprises at its 5' and 3' ends a target binding sequence capable of hybridizing to a hybridization sequence of a second proximity probe of a second proximity probe pair and an anchor sequence capable of hybridizing to a hybridization sequence of a second proximity probe of a first proximity probe pair, wherein when applied to a sample, the anchor sequence hybridizes to the hybridization sequence of the second proximity probe of the first proximity probe pair; or

[0243] (II) the two circularized oligonucleotides together form a two-part padlock probe, each circularized oligonucleotide comprising at its 5′ and 3′ ends a first target binding sequence capable of hybridizing to a hybridization sequence of a second proximity probe of a first proximity probe pair and a second target binding sequence capable of hybridizing to a hybridization sequence of a second proximity probe of a second proximity probe pair, such that when the first proximity probe and the second proximity probe are in proximity, each circularized oligonucleotide hybridizes to the nucleic acid domains of the two second proximity probes and the corresponding 5′ and 3′ ends of the two circularized oligonucleotides are juxtaposed to directly or indirectly link to each other to form a loop;

[0244] (ii) performing a gap-filling reaction in which the 5′ and 3′ ends of the padlock probes have hybridized to their respective binding sites or hybridization sequences with a gap therebetween, and ligating the hybridized padlock probes, thereby generating a first circular nucleic acid product from the first padlock probe, a second circular nucleic acid product from the second padlock probe, and a third circular nucleic acid product from the third padlock probe, wherein the first circular nucleic acid product, the second circular nucleic acid product, and the third circular nucleic acid product are distinguishable from each other by sequence (e.g., each comprises a distinguishable identifier sequence);

[0245] (iii) amplifying the first circular nucleic acid product, the second circular nucleic acid product, and the third circular nucleic acid product to produce a first amplification product, a second amplification product, and a third amplification product; and

[0246] (iv) detecting a first amplification product, a second amplification product, and a third amplification product (e.g., by detecting their identifier sequences), wherein the first amplification product indicates the first target molecule, the second amplification product indicates the second target molecule, and the third amplification product indicates an interaction between the two target molecules.

[0247] like Figure 3 In the embodiment illustrated in Figure 1A, in which proximity probe pairs are used to detect the first target molecule and the second target molecule, the first padlock probe and the second padlock probe may be capable of hybridizing to the two members of the first proximity probe pair and the second proximity probe pair, respectively. In this case, the padlock probe hybridizes to one member of the probe pair through the target binding site of the padlock probe and hybridizes to the other member of the probe pair through a sequence in the padlock probe backbone (i.e., an anchor sequence), such as Figure 3 A. In this case, the padlock probe is preferably pre-hybridized with the second member of the proximity probe pair (i.e., the proximity probe to which the padlock probe is bound via the anchor sequence) prior to applying the probes to the sample so that both proximity probes are bound to the target, which is required for padlock probe ligation and amplification. Fig.13 A similar arrangement is shown in FIG. However, in another embodiment, Figure 6 As shown, the first padlock and the second padlock hybridize to only one proximity probe of the proximity probe pair, in particular to different probes of the two proximity probe pairs, but not to the third padlock probe.

[0248] In other words, the padlock probe may bind via its target binding sequence to a proximity probe comprising a hybridization sequence, or to other proximity probes (ie, proximity probes that do not comprise a circularization sequence).

[0249] Therefore, in Figure 3 A. Figure 6 and Fig.13 In the configuration shown in, the third neighboring probe is to be made up of the second neighboring probe from the first neighboring probe and the second neighboring probe. Therefore, each of the second neighboring probes in the nucleic acid structure domain that can hybridize with the 3rd padlock probe constitutes the nucleic acid structure domain of the neighboring probe that can mediate the interaction between the nucleic acid structure domain of the third neighboring probe pair. As described above, this extra complementary region can be in the nucleic acid structure domain of the identical neighboring probe hybridized with the first padlock or the second padlock (such as Figure 3 As shown in A, or Fig.13 or it may be on the other proximity probe of the proximity probe pair (e.g. Figure 6 shown).

[0250] As mentioned above, Figure 3 A. Figure 6 and Fig.13The configuration shown in can be modified to incorporate blocking oligonucleotides on three padlocks.

[0251] As described above, in another embodiment, Figure 6 The format can be modified to replace the proximity probe pairs used to detect independent target molecules with a single probe carrying two separate nucleic acid domains, such as Figure 7 The two single probes for detecting A and B together constitute a proximity probe pair for detecting AB interaction.

[0252] Figure 3 Another embodiment is shown in B. This embodiment provides a method for detecting two target molecules (X and Y) in a sample and detecting the interaction between the two target molecules, the method comprising:

[0253] (i) contacting the sample with:

[0254] (a) a first proximity probe pair for a first target molecule, the first proximity probe pair comprising a first proximity probe and a second proximity probe, wherein the first proximity probe comprises a nucleic acid domain (depicted herein as a single-stranded nucleic acid domain) comprising a first padlock binding site (template a), and the second proximity probe comprises a single-stranded nucleic acid domain that hybridizes to the first padlock probe (padlock a), the first padlock probe comprising binding sequences at its 5' and 3' ends that are capable of hybridizing to the first padlock binding site; and

[0255] (b) a second proximity probe pair for a second target molecule, the second proximity probe pair comprising a first proximity probe and a second proximity probe, wherein the first proximity probe comprises a single-stranded nucleic acid domain comprising a second padlock binding site (template b), and the second proximity probe comprises a single-stranded nucleic acid domain hybridizing to the second padlock probe (padlock b), the second padlock probe comprising binding sequences at its 5' and 3' ends capable of hybridizing to the second padlock binding site;

[0256] wherein the binding sequence of the first padlock probe is capable of hybridizing to the second padlock binding site, and / or the binding sequence of the second padlock probe is capable of hybridizing to the first padlock site;

[0257] wherein the first padlock probe and the second padlock probe each comprise an identifier sequence, and the first padlock binding site and / or the second padlock binding site comprises a gap sequence located between sequences capable of hybridizing to the 5' and 3' ends of the respective padlock probes;

[0258] (ii) performing a gap-filling reaction in which the 5' and 3' ends of the padlock probes have hybridized to their respective binding sites with a gap therebetween, and ligating the hybridized padlock probes, thereby generating a first circular nucleic acid product from the first padlock probe hybridized to the first padlock binding site, generating a second circular nucleic acid product from the second padlock probe hybridized to the second padlock hybridization binding site, and generating a third circular nucleic acid product and optionally a fourth circular nucleic acid product from the first padlock probe hybridized to the second padlock binding site and / or the second padlock probe hybridized to the first padlock binding site, wherein the first circular nucleic acid product, the second circular nucleic acid product, the third circular nucleic acid product, and the optional fourth circular nucleic acid product are distinguishable from each other by sequence (e.g., each comprises a unique combination of an identifier sequence and an optional gap sequence);

[0259] (iii) amplifying the first circular nucleic acid product, the second circular nucleic acid product, the third circular nucleic acid product, and optionally the fourth circular nucleic acid product to produce a first amplification product, a second amplification product, a third amplification product, and optionally a fourth amplification product; and

[0260] (iv) detecting a first amplification product, a second amplification product, a third amplification product, and an optional fourth amplification product (e.g., by detecting a unique combination of their identifiers and an optional gap sequence), wherein the first amplification product indicates a first target molecule, the second amplification product indicates a second target molecule, and the third amplification product and the optional fourth amplification product indicate an interaction between the two target molecules.

