Controls for proximity detection assays

Abstract

The present invention provides a method for detecting a plurality of analytes in a sample, the method comprising performing a multiplexed proximity-based detection assay. The assay utilizes pairs of proximity probes having a common hybridization site (i.e., a hybridization site that is common between different pairs of proximity probes). Also provided are products comprising a plurality of pairs of proximity probes having a common hybridization site, which products can be used in the methods disclosed herein.

Description

Technical Field

[0001] This invention provides a method for detecting multiple analytes in a sample, the method comprising performing multiple proximity-based assays. The assays utilize pairs of proximity probes having a shared hybridization site (i.e., a hybridization site shared between different proximity probe pairs). A product comprising multiple pairs of proximity probes having a shared hybridization site is also provided, the product being used in the method disclosed herein. Background Technology

[0002] Modern proteomics methods require the ability to detect a large number of different proteins (or protein complexes) in small sample volumes. To achieve this, multiplex analysis is necessary. Common methods for multiplexing proteins in a sample include proximity extension assay (PEA) and proximity ligation assay (PLA). PEA and PLA are described in WO 01 / 61037; PEA is further described in WO 03 / 044231, WO 2004 / 094456, WO 2005 / 123963, WO 2006 / 137932, and WO 2013 / 113699.

[0003] PEA and PLA are proximity assays, relying on the principle of "proximity detection." In these methods, an analyte is detected by binding multiple (i.e., two or more, typically two or three) probes, which allow a signal to be generated when they bring the analyte (and thus the "proximity probe") into proximity through binding. Typically, at least one proximity probe contains a nucleic acid domain (or portion) linked to the probe's analyte-binding domain (or portion), and signal generation involves interactions between the nucleic acid portion and / or additional functional portions carried by other probes. Therefore, signal generation depends on interactions between probes (more particularly between nucleic acids or other functional portions / domains carried by them), and thus occurs only when the necessary probe binds to the analyte, resulting in increased specificity of the detection system.

[0004] In PEA, the nucleic acid moieties linked to the analyte-binding domains of the probe pair hybridize with each other when the probes are very close together (i.e., when bound to the target), and then extension is performed using a nucleic acid polymerase. The nucleic acid moieties of the probes in the probe pair contain complementary "hybridization sites" that hybridize with each other. The extension product forms a reporter nucleic acid, and detection of this reporter nucleic acid indicates the presence of a specific analyte (the analyte bound by the associated probe pair) in the sample of interest.

[0005] In PLA, when a probe of a probe pair binds to its target, the nucleic acid moieties linked to the analyte-binding domain of the probe pair become adjacent and can ligate together. Alternatively, they can together provide a template for the ligation of individually added oligonucleotides that are capable of hybridizing with the nucleic acid domain when they are adjacent. In the PLA method, at least one "sandwich" oligonucleotide is provided to bridge adjacent probe nucleic acid moieties. The sandwich oligonucleotide contains a sequence complementary to a "hybridization site" on the probe nucleic acid domain. The binding of the probe nucleic acid moieties to the sandwich oligonucleotide allows the two probe nucleic acid moieties to ligate together. Alternatively, as mentioned above, a second sandwich molecule can be added and linked to the first sandwich. The ligation product is then amplified and acts as a reporter nucleic acid.

[0006] Multiplex analyte detection using PEA or PLA can be achieved by including a unique identifier (ID) sequence, such as a barcode sequence or primer or probe binding site, in the nucleic acid portion of each probe. The reporter nucleic acid molecule corresponding to a specific analyte can be identified by its contained ID sequence.

[0007] In proximity assays, some "background" (i.e., false positive) signals are unavoidable. These background signals may arise from random interactions with or between the probe and unbound neighboring probes in the reaction solution. Currently, the level of background signal in proximity reactions is determined using a separate negative control. For the negative control, proximity assays are performed using only buffer (i.e., no sample), making all signals background. Comparing the experimental assay to the negative control allows for the identification of true positive signals.

[0008] This invention provides a method for multiplex proximity assays using an improved background control. In this method, different neighboring probe pairs share a hybridization site. This promotes the formation of a “background” signal among all unbound probes sharing the same hybridization site. All signals from the generated reporter nucleic acid are read together (true positives and false positives). True positive and false positive signals can be distinguished based on whether the resulting reporter nucleic acid contains paired barcode sequences (i.e., barcode sequences each corresponding to the same analyte, indicating a true positive signal) or unpaired barcode sequences (i.e., barcode sequences corresponding to different analytes, indicating a false positive signal). The level of false positive signals generated in the reaction indicates the background level, meaning that a separate negative control reaction is no longer required to determine the background level, thus simplifying the overall assay.

[0009] Using shared hybridization sites to determine background also mitigates performance differences between different hybridization sites. Different pairs of hybridization sites may interact more or less strongly than other pairs, resulting in different levels of background for each pair. Shared hybridization sites allow for the individual determination of the background level produced by each pair of hybridization sites, thus more accurately determining the background level to be calculated. Therefore, this invention provides a more direct and accurate means of controlling false positive results in adjacent assays. Summary of the Invention

[0010] Therefore, in a first aspect of the invention, a method for detecting multiple analytes in a sample is provided, the method comprising performing multiple proximity-based detection assays, the assays comprising:

[0011] (i) Contact the sample with multiple pairs of neighboring probes, wherein each pair of neighboring probes includes a first neighboring probe and a second neighboring probe, and each neighboring probe includes:

[0012] (a) An analyte-binding domain that is specific to the analyte; and

[0013] (b) Nucleic acid domains,

[0014] Each probe pair contains two probes that are specific to the same analyte and can bind to the analyte simultaneously; and each probe pair is specific to different analytes.

[0015] Each neighboring probe's nucleic acid domain comprises an ID sequence and at least a first hybridization sequence, wherein the ID sequence of each neighboring probe is different; and wherein:

[0016] In each neighboring probe pair, the first neighboring probe and the second neighboring probe contain a pairing hybridization sequence such that when the first neighboring probe and the second neighboring probe bind to their analyte, the respective pairing hybridization sequences of the first neighboring probe and the second neighboring probe hybridize to each other or hybridize to a common splice oligonucleotide containing a hybridization sequence, the hybridization sequence being complementary to each of the pairing hybridization sequences of the first neighboring probe and the second neighboring probe.

[0017] Furthermore, at least one pair of hybridization sequences is shared by at least two adjacent pairs of probes;

[0018] (ii) Hybridize the nucleic acid domains of the adjacent probes to each other or to the splint oligonucleotide to form a continuous or discontinuous duplex comprising the hybridization sequence of the first adjacent probe and the hybridization sequence of the second adjacent probe, wherein the duplex comprises at least one free 3' end.

[0019] (iii) Performing an extension and / or ligation reaction on the double strand to generate an extension and / or ligation product comprising the ID sequence of the first neighboring probe and the ID sequence of the second neighboring probe.

[0020] (iv) Amplify the extended or ligated product;

[0021] (v) Detecting the extended or linked products, wherein the detection of the extended or linked products includes identifying the ID sequence therein and determining the relative amount of each extended or linked product; and

[0022] (vi) Determine which analytes are present in the sample, wherein:

[0023] (a) Extended products and / or ligation products comprising a first ID sequence from a first neighboring probe belonging to a first neighboring probe pair and a second ID sequence from a second neighboring probe belonging to a second neighboring probe pair are considered background; and

[0024] (b) An extended product or ligation product containing a first ID sequence and a second ID sequence from a neighboring probe pair and present in an amount above the background indicates the presence of the analyte in the sample that is specifically bound by the neighboring probe pair.

[0025] In a second aspect, the present invention provides a product comprising:

[0026] (i) Multiple neighbor probe pairs, wherein each neighbor probe pair comprises a first neighbor probe and a second neighbor probe, and each neighbor probe comprises:

[0027] (a) Protein-binding domains that are specific to proteins; and

[0028] (b) Nucleic acid domains,

[0029] Each pair of probes contains a protein-binding domain that is specific to the same protein and can bind to the protein simultaneously; and each probe pair is specific to different proteins.

[0030] Each neighboring probe's nucleic acid domain comprises an ID sequence and at least a first hybridization sequence, wherein the ID sequence of each neighboring probe is different; and wherein in each neighboring probe pair, the first neighboring probe and the second neighboring probe comprise a pairing hybridization sequence; and, optionally...

[0031] (ii) A plurality of splice oligonucleotides, each splice oligonucleotide containing a hybridization sequence complementary to each of the pairing hybridization sequences of the adjacent probe pairs;

[0032] The hybridization sequence of each neighboring probe pair is configured such that when the first neighboring probe and the second neighboring probe bind to their proteins, the corresponding paired hybridization sequences of the first neighboring probe and the second neighboring probe hybridize to each other or to the splint oligonucleotide.

[0033] Furthermore, at least one pair of hybridization sequences is shared by at least two adjacent pairs of probes. Detailed Implementation

[0034] As detailed above, a first aspect of the present invention provides a method for detecting multiple analytes in a sample. As used herein, the term "analyte" means any substance (e.g., a molecule) or entity that is desired to be detected by the method of the present invention. Therefore, an analyte is the "target" of the determination method of the present invention, i.e., a substance to be detected or screened using the method of the present invention.

[0035] The analyte can therefore be any biomolecule or chemical compound that is desired to be detected, such as peptides or proteins or nucleic acid molecules or small molecules, including organic and inorganic molecules. The analyte can be a cell or microorganism, including a virus or its fragments or products. It can thus be seen that the analyte can be any substance or entity for which specific binding couplers (e.g., affinity couplers) can be developed. All that is required is that the analyte can bind to at least two binding couplers simultaneously (more specifically, at least two analyte-binding domains adjacent to the probe).

[0036] Proximity-based assays have been found to be particularly useful for detecting proteins or peptides. Therefore, analytes of particular interest include protein molecules, such as peptides, polypeptides, proteins, or prions, or any molecule or fragment thereof comprising protein or polypeptide components. In a particularly preferred embodiment of the invention, the analyte is a whole or part protein molecule, most particularly a protein. That is, it is preferred that the analyte is or contains a protein.

[0037] An analyte can be a single molecule or a complex containing two or more molecular subunits, which may or may not be covalently bonded to each other, and the molecular subunits may be the same or different. Therefore, in addition to cells or microorganisms, such complex analytes can also be protein complexes or biomolecular complexes containing proteins and one or more other types of biomolecules. Such complexes can therefore be homopolymers or heteropolymers. Aggregates of molecules (e.g., proteins) can also be target analytes, such as aggregates of the same protein or different proteins. An analyte can also be a complex between proteins or peptides and nucleic acid molecules such as DNA or RNA. Of particular interest may be the interaction between proteins and nucleic acids (e.g., regulatory factors such as transcription factors) and DNA or RNA. Therefore, in a particular embodiment, the analyte is a protein-nucleic acid complex (e.g., a protein-DNA complex or a protein-RNA complex). In another embodiment, the analyte is a non-nucleic acid analyte, meaning an analyte that does not contain nucleic acid molecules. Non-nucleic acid analytes include proteins and protein complexes, small molecules, and lipids as described above.