[0261] As described above, in this embodiment, the first padlock probe is pre-hybridized with the second proximity probe of the first proximity probe pair, and the second padlock probe is pre-hybridized with the second proximity probe of the second proximity probe pair, i.e. both padlock probes are hybridized with their respective proximity probes before the proximity probes are applied to the sample. Figure 3 As shown in B, padlock probes are prehybridized to their respective proximity probes via sequences in their backbones (ie, anchor sequences).

[0262] The method can be a homogeneous method, i.e. a method carried out in solution, or a heterogeneous method, i.e. a method carried out on a solid phase or using a solid phase. This may depend on the sample used and / or the target molecule or analyte desired to be detected. For example, the method can be carried out on a cell or tissue sample for in situ detection. This is desirable in the case of local detection. Therefore, the method can be used to determine the spatial distribution and interaction of target molecules. Alternatively, for other samples or detection assays, the method can be carried out in a solution, for example, using a liquid sample such as a plasma or serum sample, or a processed sample, etc. in a solution.

[0263] A second aspect of this paper relates to a test kit for implementing the method described in detail above. The test kit includes the component required for implementing the method. The test kit can provide the component for any experimental device. Especially, the test kit includes the first proximity probe or proximity probe pair for detecting the first target molecule, and the second proximity probe or proximity probe pair for detecting the second target molecule, wherein the probes each comprise a binding domain and a nucleic acid structure domain that can directly or indirectly hybridize with their target molecules, and wherein the first probe and the second probe or the first proximity probe one of the right probes and the second proximity probe one of the right probes form together for detecting the interactional third proximity probe pair between the first target molecule and the second target molecule.

[0264] The kit also includes at least the first nucleic acid reagent and the second nucleic acid reagent as defined and described above. In one embodiment, the kit includes the first nucleic acid substrate molecule and the second nucleic acid substrate molecule, wherein the first substrate molecule can hybridize with the first probe or the first nucleic acid domain of one of the probes adjacent to the probe pair, and the second substrate molecule can hybridize with the second probe or the second nucleic acid domain of one of the probes adjacent to the probe pair. As described in detail above, preferably, the nucleic acid substrate molecule is a padlock probe.

[0265] In one embodiment, the kit further comprises a third nucleic acid substrate molecule, particularly a third padlock probe. The third nucleic acid substrate molecule can hybridize with the reporter nucleic acid domains of the two components of the third proximity probe pair. As described in detail above, the third nucleic acid substrate molecule is preferably a padlock probe, which can be one or two parts.

[0266] Depending on the experimental setup, the first nucleic acid substrate molecule, the second nucleic acid substrate molecule and / or the third nucleic acid substrate molecule may be provided in the kit pre-hybridized with their respective proximity probes as described above in relation to the method.

[0267] The kit may also include an enzyme for the method. The kit may include a ligase for ligating the padlock probe as described in detail above. When a gap-filling padlock probe is used, the kit may include a polymerase for an extension reaction to fill the gap. Alternatively, when one or more gap-filling padlock probes are used, the kit may include one or more gap oligonucleotides capable of filling the gap by hybridizing with the gap sequence.

[0268] The kit may also include reagents for amplification and detection. For example, the kit may include a strand displacement nucleic acid polymerase for RCA, preferably phi29 polymerase. The kit may also or alternatively include components for qPCR amplification of padlock probe detection sequences. The kit may also include detection oligonucleotides suitable for detecting each nucleic acid product or its amplicon.

[0269] When detection of amplification products is performed by sequencing, components for massively parallel DNA sequencing can also be provided. For example, the kit can include components for PCR amplification of a DNA identifier / barcode sequence to be sequenced, including primers with 3' sequencing adapter sequences, polymerases, nucleotides, etc. The product can include a solid substrate to which the amplification product can be fixed for sequencing, such as a flow cell or beads.

[0270] As described above, the methods and kits herein can have many different uses and applications included in any detection assay that needs to detect or characterize an interaction. This can, for example, include detecting a target analyte, which is included for diagnostic purposes or for studying interactions. They can be used to analyze or characterize interactions. The methods and kits herein can be particularly applied to the development of bispecific binding molecules, such as bispecific antibodies, which include two different binding domains, each of which is specific to (i.e., targeting) different antigens or molecules. In the design of such molecules, it may be necessary to analyze in situ the two target molecules intended for the bispecific binding molecules to determine their respective cell expression and their cell expression when adjacent, and the methods herein are particularly suitable for such purposes. In addition, bispecific binding molecules can be used to make two target molecules, such as two proteins, more closely adjacent. The methods and kits herein can be used to determine whether such an effect occurs.

[0271] The methods herein may be further understood below with reference to the accompanying drawings and non-limiting examples. BRIEF DESCRIPTION OF THE DRAWINGS

[0272] Figure 1 Schematic diagrams of assay formats for independently detecting two target molecules, X and Y, and the interaction between them using a single proximity probe pair (including an A probe and a B probe) are depicted. Padlock probes A and B are used to detect X and Y, respectively. Padlock probe C is used to detect the interaction. (A) shows a one-part padlock probe C prehybridized with the nucleic acid domain of the A probe in its backbone region; (B) shows a two-part padlock probe C comprising c1 and c2, wherein each of c1 and c2 hybridizes with the nucleic acid domains of both the A probe and the B probe when the A probe and the B probe are adjacent (when bound to X and Y in the interaction); (C) shows a configuration in which each nucleic acid domain of the A probe and the B probe is single-stranded and a two-part padlock probe C is used - in this configuration, separate primers are used for RCA.

[0273] Figure 2 A schematic diagram of an assay format for independently detecting two target molecules, X and Y, and the interaction therebetween using a single proximity probe pair (comprising an A probe and a B probe) and two padlock probes is depicted. The A probe hybridizes to the backbone of a first padlock probe, the target binding end of which is pre-hybridized with a blocking oligonucleotide with a gap therebetween. The B probe hybridizes to the target binding end of a second padlock probe (padlock B). When X and Y interact, the A probe and the B probe are adjacent, and the first padlock is able to hybridize with its target binding site on the B probe as a template for the attachment of the padlock probe (the blocking oligonucleotide is displaced), thereby allowing detection of the XY interaction. Padlock B resolves the level of the target molecule Y. When X and Y are not interacting, the A probe and the B probe are not adjacent, and the target molecule X can be detected using the first padlock and a gap-filling reaction using a blocking oligonucleotide.

[0274] Figure 3 Describe the schematic diagram of the assay format using two adjacent probe pairs, wherein probe pairs x1 and x2 are used to detect target molecule X by dual recognition, and probe pairs y1 and y2 are used to detect target molecule Y by dual recognition, and wherein x1 and y1 form together for detecting the third adjacent probe pair of XY interaction. (A) shows three separate padlocks A, B and C for detecting X, Y and XY interaction respectively; (B) shows 2 padlocks (respectively padlocks a and b) and two connection templates for padlocks (respectively template a and b) (nucleic acid domain of a neighboring probe of each adjacent probe pair). In order to detect X, padlock a is connected to template a, in order to detect Y, padlock a is connected to template b, and in order to detect XY interaction, padlock b is connected to template a, and / or padlock a is connected to template b. Padlocks a and b include identifier sequences that can distinguish them, and gap filling reaction allows to determine the template for the connection of padlocks.

[0275] Figure 4 Depicted is a multiplexed assay format in which proximity probe pairs (A, B) and (D, F) for detecting different target molecule combinations (X, Y) and (X1, Y1) are used in combination with different padlock probes (A, B, C) and (D, E, F), respectively. The nucleic acid domains used in the corresponding proximity probe pairs may be the same or different.

[0276] Figure 5 An alternative multiplexing assay format is depicted, wherein the multiplexing is achieved by gap filling. A standard set of gap filling padlock probes A, B and C are used in combination with multiple proximity probe pairs for detecting different combinations of target molecules, whose nucleic acid domains contain gap sequences that can be used to distinguish circular padlock probes. Gap filling can be achieved by gap filling extension using the gap sequence as a template, or by hybridizing a complementary gap oligonucleotide to the gap sequence.

[0277] Figure 6 Describe the schematic diagram of another determination form of using two adjacent probes to detect the first target molecule A and the second target molecule B and the interaction (AB) therebetween, the adjacent probe is used as the secondary reagent combined with the primary binding partner that can be bonded to the target molecule.The first adjacent probe is to including the first probe combined with the primary binding partner (depicted as antibody) for the first target molecule, and the nucleic acid structure domain of the first probe of the first adjacent probe pair is detected by the first padlock probe to detect the first target molecule (A).The second adjacent probe is to including the first probe combined with the primary binding partner (depicted as antibody) for the second target molecule, and the nucleic acid structure domain of the first probe of the second adjacent probe pair is detected by the second padlock probe to detect the second target molecule (B).The first adjacent probe is to and the second adjacent probe is to each including the second probe, and the second probe is together formed for detecting the secondary reagent (" the third adjacent probe is to ") of the interaction between target molecule A and target molecule B.The third padlock probe provided with two parts is used to detect the third adjacent probe pair when two second probes have been adjacently bonded to their respective targets (primary binding partner) (this occurs when target molecules A and B are both in interaction (AB)). One end of each of the two parts of the third padlock probe hybridizes to one of the nucleic acid domains of the second probe constituting the third proximity probe pair, thereby allowing the ends of the corresponding parts of the third padlock probe to ligate together.

[0278] Figure 7 Describes the Figure 6 The modification of the assay format shown in, wherein two single probes (the first probe and the second probe, and the two probes together constitute the third proximity probe pair) are used, rather than the first proximity probe pair and the second proximity probe pair. The first probe and the second probe each carry two separate nucleic acid structure domains, and the first nucleic acid structure domain of each probe is detected by the first padlock probe and the second padlock probe respectively, to detect independent target molecules A or B. The second nucleic acid structure domain of each probe can hybridize with one end of each part of the two-part third padlock probe, to allow the third padlock probe to be used to detect the interaction (AB) between A and B.

[0279] Figure 8 Depicted Figure 1A to C of the modified schematic diagram of the embodiment shown, wherein the nucleic acid domain of two probes of the single proximity probe pair for detecting two target molecules A and B independently and detecting the interaction (AB) therebetween is double-stranded.Padlock probes A and B are respectively used to detect target molecules A and B by being combined with the first chain of the nucleic acid domain of the first probe and the second probe of the proximity probe pair respectively.The 3rd padlock probe provided in two parts is used to detect the interaction, wherein when the first probe and the second probe are adjacent (when combined with the target molecules A and B in the interaction), each of the two parts of the 3rd padlock probe hybridizes with the second chain of the nucleic acid domain of the first probe and the second probe.In this configuration, RCA does not need a separate primer, because the second chain of the nucleic acid domain as the target (that is, connection template) of the padlock probe can trigger RCA reaction.

[0280] Fig. 9 The results of the interaction between beta-catenin, E-cadherin and protein detected on MCF7 cells using the method are shown. The image panel is divided along the row based on the antibody present, and is divided along the column based on the fluorophore of imaging. The top row shows the signal produced by all different assay reactions, wherein signals are produced in three fluorophore channels, indicating that there are two independent proteins and protein interactions. The second row shows that there is only a technical control of anti-beta-catenin antibody, which means that the only channel producing positive signals is the FITC channel corresponding to anti-beta-catenin. Similarly, in the 3rd row, signals are produced only in the Texas Red channel (TxR) corresponding to the antibody present, and the interaction and beta-catenin reaction retain blanks. The bottom row without primary antibody does not produce any signal.

[0281] Fig.10 Detection of β-catenin, E-cadherin, and protein-protein interactions on FFPE skin tissue sections is shown. The image panels are divided along rows based on the fluorophore imaged, and along columns based on the antibodies present. The top row shows the case where the assay reaction requires two antibodies to generate a signal indicating an interaction (rightmost image). While in the second and third rows, signals are generated under all conditions where the assay reaction target is present, resulting in signals appearing in both images per row.

[0282] Fig.11 Detection of PDGF receptor-β phosphorylation (pPDGFR-β) on BJ-hTert cells is shown. BJ-hTert cells were starved overnight in serum-free growth medium and then stimulated with PDGFbb for 45 minutes on ice or as a control. The proximity of anti-PDGFRβ antibody and anti-pan-phosphotyrosine (pan-P-Tyr) antibody allowed the assay reaction to lead to the detection of PDGFR-β phosphorylation. At the same time, the other two assay reactions allowed the analysis of total PDGFR-β and total pan-P-Tyr in the sample. In the top row, the amount of signal detected by total phosphorylation and phosphorylated receptors increased after stimulation compared to the starved cells from the bottom row. In stimulated cells, the total receptor signal was also slightly downregulated.