[0038] The method of the present invention relates to the detection of multiple analytes in a sample. The multiple analytes can be of the same type (e.g., all analytes can be proteins or protein complexes) or of different types (e.g., some analytes can be proteins, other protein complexes, other lipids, other protein-DNA or protein-RNA complexes, or any combination of such analyte types).

[0039] As used in this disclosure, the term "multiple analytes" means more than one analyte (i.e., two or more analytes), conforming to its standard definition. The terms "multiple" and "multiple" are interchangeable. Therefore, the method of the present invention is used to detect at least two analytes in a sample. However, it is preferred to detect far more than two analytes according to the method of the present invention. Preferably, the method of the present invention detects at least 10, 20, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, or 1500 or more analytes.

[0040] The term "detecting" (or "detected") used extensively herein includes any means of determining the presence or absence of an analyte (i.e., determining whether a target analyte is present in the sample of interest). Therefore, if the method of the present invention is performed and an attempt is made to detect a specific analyte of interest in a sample, but the analyte is not detected because it is not present in the sample, the step of "detecting the analyte" is still performed because its presence or absence in the sample has been evaluated. The step of "detecting" the analyte is independent of the success of the detection, i.e., independent of the actual analyte detected.

[0041] Analytical detection may also include any form of measurement of the concentration or abundance of the analyte in the sample. The absolute concentration or relative concentration of the target analyte can be determined, and for this purpose, the concentration of the target analyte can be compared with the concentration of another target analyte (or other target analyte) in the sample or other samples.

[0042] Therefore, "detection" can include determining, measuring, evaluating, or determining the presence or absence or amount of an analyte in any way. This includes quantitative and qualitative determination, measurement, or evaluation, including semi-quantitative determination. Such determination, measurement, or evaluation can be relative (e.g., when two or more different analytes are being detected in a sample) or absolute. Therefore, the term "quantification" when used to quantify a target analyte in a sample can refer to absolute or relative quantification. Absolute quantification can be accomplished by including known concentrations of one or more control analytes and / or by referencing the detected target analyte level with reference to a known control analyte (e.g., by generating a standard curve). Alternatively, relative quantification can be achieved by comparing the detection levels or amounts of two or more different target analytes to provide a relative quantification of each of the two or more different analytes, i.e., relative to each other. Similarly, the relative levels of a particular analyte in two different samples can be quantified. Methods for achieving quantification in the methods of the present invention are further discussed below.

[0043] The method of this invention is used to detect multiple analytes in a sample. According to this invention, any sample of interest can be analyzed. That is, any sample containing or potentially containing an analyte of interest, and any sample on which one wishes to analyze it to determine whether it contains the analyte of interest and / or to determine the concentration of the analyte of interest therein.

[0044] Therefore, any biological or clinical sample can be analyzed according to the present invention, such as any cell or tissue sample from an organism or derived therefrom, or any bodily fluid or preparation derived therefrom, as well as samples such as cell cultures, cell preparations, cell lysates, etc. Environmental samples (e.g., soil and water samples) or food samples can also be analyzed according to the present invention. Samples can be freshly prepared, or they can be pretreated in any convenient manner, for example, for storage.

[0045] Therefore, representative samples include any material that may contain biomolecules or any other desired or target analytes, including, for example, food and related products, clinical and environmental samples. Samples can be biological samples, which may contain any viral or cellular material, including prokaryotic or eukaryotic cells, viruses, bacteriophages, mycoplasma, protoplasts, and organelles. Such biological material can therefore include any type of mammalian and / or non-mammal animal cells, plant cells, algae including cyanobacteria, fungi, bacteria, protozoa, etc.

[0046] Preferably, the sample is a clinical sample, such as whole blood and blood-derived products (e.g., plasma, serum, erythrocyte sedimentation rate, amber layer, and blood cells), urine, feces, cerebrospinal fluid, or any other bodily fluid (e.g., respiratory secretions, saliva, milk, etc.), tissue, and biopsy. Particularly preferred are plasma or serum samples. Therefore, the method of the present invention can be used, for example, for the detection of biomarkers, or for the determination of pathogen-derived analytes in a sample. The sample can be derived, in particular, from humans, although the method of the present invention can also be applied to samples derived from non-human animals (i.e., veterinary samples). The sample can be pretreated in any convenient or desired manner to prepare it for use in the method of the present invention, such as by cell lysis or removal.

[0047] The method of the present invention includes performing multiplex proximity-based assays. As used herein, the term "multiplex" refers to the simultaneous determination of multiple (i.e., at least two) different analytes in the same reaction mixture. However, preferably, the multiplex reaction according to the invention determines well more than two analytes. For example, a multiplex reaction can determine at least 5, 10, 15, 20, 25, 30, 40, 50, 60 or more analytes. Some multiplex reactions may determine more than this number of analytes, such as at least 70, 80, 90, 100, 110, 120, 130, 140 or 150 or more analytes.

[0048] "Proximity-based assays" are any assays that utilize proximity probes to detect analytes in a sample. Generally, a proximity probe is a probe that interacts with at least one other homologous proximity probe to generate a signal that can be detected to detect an analyte. Proximity probes are well known in the art. As used in this disclosure and invention and as defined in the claims herein, a proximity probe is an entity comprising an analyte-binding domain and a nucleic acid domain that are specific to the analyte. "Analyte-specific" means that the analyte-binding domain specifically recognizes and binds to a particular target analyte, i.e., it binds to its target analyte with a higher affinity than it binds to other analytes or portions of other analytes. The analyte-binding domain is preferably an antibody, particularly a monoclonal antibody. Antibody fragments or antibody derivatives containing an antigen-binding domain are also suitable as analyte-binding domains. Examples of such antibody fragments or derivatives include Fab, Fab', F(ab')2, and scFv molecules.

[0049] The Fab fragment consists of the antigen-binding domain of the antibody. A single antibody contains two Fab fragments, each consisting of a light chain and its attached N-terminal heavy chain portion. Therefore, a Fab fragment contains a complete light chain and the V-terminal portion of the heavy chain bound to it. H and C H 1. Domain 1. The Fab fragment can be obtained by digesting the antibody with papain.

[0050] The F(ab')2 fragment consists of two Fab fragments of the antibody plus the hinge region of the heavy chain domain (including the disulfide bond linking the two heavy chain domains together). In other words, the F(ab')2 fragment can be viewed as two covalently linked Fab fragments. The F(ab')2 fragment can be obtained by digesting the antibody with pepsin. The reduction of the F(ab')2 fragment produces two Fab' fragments, which can be viewed as Fab fragments containing additional thiol groups, which can be used to conjugate the fragments to other molecules. The ScFv molecule is a synthetic construct produced by fusing the variable domains of the antibody's light and heavy chains together. Typically, this fusion is achieved by recombinantly engineering the antibody to produce a fusion protein containing both heavy and light chain variable domains.

[0051] The nucleic acid domains of adjacent probes can be DNA domains or RNA domains. Preferably, they are DNA domains. The nucleic acid domains of adjacent probes in each pair are typically designed to hybridize with each other or with one or more common oligonucleotide molecules (with which both adjacent probe domains in a pair can hybridize). Therefore, the nucleic acid domains must be at least partially single-stranded. In some embodiments, the nucleic acid domains of adjacent probes are completely single-stranded. In other embodiments, the nucleic acid domains of adjacent probes are partially single-stranded, comprising both single-stranded and double-stranded portions.

[0052] Proximity probes are typically provided in pairs, each pair being specific for the target analyte. As mentioned above, the target analyte can be a single entity, particularly a single protein. In this embodiment, the two probes in a proximity pair bind to the target analyte (e.g., a protein), but to different epitopes. The epitopes do not overlap, such that the binding of one probe to its epitope does not interfere with or prevent the binding of the other probe to its epitope. Alternatively, as mentioned above, the target analyte can be a complex, such as a protein complex, in which case one probe in the pair binds to one member of the complex, while the other probe binds to another member of the complex. The probes bind to the protein in the complex at sites different from the protein interaction sites (i.e., the sites in the proteins where they interact with each other).

[0053] As described above, the neighboring probes are provided in pairs, each probe being specific for the target analyte. This means that within each neighboring probe pair, both probes contain an analyte-binding domain specific for the same analyte. Since the detection assays used are multiplex assays, multiple different probe pairs are used in each assay, each probe pair being specific for a different analyte. That is, the analyte-binding domain of each different probe pair is specific for a different target analyte. According to the present invention, any detection method utilizing neighboring probes can be used. As described above, particularly suitable neighbor-based detection assays are proximity extension assays (PEA) and proximity connection assays (PLA).

[0054] The method of the present invention includes a first step of contacting a sample with a plurality of paired (i.e., multiple pairs) of neighboring probes. Each neighboring probe pair comprises a first neighboring probe and a second neighboring probe, and each neighboring probe comprises: (a) an analyte-specific binding domain; and (b) a nucleic acid domain. In each neighboring probe pair, both probes contain an analyte-specific binding domain, and each probe pair is specific for different analytes (i.e., each probe pair contains an analyte-specific binding domain for different analytes).

[0055] Each neighboring probe's nucleic acid domain contains an identifier (ID) sequence. Each neighboring probe contains a unique ID sequence (i.e., a different ID sequence exists in each neighboring probe). It is important to note that this does not mean that every individual probe molecule contains a unique ID sequence. Rather, each probe species contains a unique ID sequence. "Probe species" refers to probes that contain a specific analyte-binding domain; therefore, in other words, all probe molecules containing the same analyte-binding domain contain the same unique ID sequence. Each different probe species contains a different ID sequence. As discussed further below, the ID sequence allows for the identification of the reporter nucleic acid generated in the method of this invention.

[0056] Each neighboring probe's nucleic acid domain also contains at least one (or at least a first) hybridization sequence. The first hybridization sequence (which may be the only hybridization sequence among the neighboring probes, depending on the structure of the probes used) is paired in each neighboring probe pair. "Paired hybridization sequence" means that the two hybridization sequences in the pair can interact with each other directly or indirectly, such that when the method of the present invention is performed and a pair of neighboring probes bind to their target analyte, the nucleic acid domains of the two probes are directly or indirectly connected to each other.

[0057] In a particular and preferred embodiment, the paired hybridization sequences are complementary to each other, such that they hybridize with each other. In this embodiment, the hybridization sequence of the first neighboring probe in a pair is the reverse complementary sequence of the hybridization sequence of the second neighboring probe in the pair.

[0058] In an alternative implementation, the paired hybridization sequences do not hybridize directly with each other; instead, both hybridize with a single bridging oligonucleotide (referred to herein as a sandwich oligonucleotide). The single oligonucleotide can be considered as a third oligonucleotide in this assay. However, one or more sandwich oligonucleotides may be used, and therefore there may be a third oligonucleotide or additional oligonucleotide with which the paired hybridization sequence can hybridize. In other words, the paired hybridization sequence is capable of hybridizing with a common oligonucleotide. This can be an oligonucleotide capable of providing a template for the ligation and / or extension of a nucleic acid domain, or it can be the extension and / or ligation of a third oligonucleotide and optionally additional oligonucleotides with a template provided by a nucleic acid domain.

[0059] In one such implementation, the splice oligonucleotide, together with the pair of neighboring probes, can form a third member of each neighboring assay group. The splice oligonucleotide contains two hybridization sequences: one complementary to the hybridization sequence of the first probe in the probe pair, and the other complementary to the hybridization sequence of the second probe in the probe pair. Therefore, the splice oligonucleotide is capable of hybridizing with both paired hybridization sequences of neighboring probes in its neighboring assay group. Notably, the splice oligonucleotide is capable of simultaneously hybridizing with both paired hybridization sequences of neighboring probes in its neighboring assay group. Thus, when a pair of neighboring probes binds to its analyte and becomes adjacent, the nucleic acid domains of both probes hybridize with the splice oligonucleotide, forming a complex comprising the nucleic acid domains of both probes and the splice oligonucleotide.

[0060] In the method of this invention, at least one pair of hybridization sequences is shared by at least two pairs of neighboring probes. In other words, at least two pairs of neighboring probes (bound to different analytes) have the same hybridization sequence. Probe pairs sharing a pair of hybridization sequences can hybridize with each other or form complexes together. Hybridization between nucleic acid domains is most likely to occur when both nucleic acid domains of a pair of neighboring probes are bound to their respective analytes, because the binding of the probes to the analytes brings the nucleic acid domains into close proximity. However, some interactions will inevitably form between the paired hybridization sequences of the nucleic acid domains of neighboring probes that are not bound in solution (i.e., nucleic acid domains of neighboring probes that are not bound to their analytes), or when only one neighboring probe is bound to its analyte, it may interact with another probe in solution. It is noteworthy that the nucleic acid domains of neighboring probes that are not bound in solution may also hybridize (or form complexes) with the nucleic acid domains of any neighboring probe that has a paired hybridization sequence, regardless of whether the neighboring probes are bound to the same or different analytes. The background of reporter nucleic acid formation resulting from this non-specific hybridization (i.e., hybridization between neighboring probes that are not bound in solution) is further described below.

[0061] Preferably, a significant portion of the probe pairs share their hybridization sequences with at least one other neighboring probe pair. In a particular embodiment, at least 25%, 50%, or 75% of the neighboring probe pairs share their hybridization sequences with another neighboring probe pair (i.e., with at least one other neighboring probe pair). In one particular embodiment, all neighboring probe pairs share their hybridization sequences with at least one other neighboring probe pair. However, as is apparent from the above, in another embodiment, at least one pair of hybridization sequences is unique for a single pair of neighboring probes. That is, at least one pair of neighboring probes does not share its hybridization sequence with any other neighboring probe pair. In a particular embodiment, at most 75%, 50%, or 25% of the paired neighboring probes do not share their hybridization sequences with any other neighboring probe pair.

[0062] In one embodiment of the invention, a single pair of hybridization sequences is shared among all probe pairs that share a common hybridization sequence. That is, all probe pairs that share their hybridization sequence with another probe pair have the same pair of hybridization sequences. In this embodiment, potentially, all probe pairs used in multiplex assays may have the same pair of hybridization sequences.

[0063] However, if too many probe pairs share the same pair of hybridization sequences, this can lead to excessive background interactions, thus masking true positive signals. Therefore, it may be preferable that each pair of hybridization sequences is shared by a more limited number of probe pairs. In certain embodiments, no more than 20, 15, 10, or 5 neighboring probe pairs share the same pair of hybridization sequences. Therefore, it is preferred that the multiplexing of the present invention uses multiple sets of neighboring probe pairs, each set sharing a specific pair of hybridization sequences. Thus, all neighboring probe pairs in a particular set of neighboring probe pairs share the same pair of hybridization sequences, but each different set of neighboring probe pairs uses a different pair of hybridization sequences. This enables non-specific hybridization between all probe pairs within each set of probe pairs, but prevents non-specific hybridization between probe pairs in different sets of probe pairs. Generally, each set of probe pairs includes 2 to 5 probe pairs, but larger sets can be used if preferred.

[0064] The number of probe pair sets used in any given multiplex assay depends on the total number of probe pairs used in that assay, i.e., the number of different analytes detected in that assay. Inevitably, the more probe pairs used in the assay, the larger the number of probe pair sets.

[0065] The first step of the method of the present invention involves contacting a sample with multiple pairs of neighboring probes discussed above. A pair of neighboring probes may be added to the premixed sample either in pairs or as individual neighboring probes. That is, the sample may be contacted with a pair of neighboring probes simultaneously or separately or simultaneously in the same reaction mixture. If the neighboring probes are configured such that the two probes in the probe pair hybridize with a common splice oligonucleotide (rather than hybridize with each other), various splice oligonucleotides may be included with the neighboring probe pair, or with one of the neighboring probes in the pair, or may be added separately simultaneously or after the neighboring probes. “Contacting the sample” means mixing the sample with the neighboring probe pair. The neighboring probe pair may be added to the sample, or conversely, the sample may be added to the neighboring probe pair. The sample may be diluted before contacting the neighboring probe pair. If sample dilution is required, this may be done using a suitable diluent, such as a buffer. Suitable buffers used as diluents include PBS (phosphate-buffered saline), TBS (Tris-buffered saline), HBS (HEPES-buffered saline), etc. The buffer (or other diluent) used must be prepared in a purified solvent (e.g., water) so that it is free of contaminant analytes. Therefore, the diluent should be sterile, and if water is used as the diluent or the base of the diluent, the water used is preferably ultrapure water (e.g., Milli-Q water).

[0066] After the sample comes into contact with adjacent probe pairs, the nucleic acid domains of the adjacent probes hybridize with each other, or, depending on the case, with splice oligonucleotides. Hybridization of nucleic acid domains with each other or with splice oligonucleotides results in the formation of continuous or discontinuous double strands. As used herein, a "double strand" is a segment of double-stranded nucleic acid. The double strand contains the hybridization sequence of the first adjacent probe and the hybridization sequence of the second adjacent probe. If the hybridization sequence hybridizes with a common splice oligonucleotide, rather than with each other, the double strand also contains the common splice oligonucleotide.

[0067] In this step, hybridization of nucleic acid domains with each other results in the formation of a continuous double strand, which is a single double strand containing the complete hybridization sequence of both nucleic acid domains. Hybridization of the probe nucleic acid domain with a common splice oligonucleotide results in the formation of a discontinuous double strand, comprising a first portion formed between the splice oligonucleotide of the first probe and the hybridization sequence, a second portion formed between the splice oligonucleotide of the second probe and the hybridization sequence, and a vacancy located between the first and second portions of the double strand (i.e., between the hybridization sequences of the two probes). The discontinuous double strand can be alternatively considered as two separate double strands (i.e., the first and second portions of the discontinuous double strand can be alternatively considered as separate first and second double strands). In this way, it can be seen that hybridization of the probe nucleic acid domain with a common splice oligonucleotide results in the formation of two connected double strands. The double strands are connected because they are joined together by a common splice oligonucleotide.

[0068] The duplexes produced by hybridization of nucleic acid domains to each other or to common splint oligonucleotides contain a free 3' end (or at least one free 3' end – the duplex may contain multiple free 3' ends. In some embodiments, the duplex contains two free 3' ends). The free 3' end is the 3' end of the duplex that is capable of extending the nucleic acid strand with polymerase.

[0069] In this step, hybridization typically and most often occurs between the nucleic acid domains of neighboring probes in adjacent probe pairs that bind to the target analyte. However, as mentioned above, background hybridization also occurs with or between the nucleic acid domains of unbound, unpaired probes in solution. This background hybridization occurs between the nucleic acid domains of probes from probe pairs that share a common hybridization sequence.

[0070] After the nucleic acid domains hybridize to form a double strand, the double strand is extended and / or ligated to produce an extension and / or ligation product containing the ID sequence of a first neighboring probe and the ID sequence of a second neighboring probe. The nature of the reaction depends on whether the proximity assay performed is PLA or PEA. In the case of PEA, only the extension reaction is performed, resulting in an extension product. Many PEA variants are discussed below. In the case of PLA, a ligation reaction is performed, but an extension reaction can also be performed. Variations of PLA are also discussed below. Preferably, the extension and / or ligation product is a linear extension and / or ligation product (i.e., it is not a cyclic product).

[0071] Once the extension or ligation product is generated, it is amplified. Any known nucleic acid amplification technique can be used. Preferredly, amplification is performed by PCR, but any other nucleic acid amplification method can be used, such as loop-mediated isothermal amplification (LAMP).

[0072] In a preferred embodiment, all reporter nucleic acids (i.e., extension and / or ligation products) generated in multiplex assays contain a common primer binding site. That is, all generated reporter nucleic acids contain the same pair of primer binding sites. This is advantageous because it allows all generated reporter nucleic acids to be amplified in a single amplification reaction (e.g., PCR) using a single primer pair.

[0073] Once amplified, the reporter nucleic acid is detected. Detection of the reporter nucleic acid is achieved by detecting its ID sequence. By detecting the ID sequence in the extension or ligation product, it is possible to determine which probes hybridize with each other to produce products. The relative amount of each extension or ligation product is also determined in this step. Any suitable detection method known in the art can be used.

[0074] An ID sequence can be any sequence that can distinguish or identify a neighboring probe. Therefore, it is a tag sequence through which a specific neighboring probe can be detected. The ID sequence can be directly guided, or it can provide a binding site for another entity (through which the entity can be detected), such as a specific primer, or a detection probe.

[0075] In a preferred embodiment of the invention, the ID sequence is a barcode sequence. A barcode sequence is defined as a specific nucleotide sequence corresponding to a particular analyte. If each probe carries a barcode sequence, then each reporter nucleic acid will contain two barcode sequences: one from each of the two probes that combine to produce the product. When both barcode sequences are detected, the two probes that combine to produce the reporter nucleic acid can thus be identified. If the two barcode sequences come from a pair of adjacent probes (i.e., from a pair of probes that bind the same target analyte), the reporter nucleic acid may indicate the presence of the target analyte in the sample, or it may be background. If the two barcode sequences come from unpaired adjacent probes (i.e., the two barcode sequences indicate different analytes), the reporter nucleic acid is considered background.

[0076] The barcode sequence is located within the nucleic acid domain of the probe. The barcode sequence is not located within the first hybridization sequence: as detailed above, each neighboring probe contains a different barcode sequence, while the hybridization sequence is shared among multiple different probes. The barcode sequence is also not located in a common primer binding site—as mentioned above, each probe contains a unique barcode sequence, while preferably all probes contain a common primer binding site.

[0077] Barcode sequences can be detected in several ways. First, a specific barcode sequence can be detected by sequencing all reporter nucleic acid molecules generated during multiplex assays. By sequencing all generated reporter nucleic acid molecules, all the different reporter nucleic acid molecules can be identified by their barcode sequences. Nucleic acid sequencing is the preferred method for reporter nucleic acid detection / analysis.