[0283] Fig.12 Shows verification Figure 1 Results of the format of the method depicted in A, where the proximity probes are secondary (A) or primary (B) conjugated antibodies. In (A), the proximity probe pair is used to detect E-cadherin and β-catenin located on the cell membrane and their interactions. In (B), the proximity probe pair is used to detect lamin B1 (LaminB1) and lamin A (LaminA) proteins located primarily in the nucleus and their interactions.

[0284] Fig.13 Describe the schematic diagram of the assay format using two proximity probe pairs, wherein the first proximity probe pair is used to detect the first target molecule by dual recognition, and the second proximity probe pair is used to detect the second target molecule by dual recognition, and wherein the first proximity probe and the second proximity probe pair second proximity probe member together form the third proximity probe pair for detecting the interaction between the target molecules.Three separate padlocks are used to detect the first target molecule and the second target molecule and the interaction, respectively.

[0285] Fig.14 Shown is the use of automatic slide staining machine when Figure 6 Results of the method depicted in Figure 4 to detect β-catenin, E-cadherin, and protein-protein interactions in FFPE tissue sections. Image panels are divided along rows based on the fluorophore imaged and along columns based on the antibodies present.

[0286] Fig.15 Shows the use of Figure 6 Detection of the format depicted in (A) molecule A (COX1) with a FITC label, (B) molecule (B) GM130 with a Cy3 label, and (C) molecules A and B COX1 / GM130 with a Cy5 label. In all three images, DAPI staining is also shown. Molecules A and B do not interact, and as expected, no interaction / proximity signal is detected in (C). DETAILED DESCRIPTION

[0287] Example

[0288] Example 1

[0289] Detection of β-catenin, E-cadherin and protein-protein interactions on MCF7 cells.

[0290] Experiments were conducted to confirm that Figure 6 Performance of the method of the form described in. MCF7 cells grow to high density to ensure close contact between cells and form a complex between β-catenin and E-cadherin. Cells were incubated with a blocking reagent to minimize nonspecific staining. β-catenin (target molecule A) and E-cadherin (target molecule B) were detected in MCF7 cells by incubation with primary antibodies (rabbit anti-β-catenin and mouse anti-E-cadherin). No primary antibody was used as a negative control. Subsequently, cells were contacted with proximity probes A (anti-rabbit) and B (anti-mouse). Three padlock probes were used to detect β-catenin (A), E-cadherin (B) and their interactions (AB), respectively. After hybridization with their respective targets (nucleic acid domains of proximity probes), the padlocks were made circular by connection, and RCA was performed to produce RCA products, which were detected by using fluorescently labeled detection probes. The first probe for pair A was detected by the first padlock probe (A) to detect β-catenin (FITC channel), and the first probe for pair B was detected by the second padlock probe (B) to detect E-cadherin (Texas Red (TxR) channel). The second probes for pair A and pair B were detected by the third padlock probe to detect the interaction (AB) (FarRed channel). The results are shown in Fig. 9 shown.

[0291] The top row shows the signals generated from all three padlocks using proximity probe pairs A and B. The presence of both independent proteins (columns 1 and 2) and interactions (column 3) can be detected.

[0292] The second row shows the signal generated when only the anti-β-catenin primary antibody was used (the anti-E-cadherin primary antibody was omitted). In this case, the signal was generated only from the first padlock A (column 1, FITC channel) corresponding to β-catenin; no signal was generated for E-cadherin or the interaction.

[0293] In contrast, the third row shows that a signal was detected only from the second padlock B (column 2, TxR channel) when only the anti-E-cadherin primary antibody was used (anti-β-catenin antibody was omitted), and no signal was generated for β-catenin or the interaction.

[0294] In the case where both primary antibodies were omitted, no signal was generated for any of the padlocks (bottom row).

[0295] Example 2

[0296] Detection of β-catenin, E-cadherin, and protein-protein interactions on FFPE skin tissue sections Test.

[0297] Similar to Example 1, using Figure 6 The experiment was performed in the form of a PCR amplification assay to detect the proteins β-catenin (target molecule A) and E-cadherin (target molecule B) in FFPE samples of skin tissue.

[0298] FFPE skin tissue sections were dewaxed and rehydrated for 15 minutes using the xylene dewaxing method. The slides were then autoclaved with citric acid in a pressure cooker at 125°C, washed and blocked with a blocking agent to minimize nonspecific binding. The tissue was then incubated with primary antibodies as described in Example 1, and proximity probe pairs A and B and 3 padlock probes were similarly used to detect independent proteins and their interactions as described in Example 1. Therefore, experiments were performed using two primary antibodies or only one primary antibody, and the fluorescence signals of different fluorophores used to detect independent proteins were read in three channels (FITC for β-catenin; TxR for E-cadherin, FarRed for interactions). The results are shown in Figure 1. Fig.10 shown.

[0299] Fig.10 The image panels in show in columns (1) to (3) the signals obtained in 3 channels for the corresponding fluorophores using primary antibodies: (1) anti-E-cadherin alone, (2) anti-β-catenin alone, and (3) both anti-E-cadherin and anti-β-catenin. The first row for the FarRed (interaction) channel shows the signals obtained only when two primary antibodies are present (row 1, column 3); no signal is produced when only one primary antibody is present (row 1, columns 1 and 2). The second row for the FITC (β-catenin) channel shows the signals when the anti-β-catenin primary antibody is used alone (column 2) or when used with the anti-E-cadherin primary antibody (column 3). The third row for the TxR (E-cadherin) channel shows the signals when the anti-E-cadherin primary antibody is used alone (column 1) or when used with the anti-B-catenin primary antibody (column 3). Thus, in rows 2 and 3, signal is produced in all conditions where the assay reaction target is present (as detected by the primary antibody), but no signal is produced when it is not detected (no primary antibody to the target molecule), resulting in signal in both images in each row.

[0300] Example 3

[0301] Detection of PDGF receptor-β phosphorylation (pPDGFR-β) on BJ-hTert cells

[0302] use Figure 6 The assay method described in was used to perform experiments to detect the protein PDGFRβ on BJ-hTert cells, and the phosphorylation (presence of phosphorylated tyrosine residues) after stimulating cells with PDGFR ligands. Starved and unstimulated cells were used as negative controls for the presence of phosphorylation on PDGFR. Primary antibodies for PDGFRβ (target molecule A) and for pan-phosphotyrosine (target molecule B) were used, and proximity probes for the primary antibodies for targets A and B, respectively, and padlock probes specific to the nucleic acid domains of the probes were used for detection. The first probe to A was detected by the first padlock probe (A) to detect PDGFRβ (FITC channel), and the first probe to B was detected by the second padlock probe (B) to detect phosphotyrosine Texas Red (TxR channel). The second probe to A and to B was detected by the third padlock probe to detect pPDGRβ, i.e., phosphorylation of PDGFRβ in ligand-stimulated cells and starved cells (i.e., to detect the presence of phosphate groups on the protein (AB)) (FarRed channel).