[0078] Other suitable methods for detecting barcodes in reporter nucleic acid molecules include PCR-based methods. For example, quantitative PCR can be performed using TaqMan probes. In this case, the reporter nucleic acid molecule (or at least a portion of each reporter nucleic acid molecule containing the barcode sequence) is amplified, and probes complementary to each barcode sequence are provided, with each distinct probe conjugated to a different, distinguishable fluorophore. The presence or absence of each barcode can then be determined based on whether a particular barcode has been amplified. However, it is clear that PCR-based methods, as described above, are only suitable for the simultaneous analysis of relatively small numbers of different sequences, but combined methods using probes to decode barcode sequences are known and can be used to extend multiplexing capabilities to some extent. Nucleic acid sequencing has no practical limitation on the number of sequences that can be identified in any single attempt, enabling a higher level of multiplexing than detection using PCR; therefore, sequencing is the preferred method for reporter nucleic acid molecule detection.

[0079] Preferably, a form of high-throughput DNA sequencing is used to detect barcodes in reporter nucleic acid molecules. Synthesis sequencing is a preferred DNA sequencing method. Examples of synthesis sequencing techniques include pyrosequencing, reversible dye-terminator sequencing, and ion-current sequencing, any of which can be used in the method of the present invention. Preferably, the reporter nucleic acid is sequenced using massively parallel DNA sequencing. Massively parallel DNA sequencing is particularly applicable to synthesis sequencing (e.g., reversible dye-terminator sequencing, pyrosequencing, or ion-current sequencing as described above). Massively parallel DNA sequencing using the reversible dye-terminator method is a preferred sequencing method. For example, it can be used... NovaSeq TM The system is used to perform massively parallel DNA sequencing using a reversible dye terminator method.

[0080] As is known in the art, massively parallel DNA sequencing is a technique in which multiple (e.g., thousands, millions, or more) DNA strands are sequenced in parallel (i.e., simultaneously). Massively parallel DNA sequencing requires immobilizing the target DNA molecule onto a solid surface, such as the surface of a flow cell or onto beads. Each immobilized DNA molecule is then sequenced individually. Typically, massively parallel DNA sequencing using reversible dye terminator sequencing utilizes a flow cell as the immobilization surface, while massively parallel DNA sequencing using pyrosequencing or ion-fluidic sequencing utilizes beads as the immobilization surface.

[0081] As is known to those skilled in the art, immobilizing DNA molecules onto a surface in the context of massively parallel sequencing is typically achieved by attaching one or more sequencing aptamers to the ends of the molecule. These sequencing aptamers are capable of attaching DNA molecules to a target surface. Therefore, the method of the present invention may include adding one or more sequencing aptamers (sequencing aptamers) to a reporter nucleic acid molecule, as described in more detail below.

[0082] In an alternative implementation, the ID sequence is not a barcode sequence. Instead, the ID sequence can allow for probe identification by other means. Depending on the nature of the ID sequence, any suitable method can be used to identify it. For example, the ID sequence can be a restriction site (i.e., a nucleotide sequence recognized by a restriction enzyme). In this implementation, the nucleic acid domain of each neighboring probe contains a different restriction site (making it recognized and cleaved by a different restriction enzyme). Therefore, different combinations of restriction enzymes can be applied to the reporter nucleic acid generated by multiplex assays to determine which probe combinations have interacted. If the reporter nucleic acid is cleaved by both of the applied pair of restriction enzymes, this indicates that the two probes containing the corresponding restriction sites of the enzymes have interacted to produce the reporter nucleic acid.

[0083] In another implementation, the ID sequence is the primer binding site. In this implementation, the nucleic acid domain of each neighboring probe contains a unique primer binding site. The reporter nucleic acid molecule is then amplified using different primer combinations to determine which probe combinations have interacted. If an amplification reaction with a specific primer pair yields an amplification product, this demonstrates that the two probes containing the corresponding primer binding sites have interacted to produce the reporter nucleic acid. Any other sequence used in some way to identify a specific probe can alternatively be used as the ID sequence.

[0084] Barcode sequences are particularly preferred as ID sequences because they allow the detection of all reporter nucleic acid molecules in a single sequencing reaction. Using alternative forms of ID sequences (such as unique restriction sites or primer binding sites) is less efficient because they require testing each combination of restriction enzymes or primers in a separate reaction to determine which probes have interacted to produce reporter nucleic acids. Nevertheless, this alternative type of ID sequence is preferred in certain situations.

[0085] Therefore, the detection of reporter nucleic acid molecules (i.e., extension or ligation products) includes identifying the ID sequence (preferably a barcode sequence) within each reporter nucleic acid, as detailed above. The detection step includes not only detecting the various reporter nucleic acid molecules produced but also determining the relative amount of each reporter nucleic acid molecule. This can be achieved by any suitable means. High-throughput DNA sequencing, as detailed above, is a preferred means for reporter nucleic acid detection, suitable for the relative quantification of reporter nucleic acid molecules, because the number of each specific reporter nucleic acid is quantified by the sequencing reaction. As mentioned above, quantitative PCR is another suitable means for detecting reporter nucleic acids. Detection of reporter nucleic acid molecules by quantitative PCR allows for the quantification of the relative amount of each reporter nucleic acid. Any other suitable method for quantifying the relative amount of each reporter nucleic acid can be used.

[0086] Once the reporter nucleic acid is detected, a determination step is performed to identify which analytes are present in the sample. In this step, the background level is first determined. All reporter nucleic acids generated due to non-specific probe interactions can be considered background interactions. The relative amount of each of these background interactions is determined, thereby determining the level of background interactions. "Non-specific probe interaction" refers to the interaction between unpaired probes, i.e., the interaction between probes that bind different analytes. Such a reporter nucleic acid is an extension and / or ligation product comprising a first ID sequence (e.g., a barcode sequence) from a first neighboring probe belonging to a first neighboring probe pair and a second ID sequence (e.g., a barcode sequence) from a second neighboring probe belonging to a second neighboring probe pair. This reporter nucleic acid can also be described as an extension and / or ligation product comprising a first ID sequence (e.g., a barcode sequence) from a neighboring probe specific to a first analyte and a second ID sequence (e.g., a barcode sequence) from a neighboring probe specific to a second (or different) analyte. As mentioned above, non-specific interactions between unpaired neighboring probes can occur between probes free in solution or, when only one probe binds to its analyte, due to their shared hybridization site.

[0087] The reporter nucleic acid generated through specific probe interactions is then analyzed. "Specific probe interaction" refers to the interaction between probes within a probe pair, i.e., the interaction between two probes that bind to the same analyte. This reporter nucleic acid is an extension product and / or ligation product comprising a first ID sequence and a second ID sequence (e.g., a first barcode sequence and a second barcode sequence) from a neighboring probe pair. Alternatively, this reporter nucleic acid can be described as an extension product and / or ligation product comprising a first ID sequence and a second ID sequence (e.g., a first barcode sequence and a second barcode sequence) from neighboring probes that are specific to the same analyte.

[0088] The probes within a probe pair can also interact in solution, so the reporter nucleic acids generated by specific probe interactions can also constitute background (i.e., generated due to background interactions). Therefore, the amount of each reporter nucleic acid generated by specific probe interactions is compared to the level of background interactions, just as the amount of reporter nucleic acids generated by non-specific probe interactions is determined. If the level of reporter nucleic acids generated by specific probe interactions is higher than the level of background interactions (i.e., the level of non-specific background reporter nucleic acids), this indicates the presence of analyte bound by the relevant probe pair in the sample. On the other hand, if the level of reporter nucleic acids generated by specific probe interactions is not higher than the level of non-specific background reporter nucleic acids (e.g., if the level of reporter nucleic acids generated by specific probe interactions is equal to or lower than the level of non-specific background reporter nucleic acids), then only the interactions between the relevant probe pairs are considered background. In this case, the fact that the interactions between the probes of the probe pair are merely background indicates that no analyte bound by the probe pair is present in the sample.

[0089] Alternatively, for any single target molecule, background interactions can be defined solely as non-specific interactions, including the probe that binds to that target molecule. That is, for each target molecule, background interactions can be defined as non-specific interactions between the probe that recognizes the target molecule and unpaired probes (i.e., probes that do not recognize the target molecule) that share their hybridization sites with the probe that recognizes the target molecule. Therefore, in this case, non-specific interactions between probes that neither recognize the target molecule are considered background interactions for that particular target molecule.

[0090] In a particular implementation, the background level compared to the level of specific probe interaction is the average level of the background interactions under consideration, and in particular the mean level of the background interactions under consideration.

[0091] In one particular embodiment, the first step of the method (i.e., the step of contacting the sample with multiple pairs of neighboring probes) further includes contacting the sample with one or more background probes that do not bind to an analyte, the background probes comprising: a nucleic acid domain containing an ID sequence and a hybridization sequence shared with at least one neighboring probe. The term "background probe" may also be referred to herein as "inert probe." As mentioned above, inert probes do not bind to an analyte. Nevertheless, if the inert probe is specific for an analyte known not to be present in the sample, particularly an antibody, it may contain an analyte-binding domain. An inert probe may actually contain a "binding domain" equivalent to the analyte-binding domain of a functional neighboring probe, but without performing the analyte-binding function; that is, the equivalent of the binding domain is inert. In one embodiment, the inert domain may be provided by ontological IgG. Alternatively, the inert probe may contain an inactive analyte-binding domain, i.e., a non-functional analyte-binding domain. For example, an inert probe may contain a pseudo-analyte-binding domain, such as a constant region of an antibody, or one chain of an antibody (either the heavy chain or the light chain only). Alternatively, an inert probe may contain an inert domain to which a nucleic acid domain is attached, but which is non-functional and independent of the analyte-binding domain of the active probe. The inert domain can be, for example, a protein that can be added to the assay without interfering with the assay reaction, such as serum albumin (e.g., human serum albumin or bovine serum albumin). In another alternative, the inert probe is simply a nucleic acid molecule without any non-nucleic acid domains.

[0092] Each inert probe contains an ID sequence within its nucleic acid domain. The same type of ID sequence is used in inert probes as in active (i.e., neighboring) probes. For example, if the active probe uses a barcode sequence as its ID sequence, the inert probe also uses a barcode sequence as its ID sequence. Each inert probe contains a hybridization sequence shared with at least one neighboring probe. Preferably, each inert probe contains a hybridization sequence shared with multiple neighboring probes. When using inert probes, it is possible to use only a single type of inert probe, i.e., all inert probes have the same hybridization sequence. However, it is preferable to use multiple types of inert probes, each containing a different hybridization sequence (shared with different neighboring probes or a different set of neighboring probes). It is possible that each different type of inert probe has a different, unique ID sequence. Alternatively, all inert probes of all different types can use a common inert probe ID sequence. In any case, it is clear that one or more ID sequences used in an inert probe are not shared with any neighboring probes.

[0093] Background interactions between inert probes and neighboring probes in solution are possible due to shared hybridization sites between the inert probe and certain neighboring probes. When an inert probe interacts with a neighboring probe, this results in the formation of a double-stranded structure between the nucleic acid domains of the two probes. The extension and / or ligation reactions then result in the formation of an extension and / or ligation product from the double-stranded structure formed by the two probes. This extension / ligation product, along with all other products of the assay, is amplified, processed, and detected. The extension / ligation product (i.e., the reporter nucleic acid) resulting from the interaction between the inert probe and neighboring probes is considered background in the deterministic step.

[0094] Multiplex assays for detecting analytes in a sample are preferably performed using PEA. In this embodiment, as described above, the nucleic acid domains of each adjacent probe pair contain complementary hybridization sequences that hybridize with each other to form a double strand. The formed double strand is subjected to an extension reaction to obtain an extension product. Specifically, the extension product of PEA is a linear extension product.

[0095] There are several different variants of PEA, each using slightly different neighboring probes. The design of the nucleic acid domains of each neighboring probe depends on the method used to apply the probes. Figure 1 The diagram schematically illustrates a representative sample of a proximity extension assay, and these embodiments are described in detail below. Typically, in a proximity extension assay, after a pair of adjacent probes bind to their target analyte, the nucleic acid domains of the two probes are adjacent to each other and interact (i.e., directly or indirectly hybridize with each other). The interaction between the two nucleic acid domains produces a nucleic acid duplex containing at least one free 3' end (i.e., at least one nucleic acid domain within the duplex has an extendable 3' end). The addition or activation of a nucleic acid polymerase in the assay mixture results in the extension of at least one free 3' end. Thus, using its paired nucleic acid domain as a template, at least one nucleic acid domain in the duplex is extended. The resulting extension product contains an ID sequence indicating which two probes produced the extension product.

[0096] The nucleic acid domains of adjacent probes can be single-stranded or partially double-stranded. Nucleic acid domains can hybridize with each other, and one domain can provide a template for the extension of another domain. One or two domains can be extended. In the case of a partially double-stranded nucleic acid domain, the single-stranded portions of the domains can hybridize with each other. The single-stranded portions can therefore be located at the 3' end of the strand. In the case of a partially double-stranded nucleic acid domain, one strand can conjugate with the analyte-binding domain, while the other strand can hybridize with the conjugated strand. In a particular embodiment, the single-stranded portion of a partially double-stranded domain can be part of the hybridization of that strand with the conjugated strand. As will be described in more detail below, the hybridized strand of a partially double-stranded nucleic acid domain (as opposed to the conjugated strand) can be considered a "sandwich strand" or a sandwich oligonucleotide.

[0097] Figure 1 Version 1 depicts a "conventional" proximity extension assay, in which the nucleic acid domain of each neighboring probe (shown as an arrow) is connected to the analyte-binding domain (shown as an inverted "Y") via its 5' end, leaving two free 3' ends. When the neighboring probes bind to their corresponding analytes (not shown in the figure), the nucleic acid structures of the probes (which are complementary at their 3' ends) can interact through hybridization, i.e., forming a double strand. The addition or activation of a nucleic acid polymerase in the assay mixture allows the use of the nucleic acid domain of another neighboring probe as a template to extend each nucleic acid domain, yielding the extension product.

[0098] Figure 1 Version 2 describes an alternative proximity extension assay in which the nucleic acid domain of a first proximity probe is linked to an analyte-binding domain via its 5' end, and the nucleic acid domain of a second proximity probe is linked to the analyte-binding domain via its 3' end. The nucleic acid domain of the second proximity probe thus has a free 5' end (shown as a blunt arrow), which cannot be extended using typical nucleic acid polymerases (which only extend the 3' end). The 3' end of the second proximity probe is effectively "blocked," i.e., it is not "free," and it cannot be extended because it is conjugated to the analyte-binding domain and thus blocked by it. In this embodiment, when the proximity probes bind to their respective analyte-binding targets on the analyte, the probe nucleic acid domains sharing complementary regions at their 3' ends can interact through hybridization, i.e., form a double strand. However, compared to version 1, using the nucleic acid domain of the second proximity probe as a template, only the nucleic acid domain of the first proximity probe (which has a free 3' end) can be extended, producing an extension product.

[0099] exist Figure 1 In version 3, similar to version 2, the nucleic acid domain of the first neighboring probe is linked to the analyte-binding domain via its 5' end, and the nucleic acid domain of the second neighboring probe is linked to the analyte-binding domain via its 3' end. The nucleic acid domain of the second neighboring probe thus has a free 5' end (shown as a blunt arrow) that cannot be extended. However, in this embodiment, the nucleic acid domains linked to the analyte-binding domains of the corresponding neighboring probes do not have complementary regions and therefore cannot directly form a double strand. Instead, a third nucleic acid molecule is provided, having regions homologous to the nucleic acid domains of each neighboring probe. This third nucleic acid molecule acts as a "molecular bridge" or "splint" between the nucleic acid domains. The splint oligonucleotide bridges the vacancies between the nucleic acid domains, allowing them to interact indirectly with each other; that is, each nucleic acid domain forms a double strand with the splint oligonucleotide.

[0100] Therefore, when neighboring probes bind to their corresponding analyte-binding targets on the analyte, the nucleic acid domains of each probe interact by hybridizing with the splice oligonucleotide, forming a double strand. Thus, the third nucleic acid molecule or splice can be considered as a second strand providing a partially double-stranded nucleic acid domain on one of the neighboring probes. For example, a partially double-stranded nucleic acid domain can be provided to one of the neighboring probes, which is connected to the analyte-binding domain via the 3' end of one strand, and the other (unconnected) strand has a free 3' end. Therefore, such a nucleic acid domain has a terminal single-stranded region with a free 3' end. In this embodiment, the nucleic acid domain of the first neighboring probe (which has a free 3' end) can be extended using a "splice oligonucleotide" (or a single-stranded 3' terminal region of another nucleic acid domain) as a template. Alternatively or additionally, the nucleic acid domain of the first neighboring probe can be used as a template to extend the free 3' end of the splice oligonucleotide (i.e., the unconnected strand, or the 3' single-stranded region).

[0101] As is apparent from the above description, in one embodiment, the splint oligonucleotide can be provided as a separate component of the assay. In other words, it can be added separately to the reaction mixture (i.e., separately to the adjacent probe to be added to the sample containing the analyte). Nevertheless, since it hybridizes with, and will hybridize upon contact with, the nucleic acid molecule that is part of the adjacent probe, it can still be considered as a strand of a partially double-stranded nucleic acid domain, even though it is added separately. Alternatively, the splint can be pre-hybridized with one of the nucleic acid domains of the adjacent probe, i.e., hybridized before the adjacent probe comes into contact with the sample. In this embodiment, the splint oligonucleotide can be directly considered as part of the nucleic acid domain of the adjacent probe, i.e., wherein the nucleic acid domain is a partially double-stranded nucleic acid molecule, for example, the adjacent probe can be prepared by concatenating a double-stranded nucleic acid molecule with an analyte-binding domain (preferably the nucleic acid domain is concatenated with the analyte-binding domain by a single strand) and modifying the nucleic acid molecule to produce a partially double-stranded nucleic acid domain (having a single-stranded overhang capable of hybridizing with the nucleic acid domain of another adjacent probe).

[0102] Therefore, the extension of the nucleic acid domain of the neighboring probe, as defined herein, also encompasses the extension of the "sandwich" oligonucleotide. Advantageously, when the extension product originates from the extension of the sandwich oligonucleotide, the resulting extended nucleic acid chain couples to the neighboring probe pair solely through interactions between the two strands of the nucleic acid molecule (via hybridization between the two nucleic acid chains). Thus, in these embodiments, the extension product can be dissociated from the neighboring probe pair using denaturing conditions (e.g., increased temperature, decreased salt concentration, etc.).

[0103] Although Figure 1The splint oligonucleotide described in version 3 is shown to be complementary to the full-length nucleic acid domain of the second neighboring probe, but this is only one example and is sufficient to enable the splint to form a double strand with the end (or near the end) of the nucleic acid domain of the neighboring probe, i.e., to form a bridge between the nucleic acid domains of the two probes.

[0104] In another embodiment, the splint oligonucleotide can be provided as the nucleic acid domain of the third neighboring probe, as described in WO2007 / 107743, which is incorporated herein by reference, suggesting that this can further improve the sensitivity and specificity of the neighboring probe assay.

[0105] Figure 1 Version 4 is a modification of version 1, wherein the nucleic acid domain of the first neighboring probe contains a sequence at its 3' end that is not perfectly complementary to the nucleic acid domain of the second neighboring probe. Therefore, when the neighboring probes bind to their respective analytes, the nucleic acid domains of the probes can hybridize, i.e., form a double strand, but the 3' end of the nucleic acid domain of the first neighboring probe (the portion of the nucleic acid molecule containing the free 3' hydroxyl group) cannot hybridize with the nucleic acid domain of the second neighboring probe, and thus exists as a single-stranded, unhybridized "flap". When adding or activating a nucleic acid polymerase, the nucleic acid domain of the first neighboring probe can be used as a template to extend only the nucleic acid domain of the second neighboring probe. Therefore, in this embodiment, only the 3' end of the nucleic acid domain of the second neighboring probe is "free"—the 3' end of the nucleic acid domain of the first neighboring probe is not "free" because it is not complementary to the nucleic acid domain of the second neighboring probe, and therefore does not hybridize with it and cannot be extended.

[0106] Figure 1 Version 5 can be seen as a modification of version 3. However, compared to version 3, the nucleic acid domains of two adjacent probes are connected to their respective analyte-binding domains via their 5' ends. In this implementation, the 3' ends of the nucleic acid domains are not complementary, so the nucleic acid domains of adjacent probes cannot directly interact or form double strands. Instead, a third nucleic acid molecule is provided, which has regions homologous to the nucleic acid domains of each adjacent probe. This third nucleic acid molecule acts as a "molecular bridge" or "splint" between the nucleic acid domains. This "splint" oligonucleotide bridges the vacancies between the nucleic acid domains, allowing them to interact indirectly with each other, i.e., each nucleic acid domain forms a double strand with the splint oligonucleotide. Therefore, when adjacent probes bind to their respective analytes, the nucleic acid domains of the probes interact with each other by hybridizing with the splint oligonucleotide, i.e., forming a double strand.

[0107] According to version 3, it can be seen that the third nucleic acid molecule or splice can be considered as a second strand providing a partially double-stranded nucleic acid domain on one of the adjacent probes. In a preferred embodiment, a partially double-stranded nucleic acid domain can be provided to one of the adjacent probes, which is connected to the analyte-binding domain via the 5' end of one strand, and wherein the other (unconnected) strand has a free 3' end. Thus, such a nucleic acid domain has a terminal single-stranded region with at least one free 3' end. In this embodiment, the nucleic acid domain of the second adjacent probe (which has a free 3' end) can be extended using a "splice oligonucleotide" as a template. Alternatively or additionally, the nucleic acid domain of the second adjacent probe can be used as a template to extend the free 3' end of the splice oligonucleotide (i.e., the unconnected strand, or the 3' single-stranded region of the first adjacent probe).