[0303] BJ-hTert cells were starved overnight in serum-free growth medium and then stimulated with PDGFbb for 45 minutes on ice or as a control. The cells were then blocked and exposed to one or two primary antibodies, followed by assay reactions with proximity probe pairs A and B and three padlock probes to determine the presence of the primary antibodies. The RCA reaction products of the ligated and circularized padlock probes were detected as described above. The results are shown in Fig.11 shown.

[0304] from Fig.11 As can be seen, PDGFR-β phosphorylation is detected by detecting the presence of adjacent anti-PDGFRβ antibodies and anti-pan-phosphotyrosine (pan-P-Tyr) antibodies, i.e., by detecting the interaction (column 3). Detection of anti-PDGFRβ alone (column 1) or phosphotyrosine alone (column 2) allows analysis of total PDGFR-β and total pan-P-Tyr in the sample. Under stimulated conditions (top row), the amount of signal for total phosphorylation and phosphorylated receptor detection increases compared to starved cells (bottom row). In stimulated cells, the total receptor signal is also slightly downregulated.

[0305] Example 4

[0306] Protein β-catenin and E-cadherin on MCF7 cells and their interactions, protein nuclear Detection of lamin A and lamin B and their interactions

[0307] This experiment was performed to demonstrate the detection of proteins and their interactions using different assay formats. In particular, this example demonstrates that Figure 1The assay format depicted in A.

[0308] Two pairs of interacting proteins were selected as targets: β-catenin (X) and E-cadherin (Y), which are located on the cell membrane or in the nucleus, respectively. Lamin A(X) and Lamin B1(Y). Probes A and B and padlocks A and B are used to detect proteins X and Y independently, respectively. Figure 1 Depicted in A. Probes A and B are used together as a proximity probe pair, together with padlock C to detect the interaction between proteins X and Y.

[0309] right Figure 1 The design of A was modified to incorporate the use of a blocking oligonucleotide that hybridizes to padlock C. This was done to prevent premature interaction of the padlock with its ligation template (i.e., with its target) during probe incubation, thereby preventing nonspecific signal. The blocking oligonucleotide was displaced by the padlock target (the nucleic acid domain of probe B).

[0310] Probe A was prepared by hybridizing padlock A and an adjacent padlock (padlock C) to the nucleic acid domain of probe A. Probe B was prepared by hybridizing (i) padlock B and (ii) a ligation template for padlock C to the nucleic acid domain of probe B to generate a partially double-stranded nucleic acid domain of probe B. The padlock and ligation template for padlock C were hybridized at a 5:1 ratio to ensure maximum hybridization.

[0311] The detection of E-cadherin and β-catenin used probes A and B as secondary reagents. MCF7 cell samples were incubated with anti-E-cadherin (from mouse) and anti-β-catenin (from rabbit) antibodies targeting E-cadherin and β-catenin in cells. Probes A and B probes, which contain nucleic acid domains conjugated with secondary antibodies against primary antibodies (3 nM each) and hybridized to padlock or ligation templates, were applied to detect the individual proteins and their interactions. Results are shown in Fig.12 As shown in A.

[0312] Use probes A and B as a primary reagent pair Lamin A and Lamin B for detection. Lamin B1 (from mouse) and anti Lamin A (from rabbit) is conjugated to the nucleic acid domain and hybridized to the padlock or ligation template. Detection using 20 nM of A and B probes Lamin B1. Lamin A and its interactions. The results are as follows Fig.12 As shown in B.

[0313] In both cases, after probes A and B bind to their targets and excess reagents are removed by washing, a ligation reaction is initiated, followed by rolling circle amplification. Fluorophore-labeled oligonucleotides complementary to each of the three circular padlocks are used to detect the RCA products, resulting in three different signals: two representing the expression of each participating protein and one revealing the level of interaction.

[0314] Example 5

[0315] Demonstration of Assay Automation

[0316] This example demonstrates that Figure 6 The assay format is described in and is run using an automated slide stainer instrument.

[0317] β-Catenin (target molecule A), E-cadherin (target molecule B) and their interaction (interaction AB) were detected in healthy human FFPE colon tissue sections on the Leica BondRX FullyAutomated Research Stainer.

[0318] Tissue sections were placed in a Leica Bond instrument and dewaxed using the Bond Dewax Leica protocol. Next, antigen retrieval was performed using Leica Bond Epitope Retrieval 2 (RE2) at 100°C for 40 minutes.

[0319] The proteins β-catenin and E-cadherin were detected by incubation with anti-rabbit and anti-mouse primary antibodies, respectively. No primary antibody was used as a negative control. Three padlock probes were used to detect β-catenin (A), E-cadherin (B), and their interaction (AB). After hybridization with their respective targets (nucleic acid domains of the proximity probes), the padlocks were circularized by ligation, RCA was performed to produce RCA products, and detected with fluorescently labeled detection probes complementary to their corresponding RCA products. The first probe for A was detected by the first padlock probe (A) to detect B-catenin (FITC channel), and the first probe for B was detected by the second padlock probe (B) to detect E-cadherin (Cy3 channel). The second probes for A and B were detected with the third padlock probe to detect the interaction (AB) (Cy5 channel). The DAPI stain and slide mount were removed from the instrument before image scanning. The results are shown in Fig.14 shown.

[0320] Example 6

[0321] Detection of proteins when no protein-protein interactions are expected

[0322] By means of this example it was demonstrated that an interaction signal is only generated when the interaction partners are in close proximity. Fig.15 Shows the use of Figure 6 The format depicted in the figure was used to detect the mitochondrial protein COX1 (target molecule A; FITC channel) in MCF-7 cells ( Fig.15 A) and Golgi protein GM130 (target molecule B; Cy3 channel) ( Fig.15 B). No interaction signal (AB) is expected, since the proteins are located in different subcellular compartments and the separation distance is likely greater than the assay limit. In fact, no AB interaction signal was observed (Cy5 channel) ( Fig.15 C). Each image ( Fig.15 A, B, and C) all contain DAPI staining to guide the expected locations of other targets.