[0108] As discussed above in conjunction with Version 3, the splint oligonucleotide can be provided as a separate component of the assay. Alternatively, it can be considered as a strand of a partially double-stranded nucleic acid domain, even though it is added separately, because it hybridizes with and will hybridize upon contact with a nucleic acid molecule that is part of a neighboring probe. Alternatively, the splint can be pre-hybridized with one of the nucleic acid domains of a neighboring probe, i.e., hybridized before the neighboring probe comes into contact with the sample. In this embodiment, the splint oligonucleotide can be directly considered as part of a nucleic acid domain of a neighboring probe, i.e., wherein the nucleic acid domain is a partially double-stranded nucleic acid molecule, for example, the neighboring probe can be prepared by concatenating a double-stranded nucleic acid molecule with an analyte-binding domain (preferably the nucleic acid domain is concatenated with the analyte-binding domain via a single strand) and modifying the nucleic acid molecule to produce a partially double-stranded nucleic acid domain (having a single-stranded overhang capable of hybridizing with the nucleic acid domain of another neighboring probe).

[0109] Therefore, the extension of the nucleic acid domain of the neighboring probe, as defined herein, also encompasses the extension of the "sandwich" oligonucleotide. Advantageously, when the extension product originates from the extension of the sandwich oligonucleotide, the resulting extended nucleic acid chain couples to the neighboring probe pair solely through interactions between the two strands of the nucleic acid molecule (via hybridization between the two nucleic acid chains). Thus, in these embodiments, the extension product can be dissociated from the neighboring probe pair using denaturing conditions (e.g., increased temperature, decreased salt concentration, etc.).

[0110] Although Figure 1 The splint oligonucleotide described in version 5 is shown to be complementary to the full-length nucleic acid domain of the first neighboring probe, but this is only one example and is sufficient to enable the splint to form a double strand with the end (or near the end) of the nucleic acid domain of the neighboring probe, i.e., to form a bridge between the nucleic acid domains of the neighboring probe.

[0111] In another embodiment, the splint oligonucleotide can be provided as the nucleic acid domain of the third neighboring probe, as described in WO2007 / 107743, which is incorporated herein by reference, suggesting that this can further improve the sensitivity and specificity of the neighboring probe assay.

[0112] Figure 1 Version 6 is the most preferred embodiment of the invention. As depicted, both probes in a pair are conjugated to a partial single-stranded nucleic acid molecule. In each probe, a short nucleic acid strand is conjugated to an analyte-binding domain via its 5' end. The short nucleic acid strand conjugated to the analyte-binding domain does not hybridize with each other. Instead, each short nucleic acid strand hybridizes with a longer nucleic acid strand having a single-stranded overhang at its 3' end (that is, the 3' end of the longer nucleic acid strand extends beyond the 5' end of the shorter strand conjugated to the analyte-binding domain). The overhangs of the two longer nucleic acid strands hybridize with each other to form a double strand. The hybridized longer nucleic acid strands are referred to herein as "hybrid oligonucleotides". If the 3' ends of the two longer nucleic acid molecules hybridize completely with each other, as shown, the double strand contains two free 3' ends, although the 3' ends of the longer nucleic acid molecules can be designed as in Version 4 such that the 3' end of one of the longer nucleic acid molecules is not complementary to the other, forming an accessory wing, meaning the double strand contains only one free 3' end.

[0113] Therefore, it can be seen that when using PEA to perform the method of the present invention, in some embodiments, at least one nucleic acid domain in each adjacent probe pair is partially double-stranded. It is possible that one nucleic acid domain in each adjacent probe pair is partially double-stranded (as in versions 3 and 5). Preferably, both nucleic acid domains in each adjacent probe pair are partially double-stranded, as in version 6.

[0114] As detailed in version 6, it is preferred that the partially double-stranded nucleic acid domains contain:

[0115] (i) a first oligonucleotide conjugated to the binding domain of the analyte; and

[0116] (ii) A hybrid oligonucleotide comprising the first hybridization sequence, the ID sequence and the second hybridization sequence, wherein the first hybridization sequence is located at the 3' end of the hybrid oligonucleotide;

[0117] The double-stranded portion of the nucleic acid domain comprises a double strand between the second hybridization sequence of the hybrid oligonucleotide and the first oligonucleotide, and the single-stranded portion of the nucleic acid domain comprises the first hybridization sequence of the hybrid oligonucleotide.

[0118] In one particular embodiment, the hybrid oligonucleotide comprises the second hybrid sequence, the ID sequence, and the first hybrid sequence from 5' to 3', and the ID sequence (preferably a barcode sequence) is located in the single-stranded portion of the nucleic acid domain.

[0119] The situation might be such that all neighboring probes contain the same first oligonucleotide and the same second hybridization sequence (within the hybridization oligonucleotide). In other words, all neighboring probes can share a common first oligonucleotide and second hybridization sequence. This could lead to a more straightforward probe manufacturing process.

[0120] As detailed above, the first oligonucleotide and the second hybridization sequence are complementary to each other, enabling the two sequences to hybridize. In one particular embodiment, the second hybridization site is complementary to the entire first oligonucleotide, such that the duplex formed between them contains the entire first oligonucleotide. However, this is not necessary, and it is possible that the second hybridization site is complementary to only a portion of the first oligonucleotide, such that the duplex formed between them contains only a portion of the first oligonucleotide. Also as mentioned above, the first hybridization sequence is located at the 3' end of the hybridization oligonucleotide, such that when two probes with complementary first hybridization sequences become adjacent, the 3' ends of their hybridization oligonucleotides hybridize with each other. "Located at the 3' end" can mean that the first hybridization sequence extends to the 3' end of each hybridization oligonucleotide, i.e., the first hybridization sequence may include the 3' nucleotide of each hybridization oligonucleotide. However, this is not necessary, and the first hybridization sequence may alternatively extend only to the 3' end of one hybridization oligonucleotide in each probe pair. Therefore, the nucleic acid domains used in PEA version 6 can be designed in the same way as version 4, such that one of the hybrid oligonucleotides in each probe pair contains a sequence at its 3' end that is not completely complementary to the hybrid oligonucleotide of the other neighboring probe, thus forming a single-stranded, unhybridized "flap".

[0121] Therefore, after the two nucleic acid domains hybridize with each other, at least one hybrid oligonucleotide is extended to produce an extension product (in other words, one or both hybrid oligonucleotides are extended to produce an extension product). If the first hybridization sequence extends to the 3' end of both hybrid oligonucleotides in each probe pair, then both hybrid oligonucleotides may be extended to produce an extension product. On the other hand, if one of the hybrid oligonucleotides contains an unhybridized flap at its 3' end (as detailed above), then only one hybrid oligonucleotide is extended to produce an extension product (i.e., a hybrid oligonucleotide without a flap).

[0122] When the multiplex assay being performed is PEA, it is preferable to perform the extension reaction in conjunction with PCR amplification, or in other words, to perform a single reaction including PCR amplification to extend the adjacent probe nucleic acid domains, thereby generating a reporter nucleic acid molecule and amplifying the resulting reporter nucleic acid molecule. In this embodiment, the reaction does not begin with a denaturation step (as is typically the case in PCR), but rather with an extension step, during which the reporter nucleic acid molecule is generated. Subsequently, starting with the denaturation of the reporter molecule, standard PCR is performed to amplify the reporter nucleic acid molecule. As detailed above, PCR is performed using common primers that bind to a common sequence at the ends of the reporter nucleic acid molecule. As detailed below, one or both of the primers may also contain sequencing aptamers. In other words, the extension and amplification steps of the method of the present invention can be performed in a single reaction.

[0123] In another embodiment, the multiplex assay used to detect an analyte in a sample is a PLA. The PLA used can be a “standard” PLA. This means that the PLA uses a single splint oligonucleotide to bind the nucleic acid domains of two adjacent probes. In a standard PLA, the nucleic acid domain of each adjacent probe pair contains a pairing hybridization sequence that hybridizes with the splint oligonucleotide to form a double strand. The nucleic acid domains of the adjacent probe pair are conjugated to their respective probes such that, in each pair, one adjacent probe has a nucleic acid domain with a free 3' end, and the other adjacent probe has a nucleic acid domain with a free 5' end, allowing the free ends of the nucleic acid domains of the two probes to join together. The splint oligonucleotide may contain an extension blocker at its 3' end, preventing it from being extended. After the double strand is formed, the nucleic acid domains of the two adjacent probes are directly or indirectly linked to each other to produce a ligation product containing the ID sequence of the first adjacent probe and the ID sequence of the second adjacent probe.

[0124] When multiplexing is PLA, it is preferable that the nucleic acid domains of adjacent probe pairs hybridize with the splice oligonucleotides, resulting in a break in the double strand between the two nucleic acid domains, but no vacancies. In other words, the 3' end of one nucleic acid domain can hybridize with a splice nucleotide, which is directly adjacent to the splice nucleotide that hybridizes with the 5' end of the other nucleic acid domain. This allows the two nucleic acid domains to be directly linked to each other.

[0125] Alternatively, the nucleic acid domains of adjacent probe pairs can hybridize with the splice oligonucleotide, resulting in a vacancy between the 3' end of one nucleic acid domain and the 5' end of the other. In this embodiment, the double-stranded structure formed between the splice oligonucleotide and the two probe nucleic acid domains comprises a single-stranded nucleic acid of a certain length from the splice oligonucleotide, separating the two parts of the double-stranded structure. The length of the single-stranded vacancy can be any number of nucleotides. In this embodiment, a vacancy-filling extension reaction is performed to fill the vacancy between the ends of the two probe nucleic acid domains (i.e., extending the probe nucleic acid domain containing the free 3' end to fill the vacancy). After vacancy filling, a ligase is used to ligate the nucleic acid domains of the two splice oligonucleotides to each other. This ligation of nucleic acid domains to each other after vacancy filling is referred to herein as “indirect ligation” of nucleic acid domains to each other.

[0126] Vacancies-filling extension reactions are performed using polymerases lacking strand displacement activity, so that extension ends when the vacancy is filled, rather than by displacing the free hybrid nucleic acid domain downstream of the 3' end. Non-displacement polymerases include T4 DNA polymerase. Other such polymerases are known in the art.

[0127] After ligation, the ligation product is amplified (e.g., by PCR) and detected as described above. These PLA implementations yield linear ligation (or extension and ligation) products. Preferably, the ligation product, or extension and ligation product, generated in the method of the present invention is linear.

[0128] Alternatively, the PLA used can be rolling circle amplification PLA (PLA-RCA). This PLA format uses two splice oligonucleotides that are linked together to produce a ligation product. PLA-RCA, for example... As described in Nature Methods 3(12):995-1000 (2006), in this embodiment, neighboring probes each comprise a nucleic acid domain containing two hybridization sequences. The first hybridization sequence is complementary to the hybridization sequence on a first clip oligonucleotide, and the second hybridization sequence is complementary to the hybridization sequence on a second clip oligonucleotide. The first hybridization sequences are paired, as described above, such that the same pair of first hybridization sequences is shared by multiple neighboring probe pairs. As detailed above, these hybridize with a specific first clip oligonucleotide. The second hybridization sequences may also be paired, but more preferably, these are universal sites shared by all neighboring probe nucleic acid domains in multiplex assays, such that all neighboring probe pairs require only a single second clip oligonucleotide.

[0129] In PLA-RCA, the ID sequence (preferably a barcode sequence) of the probe nucleic acid domain is located between the first and second hybridization sites. After the two splice oligonucleotides bind to the probe nucleic acid domain, a vacancy-filling extension reaction is performed, as described above. The two splice oligonucleotides are then linked together to form a circular molecule, which is amplified by rolling circle amplification and detected.

[0130] As mentioned above, it is preferable to detect reporter nucleic acids through massively parallel DNA sequencing, which typically requires adding sequencing aptamers to the DNA molecule to be sequenced. As described above, sequencing aptamers serve to immobilize the DNA molecule on a surface.

[0131] Therefore, the method of the present invention may include adding one or more aptamers for sequencing (sequencing aptamers) to the reporter nucleic acid.

[0132] Typically, sequencing aptamers are nucleic acid molecules (particularly DNA molecules). In this case, a short oligonucleotide complementary to the aptamer sequence is conjugated to a stationary surface (e.g., a bead or the surface of a flow cell), allowing the target DNA molecule to anneal to the surface via the aptamer sequence. Alternatively, any other binding pair can be used to conjugate the target DNA molecule to the stationary surface, such as biotin and avidin / streptavidin. In this case, biotin can be used as the sequencing aptamer, and avidin or streptavidin conjugates to the stationary surface to bind the biotin-based sequencing aptamer, and vice versa.

[0133] Therefore, sequencing aptamers can be short oligonucleotides (preferably DNA), typically 10-30 nucleotides long (e.g., 15-25 or 20-25 nucleotides long). As detailed above, the purpose of sequencing aptamers is to enable the target DNA molecule to anneal to the immobilization surface; therefore, the nucleotide sequence of the nucleic acid aptamer is determined by the sequence of its binding partner that conjugates to the immobilization surface. Apart from this, there are no particular restrictions on the nucleotide sequence of nucleic acid sequencing aptamers.

[0134] The sequencing aptamer can be added to the reporter nucleic acid of the present invention during PCR amplification. In the case of a nucleic acid sequencing aptamer, this can be achieved by including the sequencing aptamer nucleotide in one or both primers. Alternatively, if the sequencing aptamer is a non-nucleic acid sequencing aptamer (e.g., a protein / peptide or small molecule), the aptamer can be conjugated to one or both PCR primers. Alternatively, the sequencing aptamer can be ligated to the reporter nucleic acid molecule directly or conjugated. Preferably, one or more sequencing aptamers used in the method of the present invention are nucleic acid sequencing aptamers.

[0135] Therefore, one or more nucleic acid sequencing aptamers can be added to the reporter nucleic acid in one or more ligation and / or amplification steps. Thus, for example, if two sequencing aptamers are added to the reporter nucleic acid molecule (one at each end), these aptamers can be added in a single step (e.g., by PCR amplification using a pair of primers both containing the sequencing aptamer) or in two steps. The two steps can be performed using the same or different methods; for example, the first sequencing aptamer can be added to the reporter nucleic acid molecule by ligation, and the second sequencing aptamer can be added by PCR amplification, or vice versa; or a first amplification reaction can be performed to add the first sequencing aptamer to the reporter nucleic acid molecule, followed by a second amplification reaction to add the second sequencing aptamer to the reporter nucleic acid molecule.

[0136] As described above, one or more sequencing aptamers can be added to a reporter nucleic acid molecule. This means one or two sequencing aptamers—because sequencing aptamers are added to the ends of DNA molecules, the maximum number of sequencing aptamers that can be added to a single DNA molecule (e.g., a reporter nucleic acid) is two. Therefore, a single sequencing aptamer can be added to one end of a reporter nucleic acid molecule, or two sequencing aptamers can be added to the reporter nucleic acid molecule, one to each end. In a particular embodiment, Illumina P5 and P7 aptamers are used, i.e., the P5 aptamer is added to one end of the reporter nucleic acid molecule and the P7 aptamer is added to the other end. The sequence of the P5 aptamer is shown in SEQ ID NO:1 (AAT GATACG GCG ACC ACC GA), and the sequence of the P7 aptamer is shown in SEQ ID NO:2 (CAA GCA GAA GAC GGCATA CGA GAT).

[0137] PCR amplification can therefore be combined with the addition of one or more sequencing aptamers to a reporter nucleic acid molecule. This can be achieved by amplifying the reporter nucleic acid molecule using a primer pair containing at least one sequencing aptamer. In this case, at least one primer in the primer pair contains a sequencing aptamer upstream of the sequence that binds to the reporter nucleic acid molecule. Therefore, the sequencing aptamer is typically located at the 5' end of any primer that contains it.

[0138] In a particular implementation, an amplification step is performed using a primer pair containing a primer with a sequencing aptamer, such that a single sequencing aptamer is added to one end of a reporter nucleic acid molecule.

[0139] In another implementation, an amplification step is performed using a primer pair in which both primers contain a sequencing aptamer, such that the sequencing aptamer is added to each end of the reporter nucleic acid molecule in a single amplification step.

[0140] In another implementation, two separate amplification reactions are performed to add sequencing aptamers to each end of the reporter nucleic acid molecule, wherein each amplification step adds a different sequencing aptamer to a different end of the molecule.

[0141] In another implementation, an initial amplification step is performed using primers that do not contain sequencing aptamers. The amplified reporter nucleic acid molecule is then subjected to one or more additional amplification reactions to add sequencing aptamers to each end of the molecule, as described above.

[0142] Preferably, the reporter nucleic acid (i.e., the extension and / or ligation product) is amplified in two PCR steps. In the first PCR reaction, a first sequencing aptamer is added to one end of the extension or ligation product. Then, in the second PCR reaction, the product of the first PCR reaction is amplified, wherein a second sequencing aptamer is added to the other end of the reporter nucleic acid. In a particular embodiment, the first PCR reaction is performed with a nucleic acid polymerase that also has 3' to 5' exonuclease activity, and the second PCR reaction is performed with a nucleic acid polymerase lacking 3' to 5' exonuclease activity, as described in WO 2012 / 104261. Suitable nucleic acid polymerases with 3' to 5' exonuclease activity include T4 DNA polymerase, T7 DNA polymerase, Phi29 (Φ29) DNA polymerase, DNA polymerase I, the Klenow fragment of DNA polymerase I, Pyrococcus furiosus (Pfu) DNA polymerase, and Pyrococcus woesei (Pwo) DNA polymerase. Suitable nucleic acid polymerases lacking 3' to 5' exonuclease activity include the α subunit of DNA polymerase III, the Klenow exo(-) fragment of DNA polymerase I, Taq polymerase, and Pfu(exo) - DNA polymerase and Pwo (exo) - DNA polymerase.

[0143] In another implementation, the same polymerase can be used for both PCR steps, such as Pwo or Pfu polymerase.

[0144] The method of this invention can be used to simultaneously measure multiple samples. In this case, each sample is individually measured multiple times as described above. Once extension and / or ligation products are generated, a sample index sequence is added. The sample index is a nucleotide sequence that identifies the source sample from which the extension and / or ligation products are derived. Therefore, different nucleotide sequences are used as sample index sequences for extension / ligation products derived from each different sample. Conversely, all extension and / or ligation products from a particular sample are labeled with the same sample index sequence.

[0145] Once all products are tagged with a sample index, products from multiple samples can be pooled and analyzed. When sequencing reporter nucleic acids, the sample index indicates which sample each individual reporter nucleic acid molecule originated from. Any nucleotide sequence can be used as a sample index. The sample index sequence can be of any length, but is preferably relatively short, such as 3–12, 4–10, or 4–8 nucleotides.

[0146] The sample index sequence can be added to the extension / ligation product by any suitable method, such as adding it during the amplification reaction (e.g., by PCR) or the ligation reaction. It is important to note that if the reporter nucleic acid molecule is to be analyzed by massively parallel DNA sequencing and requires sequencing aptamers at both ends, the sample index sequence should not be added so that it ends up at the end of the reporter nucleic acid molecule.

[0147] In a preferred embodiment, the sample index sequence is added to the extension / ligation product during PCR amplification. As described above, the sequencing aptamer can also be added to the extension / ligation product during PCR amplification. In one particular embodiment, a dedicated amplification step can be performed specifically to add the sample index to the reporter nucleic acid molecule. In another embodiment, the sample index can be added simultaneously with one or more sequencing aptamers during PCR amplification. For example, if a single PCR amplification is performed to add a sequencing aptamer to both ends of the reporter nucleic acid molecule, the sample index can be added simultaneously. Alternatively, if the sequencing aptamer is added to each end of the reporter nucleic acid molecule in two consecutive PCR amplifications, the sample index can be added during either PCR amplification. The sample index can be added during the first PCR amplification, in which case it can be added to the same end (within the sequencing aptamer) or the opposite end of the reporter nucleic acid. Alternatively, the sample index can be added during the second PCR amplification, in which case it must be added to the same end (within the sequencing aptamer) of the reporter nucleic acid. Alternatively, a ligation step can be performed to add the sample index to the end of each reporter nucleic acid molecule prior to amplification.

[0148] A second aspect of the present invention is a product that can be used to perform the method of the first aspect of the present invention. Specifically, as described above, the product comprises:

[0149] (i) Multiple neighbor probe pairs, wherein each neighbor probe pair comprises a first neighbor probe and a second neighbor probe, and each neighbor probe comprises:

[0150] (a) Protein-binding domains that are specific to proteins; and

[0151] (b) Nucleic acid domains,

[0152] Each pair of probes contains a protein-binding domain that is specific to the same protein and can bind to the protein simultaneously; and each probe pair is specific to different proteins.

[0153] Each neighboring probe's nucleic acid domain comprises an ID sequence and at least a first hybridization sequence, wherein the ID sequence of each neighboring probe is different; and wherein in each neighboring probe pair, the first neighboring probe and the second neighboring probe comprise a pairing hybridization sequence; and, optionally...

[0154] (ii) A plurality of splice oligonucleotides, each splice oligonucleotide containing a hybridization sequence complementary to each of the pairing hybridization sequences of the adjacent probe pairs;

[0155] The hybridization sequence of each neighboring probe pair is configured such that when the first neighboring probe and the second neighboring probe bind to their proteins, the corresponding paired hybridization sequences of the first neighboring probe and the second neighboring probe hybridize to each other or to the splint oligonucleotide.

[0156] Furthermore, at least one pair of hybridization sequences is shared by at least two adjacent pairs of probes.

[0157] The various features in this aspect are identical to the equivalent features in the first aspect (e.g., neighboring probes, ID sequences, splint oligonucleotides, hybridization sequences, etc.). It is noteworthy that in this aspect of the invention, both probes within each probe pair contain protein-binding domains specific for the same protein. In other words, in each probe pair of the product, the two probes bind to the same protein. As detailed above, the two probes in each probe pair bind their target protein to different epitopes, such that they do not interfere with each other's binding to the target. The probes of the present invention can be designed for any pattern or variation of PEA or PLA as described above.