Claims

1. A method for detecting two target molecules in a sample and detecting the interaction between the two target molecules, the method comprising: include: (i) contacting the sample with (a) a first probe or a first proximity probe pair for detecting a first target molecule and (b) a second probe or a second proximity probe pair for detecting a second target molecule; wherein each of the probes comprises a binding domain and a nucleic acid domain capable of binding directly or indirectly to their target molecules, and the first probe and the second probe or one of the probes of the first proximity probe pair and one of the probes of the second proximity probe pair together form a third proximity probe pair for detecting the interaction between the first target molecule and the second target molecule; (ii) performing a first assay reaction to detect the nucleic acid domain of the first probe or the nucleic acid domain of at least one member of the first proximity probe pair, thereby detecting the first target molecule; (iii) performing a second assay reaction to detect the nucleic acid domain of the second probe or the nucleic acid domain of at least one member of the second proximity probe pair, thereby detecting the second target molecule; (iv) performing a third assay reaction to detect the interaction between the first target molecule and the second target molecule, wherein the third assay is a proximity assay using the third proximity probe pair, wherein when the first target molecule and the second target molecule are present in proximity in an interactive manner, the nucleic acid domains of the third proximity probe pair interact with each other directly or indirectly to produce a nucleic acid product, and detecting the product to detect the interaction between the first target molecule and the second target molecule. 2 . The method of claim 1 , wherein the first determination and the second determination comprise quantifying the amount of the detected target molecule, and the third determination comprises quantifying the amount of the target molecule present in the interaction.

3. The method according to claim 1 or claim 2, in: (a) the first assay reaction utilizes a first nucleic acid reagent capable of hybridizing to the nucleic acid domain of the first probe or the nucleic acid domain of at least one member of the first proximity probe pair, and detecting the first reagent or hybridization thereof; (b) the second assay reaction utilizes a second nucleic acid reagent capable of hybridizing to the nucleic acid of the second probe or the nucleic acid of at least one member of the second proximity probe pair, and detecting the second reagent or hybridization thereof; (c) the third assay reaction either (i) utilizes a third nucleic acid reagent that is capable of hybridizing to both nucleic acid domains of the third proximity probe pair to produce a proximity probe-interactive nucleic acid product, or (iii) utilizes one of the first nucleic acid reagent or the second nucleic acid reagent that is also capable of hybridizing to the nucleic acid domain of the other member of the third proximity probe pair to produce a third proximity probe pair-interactive nucleic acid product.

4. according to the method described in any one of claims 1 to 3, the nucleic acid structure territory of the wherein said first probe or the nucleic acid structure territory of the wherein said first adjacent probe right member comprises the first label sequence being detected, to detect the first target molecule; The nucleic acid structure territory of the wherein said second probe or the nucleic acid structure territory of the wherein said second adjacent probe right member comprises the second label sequence being detected, to detect the second target molecule; and the nucleic acid structure territory constituting the wherein said first adjacent probe right and the wherein said second probe or the wherein said first adjacent probe right and the wherein said second adjacent probe right member each also comprises a separate complementary region capable of mediating the interaction between the nucleic acid structure territory of the wherein said third adjacent probe right. 5 . The method according to claim 4 , wherein the first tag sequence and the second tag sequence constitute binding sites for the first nucleic acid agent and the second nucleic acid agent, respectively.

6. The method according to any one of claims 3 to 5, wherein in the first assay, a first nucleic acid product is produced from or using the first nucleic acid reagent, in the second assay, a second nucleic acid product is produced from or using the second nucleic acid reagent, and in the third assay, a third nucleic acid product is produced, wherein the first nucleic acid product, the second nucleic acid product and the third nucleic acid product can be distinguished from each other by sequence and are detected to detect the first target molecule and the second target molecule and the interaction between them.

7. The method of claim 6, wherein the first nucleic acid product, the second nucleic acid product, and the third nucleic acid product are amplified and the resulting amplicons are detected.

8. The method according to any one of claims 3 to 5, wherein an extension product, a ligation product, a hybridization product or an amplification product of the nucleic acid reagent is generated and detected.

9. The method according to claim 1, include: In step (i) or after step (i), the probe is contacted with at least a first nucleic acid substrate molecule and a second nucleic acid substrate molecule, wherein the first substrate molecule hybridizes with the nucleic acid domain of one of the first probe or the first proximity probe pair, and the second substrate molecule hybridizes with the nucleic acid domain of one of the second probe or the second proximity probe pair; In step (ii), a first nucleic acid product is produced from the first nucleic acid substrate; In step (iii), a second nucleic acid product is produced from the second nucleic acid substrate; wherein the product of step (iv) is a third nucleic acid product, wherein the first nucleic acid product, the second nucleic acid product and the third nucleic acid product are distinguishable from each other by sequence; In step (v), optionally generating an amplification product from the first nucleic acid product, the second nucleic acid product, and the third nucleic acid product; as well as In step (vi), the nucleic acid products or amplification products are detected, wherein the first nucleic acid product or amplification product indicates the first target molecule, the second nucleic acid product or amplification product indicates the second target molecule, and the third nucleic acid product or amplification product indicates the interaction between the first target molecule and the second target molecule.

10. The method of claim 9, wherein the relative levels of the first nucleic acid product or amplification product, the second nucleic acid product or amplification product, and the third nucleic acid product or amplification product respectively indicate the relative levels of two target molecules and the ratio of the two target molecules that interact with each other.

11. The method of claim 9 or 10, wherein a third substrate nucleic acid molecule contacts the probe, the third substrate nucleic acid molecule hybridizes to both nucleic acid domains of the third proximity probe pair, and the third nucleic acid product is generated from the third substrate nucleic acid molecule.

12. The method according to any one of claims 9 to 11, wherein the third nucleic acid molecule is generated by the first substrate molecule or the second substrate molecule when it interacts with the nucleic acid domain of the proximity probe of the third proximity probe pair.

13. The method according to any one of claims 9 to 12, wherein the first nucleic acid substrate molecule and the second nucleic acid substrate molecule and, where present, the third nucleic acid substrate molecule are padlock probes provided in one or more parts.

14. The method according to any one of claims 9 to 13, wherein the first amplification product, the second amplification product and the third amplification product are RCA products (RCPs).

15. The method according to any one of claims 9 to 14, wherein the first nucleic acid product, the second nucleic acid product and the third nucleic acid product are generated by ligation to form a circular nucleic acid molecule.

16. The method of any one of claims 9 to 15, wherein the first nucleic acid product, the second nucleic acid product, and the third nucleic acid product are produced by direct or indirect ligation of padlock probes, wherein the padlock probes are capable of hybridizing to the nucleic acid domains of the probes, and the ligation is templated by the nucleic acid domains of the probes.

17. The method according to any one of claims 9 to 16, wherein the two target molecules are complexed with each other, or wherein the first target molecule is a protein and the second target molecule is a modification group added post-translationally.