[0158] In one embodiment, the product of the present invention further comprises one or more background probes (or inert probes) that do not bind to the analyte, as described above. As in the method of the present invention, it is preferred that a large proportion of probe pairs share their hybridization sequences with at least one other neighboring probe pair. In a particular embodiment, at least 25%, 50%, or 75% of neighboring probe pairs share their hybridization sequences with another neighboring probe pair (i.e., with at least one other neighboring probe pair), as in the method. In a particular embodiment, all neighboring probe pairs share their hybridization sequences with at least one other neighboring probe pair. However, as is apparent from the above, in another embodiment, at least one pair of hybridization sequences is unique for a single pair of neighboring probes. That is, at least one pair of neighboring probes does not share its hybridization sequence with any other neighboring probe pair. In a particular embodiment, at most 75%, 50%, or 25% of paired neighboring probes do not share their hybridization sequences with any other neighboring probe pair. As in the method, in some embodiments, no more than 20, 15, 10, or 5 neighboring probe pairs in the product share the same pair of hybridization sequences.

[0159] The product of this invention can be supplied as a single composition comprising all neighboring probes (and, if present, splice oligonucleotides and / or inert probes). Alternatively, all components of the product can be supplied in separate containers. For example, neighboring probe pairs, splice oligonucleotides, and inert probes can all be supplied in separate containers. If desired, each probe pair or even each individual neighboring probe can be supplied in a separate container, as can each different splice oligonucleotide and each different inert probe.

[0160] The product may also contain additional components for use in the methods of this invention. For example, the product may contain one or more nucleic acid polymerases for extension and / or amplification steps, and / or ligases if a ligation step is required. The product may contain primers for amplification. As detailed above, primers for the amplification step may contain sequence aptamers and / or sample index sequences for nucleic acid sequencing. The product may also contain nucleotides (e.g., dATP, dCTP, dGTP, and dTTP) for the extension / amplification reaction. The product may contain solid bases on which the reporter nucleic acid can be immobilized for sequencing, such as in a flow cell or beads.

[0161] The invention can be further understood by referring to the following non-limiting embodiments and accompanying drawings. Attached Figure Description

[0162] Figure 1 Schematic illustrations of six different versions of proximity extension assays are shown, as detailed above. The inverted "Y" shape represents the antibody, serving as an exemplary proximity probe analyte binding domain.

[0163] Figure 2 The results obtained by multiple PEA determination of expression levels of four analytes in six different samples using conventional negative controls or shared hybridization site negative controls are shown.

[0164] Figure 3 Six different samples (and samples used for) were identified by multiple PEA using either a conventional negative control or a shared hybridization site negative control. Figure 2 A comparison of the results obtained for the expression levels of five analytes in the same sample.

[0165] Example

[0166] Plasma samples were obtained from six donors: three healthy subjects, one subject diagnosed with breast cancer, one subject diagnosed with rheumatoid arthritis (RA), and one subject diagnosed with inflammatory bowel disease (IBD).

[0167] Multiplex PEA (using probes containing antibodies conjugated to nucleic acid domains having the structures described in Version 6 above) was performed to detect nine proteins in the sample: NPDC1 (UniProt Q9NQX5); AHCY (UniProt P23526); TM (UniProt P07204); ANGPTL1 (UniProt O95841); LOX-1 (UniProt P78380); SEMA3F (UniProt Q13275); CDH2 (UniProt P19022); CANT1 (UniProt Q8WVQ1); and CA13 (UniProt Q8N1Q1). Probes targeting NPDC1, AHCY, TM, and ANGPTL1 all share a single pair of hybridization sites; while probes targeting LOX-1, SEMA3F, CDH2, CANT1, and CA13 all share a single pair of distinct hybridization sites. Each probe contains a unique barcode sequence. A negative control was also used, which consisted of phosphate-buffered saline containing 1% bovine serum albumin and no sample.

[0168] PEA was performed as described above. During the amplification of the extension products, along with unique sample indexes of the reporter nucleic acid from each different sample, P5 and P7 sequencing aptamers were added to each end of the product, and all extension products were sequenced by massively parallel DNA sequencing using reversible dye terminator sequencing technology on the Illumina NovaSeq platform.

[0169] The background from the standard negative control of the target is determined by the pairing barcode interactions of the target probes. For each sample, the background from the shared hybridization site of the target is determined by the average of the mismatch interactions (determined by the mismatch barcode) between each corresponding probe of the target probe pair and other probes within that group (i.e., probes that share a hybridization site with the target probe). In other words, for each target background from a shared hybridization site, it is defined as the nonspecific interactions between each probe of the target and other probes that share a shared hybridization site. Nonspecific interactions between probes that do not bind to the target are not included in the background calculation.

[0170] The following results were obtained from the two sets of target analytes:

[0171] Group 1 - Linear Analysis

[0172] Signal above background from negative control:

[0173]

[0174]

[0175] Signal above background from shared hybridization sites:

[0176]

[0177] Group 1 - Logarithmic Analysis (Base 2)

[0178] Signal above background from negative control:

[0179]

[0180] Signal above background from shared hybridization sites:

[0181]

[0182]

[0183] Logarithmic results in Figure 2 As shown in the image.

[0184] Group 2 - Linear Analysis

[0185] Signal above background from negative control:

[0186]

[0187] Signal above background from shared hybridization sites:

[0188]

[0189] Group 2 - Logarithmic Analysis (Base 2)

[0190] Signal above background from negative control:

[0191]

[0192]

[0193] Signal above background from shared hybridization sites:

[0194]

[0195] Logarithmic results in Figure 3 As shown in the image.

[0196] Although the actual values ​​of the above-background signal may differ between the two types of controls (negative control and common hybridization site control) for both groups of analytes, the changes in value are roughly the same for each analyte per sample (i.e., parallel changes exist). The results obtained for each analyte are characterized by a high Rw. 2 The values ​​demonstrate this, showing a very high correlation between the above-background signal levels determined by each of the two methods. These results prove that using a shared hybridization site is an effective alternative to a standard negative control, as the relative signal levels of the analyte are maintained between samples. Background determination results from the shared hybridization site show similar differences between samples as when using a standard negative control. SEQUENCE LISTING <110> Orinco Protein Company <120> Control for proximity detection assay <130> P22116653WP <150> GB2004469.9 <151> 2020-03-27 <160> 2 <170> PatentIn version 3.5 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> P5 aptamer <400> 1 aatgatacgg cgaccaccga 20 <210> 2 <211> twenty four <212> DNA <213> Artificial Sequence <220> <223> P7 aptamer <400> 2 caagcagaag acggcatacg agat 24

Claims

1. A product comprising: (i) Multiple neighbor probe pairs, wherein each neighbor probe pair comprises a first neighbor probe and a second neighbor probe, and each neighbor probe comprises: (a) Protein-binding domains that are specific to proteins; and (b) Nucleic acid structural domains, Each pair of probes contains a protein-binding domain that is specific to the same protein and can bind to the protein simultaneously; and each probe pair is specific to different proteins. Each neighboring probe's nucleic acid domain includes an ID sequence and at least a first hybridization sequence, wherein the ID sequence of each neighboring probe is a barcode sequence corresponding to a specific analyte; and wherein in each neighboring probe pair, the first neighboring probe and the second neighboring probe include a pairing hybridization sequence; The hybridization sequence of each neighboring probe pair is configured such that, when the first neighboring probe and the second neighboring probe bind to their proteins, the corresponding paired hybridization sequences of the first neighboring probe and the second neighboring probe hybridize to each other or to a splint oligonucleotide, the splint oligonucleotide comprising a hybridization sequence complementary to each of the paired hybridization sequences of the neighboring probe pair. Furthermore, at least one pair of hybridization sequences is shared by at least two adjacent pairs of probes.

2. The product of claim 1, wherein the product further comprises (ii) a plurality of splice oligonucleotides, each splice oligonucleotide comprising a hybridization sequence complementary to each of the pairing hybridization sequences of the neighboring probe pairs; and wherein the hybridization sequence of each neighboring probe pair is configured such that, when the first neighboring probe and the second neighboring probe bind to their proteins, the corresponding pairing hybridization sequence of the first neighboring probe and the second neighboring probe hybridizes with the splice oligonucleotide.

3. The product of claim 1, wherein the hybridization sequence of each neighboring probe pair is configured such that when the first neighboring probe and the second neighboring probe bind to their proteins, the corresponding paired hybridization sequences of the first neighboring probe and the second neighboring probe hybridize with each other.

4. The product of claim 1, wherein the protein binding domain is an antibody or a fragment thereof.

5. The product of claim 1, further comprising one or more background probes that do not bind to the analyte, the background probes comprising: a nucleic acid domain comprising an ID sequence and a hybridization sequence shared with at least one neighboring probe.

6. The product of claim 1, wherein at least one pair of hybridization sequences is unique for a single pair of neighboring probes.

7. The product of claim 1, wherein no more than 10 adjacent probe pairs share the same pair of hybridization sequences.

8. The product of claim 1, wherein at least 75% of the neighboring probe pairs share a pair of hybridization sequences with another neighboring probe pair.

9. The product of claim 1, wherein the nucleic acid domain of each adjacent probe pair comprises a complementary hybridization sequence that hybridizes with each other to form a double strand.

10. The product of claim 1, wherein in each adjacent probe pair, at least one nucleic acid domain is partially double-stranded.

11. The product of claim 1, wherein in each adjacent probe pair, both nucleic acid domains are partially double-stranded.

12. The product of claim 11, wherein the partially double-stranded nucleic acid domain comprises: (i) a first oligonucleotide conjugated to the binding domain of the analyte; and (ii) A hybrid oligonucleotide comprising the first hybridization sequence, the ID sequence, and the second hybridization sequence, wherein the first hybridization sequence is located at the 3' end of the hybrid oligonucleotide; The double-stranded portion of the nucleic acid domain comprises a double strand between the second hybridization sequence of the hybrid oligonucleotide and the first oligonucleotide, and the single-stranded portion of the nucleic acid domain comprises the first hybridization sequence of the hybrid oligonucleotide.

13. The product of claim 12, wherein the hybrid oligonucleotide comprises the second hybrid sequence, the ID sequence, and the first hybrid sequence from 5' to 3', and the ID sequence is located in the single-stranded portion of the nucleic acid domain.

14. The product of claim 1, wherein the nucleic acid domain is a DNA domain.

15. The product of claim 1, wherein the ID sequence of each neighboring probe is different.

16. The product as claimed in claim 1, wherein, The nucleic acid domains of the two adjacent probes in a pair are both conjugated to the protein-binding domain via their 5' ends.

17. The product of claim 2, wherein the nucleic acid domains of the adjacent probe pair hybridize with the splice oligonucleotide such that a vacancy exists between the 3' end of one nucleic acid domain and the 5' end of the other nucleic acid domain.

18. The product of claim 1, further comprising a plurality of sample index oligonucleotides having nucleotide sequences that identify the source sample.

19. The product of claim 1, comprising at least 25 adjacent probe pairs.

20. The product of claim 1, comprising at least 50 adjacent probe pairs.

21. The product of claim 1, comprising at least 100 adjacent probe pairs.

22. The product of claim 1, wherein no more than 20 adjacent probe pairs share the same hybridization sequence.

23. The product of claim 1, wherein no more than 15 adjacent probe pairs share the same hybridization sequence.

24. The product of claim 1, wherein no more than 5 adjacent probe pairs share the same hybridization sequence.

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