18. The method according to any one of claims 1 to 11 or 13 to 17, wherein the method include: (i) contacting the sample with: (a) a first proximity probe for the first target molecule, wherein the first proximity probe comprises a first nucleic acid domain comprising one or more single-stranded regions, wherein the first nucleic acid domain comprises a first padlock binding site for a first padlock probe and a hybridization sequence capable of hybridizing to a third padlock probe, both the first padlock binding site and the hybridization sequence being located within the one or more single-stranded regions; (b) a second proximity probe for the second target molecule, wherein the second proximity probe comprises a second nucleic acid domain comprising one or more single-stranded regions, wherein the second nucleic acid domain comprises a second padlock binding site for a second padlock probe and a hybridization sequence capable of hybridizing to the third padlock probe, both the second padlock binding site and the hybridization sequence being located within the one or more single-stranded regions; (c) a first padlock probe comprising target binding sequences at its 5' and 3' ends capable of hybridizing to the first padlock binding site of the first proximity probe; (d) a second padlock probe comprising target binding sequences at its 5' and 3' ends capable of hybridizing to the second padlock binding site of the second proximity probe; and (e) a third padlock probe, the third padlock probe comprising: (I) a single circularisable oligonucleotide comprising at its 5' and 3' ends a target binding sequence capable of hybridising to the hybridising sequence of the second proximity probe and comprising in the backbone region between the 5' and 3' ends an anchor sequence capable of hybridising to the hybridising sequence of the first proximity probe, and wherein when applied to the sample, the anchor sequence hybridises to the hybridising sequence of the first proximity probe; or (II) two circularized oligonucleotides, said two circularized oligonucleotides together forming a two-part padlock probe, Each circularized oligonucleotide comprises at its 5' and 3' ends a first target binding sequence capable of hybridizing to the hybridization sequence of the first proximity probe and a second target binding sequence capable of hybridizing to the hybridization sequence of the second proximity probe, such that when the first proximity probe and the second proximity probe are in proximity, each circularisation oligonucleotide hybridises to the nucleic acid domains of both proximity probes and the corresponding 5' and 3' ends of the two circularisation oligonucleotides are juxtaposed to directly or indirectly link to each other to form a circle; (ii) in the case where the 5′ and 3′ ends of the padlock probe have hybridized to their respective binding sites or hybridization sequences with a gap therebetween, performing a gap-filling reaction and ligating the hybridized padlock probe and one or two circularized oligonucleotides, thereby generating a first circular nucleic acid product from the first padlock probe, a second circular nucleic acid product from the second padlock probe, and a third circular nucleic acid product from the third padlock probe, wherein the first circular nucleic acid product, the second circular nucleic acid product and the third circular nucleic acid product are distinguishable from each other by sequence, (iii) amplifying the first circular nucleic acid product, the second circular nucleic acid product and the third circular nucleic acid product by rolling circle amplification (RCA) to produce a first RCA product (RCP), a second RCA product and a third RCA product; as well as (iv) detecting the first RCP, the second RCP, and the third RCP, wherein the first RCP indicates the first target molecule, the second RCP indicates the second target molecule, and the third RCP indicates an interaction between the two target molecules.

19. The method according to any one of claims 1 to 11 or 13 to 17, wherein the method include: (i) contacting the sample with: (a) a first proximity probe pair for the first target molecule, the first proximity probe pair comprising a first proximity probe and a second proximity probe, each proximity probe comprising a nucleic acid domain comprising one or more single-stranded regions, wherein the nucleic acid domain of the first proximity probe comprises a first padlock binding site for a first padlock probe, and the nucleic acid domain of the second proximity probe comprises a hybridization sequence capable of hybridizing to a third padlock probe, both the first padlock binding site and the hybridization sequence being located within the one or more single-stranded regions; (b) a second proximity probe pair for the second target molecule, the second proximity probe pair comprising a first proximity probe and a second proximity probe, each proximity probe comprising a nucleic acid domain comprising one or more single-stranded regions, wherein the nucleic acid domain of the first proximity probe comprises a second padlock binding site for a second padlock probe, and the nucleic acid domain of the second proximity probe comprises a hybridization sequence capable of hybridizing to the third padlock probe, both the second padlock binding site and the hybridization sequence being located within the one or more single-stranded regions; (c) a first padlock probe comprising target binding sequences at its 5' and 3' ends capable of hybridizing to the first padlock binding site of the first proximity probe of the first proximity probe pair; (d) a second padlock probe comprising target binding sequences at its 5' and 3' ends capable of hybridizing to the second padlock site of the first proximity probe of the second proximity probe pair; (e) a third padlock probe, the third padlock probe comprising: (I) a single circularisable oligonucleotide comprising at its 5' and 3' ends a target binding sequence capable of hybridising to the hybridisation sequence of the second proximity probe of the second proximity probe pair and an anchor sequence capable of hybridising to the hybridisation sequence of the second proximity probe of the first proximity probe pair, and wherein when applied to the sample, the anchor sequence hybridises to the hybridisation sequence of the second proximity probe of the first proximity probe pair; or (II) two circularized oligonucleotides, said two circularized oligonucleotides together forming a two-part padlock probe, each circularizing oligonucleotide comprises at its 5' and 3' ends a first target binding sequence capable of hybridizing to the hybridization sequence of the second proximity probe of the first proximity probe pair and a second target binding sequence capable of hybridizing to the hybridization sequence of the second proximity probe of the second proximity probe pair, such that when the first proximity probe and the second proximity probe are in proximity, each circularizing oligonucleotide hybridizes to the nucleic acid domains of the two second proximity probes and the corresponding 5' and 3' ends of the two circularizing oligonucleotides are juxtaposed to directly or indirectly link to each other to form a loop; (ii) performing a gap-filling reaction in the case where the 5′ and 3′ ends of the padlock probes have hybridized to their respective binding sites or hybridization sequences with a gap therebetween, and ligating the hybridized padlock probes, thereby generating a first circular nucleic acid product from the first padlock probe, a second circular nucleic acid product from the second padlock probe, and a third circular nucleic acid product from the third padlock probe, wherein the first circular nucleic acid product, the second circular nucleic acid product, and the third circular nucleic acid product are distinguishable from each other by sequence; (iii) amplifying the first circular nucleic acid product, the second circular nucleic acid product and the third circular nucleic acid product by rolling circle amplification (RCA) to produce a first RCA product (RCP), a second RCA product and a third RCA product; as well as (iv) detecting the first RCP, the second RCP, and the third RCP, wherein the first RCP indicates a first target molecule, the second RCP indicates a second target molecule, and the third RCP indicates an interaction between two target molecules.

20. The method according to any one of claims 1 to 3, 6 to 10 or 12 to 17, wherein the method include: (i) contacting the sample with: (a) a first proximity probe for the first target molecule, wherein the first proximity probe comprises a nucleic acid domain (e.g., a single-stranded nucleic acid domain) that hybridizes to a first padlock probe, wherein the 5' and 3' ends of the first padlock probe are hybridized to a blocking oligonucleotide; (b) a second proximity probe for the second target molecule, wherein the second proximity probe comprises a nucleic acid domain comprising one or more single-stranded regions, and wherein the nucleic acid domain comprises a first padlock binding site capable of hybridizing to the 5' and 3' ends of the first padlock probe and a second padlock binding site for the second padlock probe, both of the padlock binding sites being located within the one or more single-stranded regions; as well as (c) a second padlock probe comprising target binding sequences at its 5' and 3' ends capable of hybridizing to the second padlock binding site of the second proximity probe; such that when the first proximity probe and the second proximity probe are in proximity, the blocking oligonucleotide is displaced from the first padlock probe by the single stranded region of the second proximity probe comprising the first padlock binding site, wherein the first padlock binding site of the blocking oligonucleotide and / or the second proximity probe comprises a gap sequence located between complementary binding sites capable of hybridizing to the 5′ and 3′ ends of the first padlock probe, such that the hybridized 3′ and 5′ ends of the first padlock probe are separated by a gap; (ii) when the 5' and 3' ends of the padlock probe have hybridized to their respective binding sites with a gap therebetween, performing a gap-filling reaction and ligating the hybridized padlock probes, thereby generating a first circular nucleic acid product from the first padlock probe hybridized to the blocking oligonucleotide, a second circular nucleic acid product from the second padlock probe, and a third circular nucleic acid product from the first padlock probe hybridized to the first padlock binding site of the second proximity probe, wherein the first circular nucleic acid product, the second circular nucleic acid product and the third circular nucleic acid product are distinguishable from each other by sequence; (iii) amplifying the first circular nucleic acid product, the second circular nucleic acid product and the third circular nucleic acid product by rolling circle amplification (RCA) to produce a first RCA product (RCP), a second RCA product and a third RCA product; as well as (iv) detecting the first RCP, the second RCP, and the third RCP, wherein the first RCP indicates the first target molecule that has not interacted with the second target molecule, the second RCP indicates the second target molecule, and the third RCP indicates an interaction between two target molecules.

21. The method according to any one of claims 1 to 3, 6 to 10 or 12 to 17, wherein the method include: (i) contacting the sample with: (a) a first proximity probe pair for the first target molecule, the first proximity probe pair comprising a first proximity probe and a second proximity probe, wherein the first proximity probe comprises a nucleic acid domain comprising a first padlock binding site, and the second proximity probe comprises a nucleic acid domain that hybridizes to the first padlock probe, the first padlock probe comprising binding sequences at its 5' and 3' ends that are capable of hybridizing to the first padlock binding site; and (b) a second proximity probe pair for the second target molecule, the second proximity probe pair comprising a first proximity probe and a second proximity probe, wherein the first proximity probe comprises a nucleic acid domain comprising a second padlock binding site, and the second proximity probe comprises a nucleic acid domain that hybridizes to a second padlock probe, the second padlock probe comprising binding sequences at its 5' and 3' ends that are capable of hybridizing to the second padlock binding site; wherein the binding sequence of the first padlock probe is also capable of hybridizing to the second padlock binding site, and / or the binding sequence of the second padlock probe is also capable of hybridizing to the first padlock binding site; wherein the first padlock probe and the second padlock probe each comprise an identifier sequence, and the first padlock binding site and / or the second padlock binding site comprises a gap sequence located between complementary binding sites capable of hybridizing to the 5' and 3' ends of the respective padlock probes; (ii) performing a gap-filling reaction in which the 5′ and 3′ ends of the padlock probes have hybridized to their respective binding sites with a gap therebetween, and ligating the hybridized padlock probes, thereby generating a first circular nucleic acid product from the first padlock probe hybridized to the first padlock binding site, a second circular nucleic acid product from the second padlock probe hybridized to the second padlock binding site, and a third circular nucleic acid product and optionally a fourth circular nucleic acid product from the first padlock probe hybridized to the second padlock binding site and / or the second padlock probe hybridized to the first padlock binding site, wherein the first circular nucleic acid product, the second circular nucleic acid product, the third circular nucleic acid product and optionally the fourth circular nucleic acid product are distinguishable from each other by sequence; (iii) amplifying the first circular nucleic acid product, the second circular nucleic acid product, the third circular nucleic acid product and optionally the fourth circular nucleic acid product by rolling circle amplification (RCA) to produce a first RCA product (RCP), a second RCA product, a third RCA product and optionally a fourth RCA product; as well as (iv) detecting the first RCP, the second RCP, the third RCP and optionally the fourth RCP, wherein the first RCP indicates the first target molecule, the second RCP indicates the second target molecule, and the third RCP and optionally the fourth RCP indicate an interaction between two target molecules.

22. A kit for carrying out the method according to any one of claims 1 to 21, said kit include: (i) a first probe or a proximity probe pair for detecting a first target molecule, and a second probe or a proximity probe pair for detecting a second target molecule, wherein the probes each comprise a binding domain capable of hybridizing directly or indirectly to their target molecules and a nucleic acid domain, and wherein the first probe and the second probe or one of the probes of the first proximity probe pair and one of the probes of the second proximity probe pair together form a third proximity probe pair for detecting an interaction between the first target molecule and the second target molecule; as well as (ii) a first nucleic acid agent and a second nucleic acid agent, wherein the first agent is capable of hybridizing to the nucleic acid domain of the first probe or one of the probes of the first proximity probe pair, and the second agent is capable of hybridizing to the nucleic acid domain of the second probe or one of the probes of the second proximity probe pair; Optionally, wherein the first reagent and / or the second reagent is further capable of hybridizing to the nucleic acid domain of another member of the third proximity probe pair.

23. The kit of claim 22, wherein the first nucleic acid agent and the second nucleic acid agent are substrate molecules capable of producing distinguishable first nucleic acid products and second nucleic acid products.

24. A kit according to claim 22 or claim 23, further comprising a third nucleic acid substrate molecule capable of hybridising to the two nucleic acid domains of the third proximity probe pair.

25. The kit according to any one of claims 22 to 24, wherein the first and second nucleic acid substrate molecules and, where present, the third nucleic acid substrate molecule are padlock probes provided in one or more parts.

26. A kit according to any one of claims 22 to 25, in: (i) at least the binding domain of the first probe or at least the binding domain of the first proximity probe pair is an antibody or an antigen-binding fragment thereof; and / or (ii) the kit further comprises a ligase; and / or (iii) The kit further comprises a strand-displacing nucleic acid polymerase, preferably phi29 polymerase.

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