System and method for detecting analytes in a sample

Abstract

The invention relates to a system comprising: a) at least one pair of oligonucleotides, wherein the first member and the second member of the oligonucleotide pair have complementary nucleotide sequences at their respective 3'-ends and are able to hybridize to each other at the 3'-ends, the first and / or second member comprising an identification sequence unique to the individual member or unique to the oligonucleotide pair; b) optionally, at least one index oligonucleotide, comprising an index sequence and a member association sequence; and c) a polymerase having 3'-exonuclease activity; wherein the first or the second member of the oligonucleotide pair of (a) is, at its 3'-end, blocked from being extended and / or from being degraded by the polymerase, and / or the index oligonucleotide of (b) is, if present, at its 3'-end, blocked from being extended and / or from being degraded by the polymerase The invention also relates to methods for detecting analytes.

Description

[0001] SYSTEM AND METHOD FOR DETECTING ANALYTES IN A SAMPLE

[0002] The present application claims priority from Swedish patent application 2450669-3 filed 18 June 2024, which application is incorporated by reference herein.

[0003] Field of the invention

[0004] The present invention relates to tools for biotechnological research, and in particular is directed to products and methods that are useful in improving the signal-to-noise ratio in proximity assays for detecting analytes in a sample.

[0005] Background

[0006] Modern proteomics methods require the ability to detect a large number of different proteins (or protein complexes) in a small sample volume. To achieve this, multiplex analysis must be performed. Common methods by which multiplex detection of proteins in a sample may be achieved include proximity extension assays (PEA) and proximity ligation assays (PLA). PEA and PLA are described in US 7,306,904; multiplexed PLA is described in Lundberg et al., Molecular & Cellular Proteomics 2011, 10, DOI 10.1074 / mcp.M110.004978; aspects of PEA is further described in US 8,013,134, US 8,580,504, US 9,902,993, US 2023 / 0159983, US2023 / 0323424, US2022 / 0162589; Assarsson et al., PLoS 1, 2014, 9(4), e95192;

[0007] Lundberg et al., Nucleic Acids Research, 2011, Vol. 39, No. 15 e102; and Wik et al., 2021 , Mol Cell Proteomics 20, 100168, all incorporated herein by reference in their entirety.

[0008] WO 2014 / 144371 describes methods and reagents for detection and analysis of nucleic acids. The methods employ proximity extension assays for detection of a target nucleic acid of interest, e.g., a target RNA, and mentions protein proximity extension assays to detect protein.

[0009] PEA and PLA are proximity assays, which rely on the principle of “proximity probing”. In these methods, an analyte is detected by the binding of multiple (i.e. two or more, generally two or three) probes, which when brought into proximity by binding to the analyte (hence "proximity probes") allow a signal to be generated. Typically, at least one of the proximity probes comprises a nucleic acid domain (or moiety) linked to the analyte-binding domain (or moiety) of the probe, and generation of the signal involves an interaction between the nucleic acid moieties and / or a further functional moiety which is carried by the other probe(s). Thus, signal generation is dependent on an interaction between the probes (more particularly between the nucleic acid or other functional moieties / domains carried by them) and hence only occurs when the necessary probes have bound to the analyte, thereby lending improved specificity to the detection system.

[0010] In PEA, nucleic acid moieties linked to the analyte-binding domains of a probe pair hybridise to one another when the probes are in close proximity (i.e. when bound to the same target molecule, or to target molecules which are in close proximity, for example in a complex, interaction, or aggregate, or when two molecules are closely co-located), and are then extended using a nucleic acid polymerase. The extension product forms a reporter nucleic acid, detection of which demonstrates the presence of a particular analyte (the analyte bound by the relevant probe pair) in a sample of interest. The reporter molecule can be detected through qPCR (Assarsson et al.) or by sequencing PCR-amplified DNA reporter using Next Generation Sequencing, NGS (Wik et al.). In PLA, nucleic acid moieties linked to the analyte-binding domains of a probe pair come into proximity when the probes of the probe pair bind their target, and may be ligated together, or alternatively they may together template the ligation of separately added oligonucleotides which are able to hybridise to the nucleic acid domains when they are in proximity. The ligation product is then amplified, acting as a reporter nucleic acid. Multiplex analyte detection using PEA or PLA may be achieved by including a unique barcode sequence in the nucleic acid moiety of each probe. A reporter nucleic acid molecule corresponding to a particular analyte may be identified by the barcode sequences it contains.

[0011] Panels of proximity assays, as described above, are commercially available from Olink Proteomics AB (Uppsala, Sweden) under the trademark Olink® Target, Olink® Focus, and Olink® Explore. These are fixed panels of up to 92 assays (Olink® Target and Focus) or up to -5,400 assays (Olink® Explore). Each panel generally includes assays for proteins that have known functions within certain biological or physiological areas, pathways or organs in the body, such as inflammation, organ-specific proteins, cardiovascular, neurology etc.

[0012] Enroth et al., Communications Biology 2(1), 2019, pages 1-12 (DOI: 10.1038 / s42003-019- 0464-9) describes the use of Olink Panels, and custom-designed panels, in PEA assays to identify a novel high accuracy plasma protein biomarker signature for ovarian cancer.

[0013] The signal signifying the presence of a protein is carried by correctly amplified reporter molecules. However, amplification of the reporter molecules is a complex process that introduces noise in the form of incorrectly or incompletely amplified reporter molecules.

[0014] Thus, there is a continuous need in the art for products and methods providing an improved signal-to-noise ratio in proximity assays. Summary

[0015] The above objective to provide products and methods for improved signal-to-noise ratio in proximity assays is achieved by the present invention, which is directed to systems comprising modified oligonucleotides and uses thereof in methods for detecting analytes in a sample. The products and methods of the present invention find particular utility in multiplex proximity extension assays (PEA) methods.

[0016] More particularly, the present invention provides a system comprising: a) at least one pair of oligonucleotides, wherein the first member and the second member of the oligonucleotide pair have complementary nucleotide sequences at their respective 3’-ends and are able to hybridize to each other at the 3’-ends, the first and / or second member comprising an identification sequence unique to the individual member or unique to the oligonucleotide pair; b) at least one index oligonucleotide, comprising an index sequence and a member association sequence; and c) a polymerase having 3’-exonuclease activity; characterized in that the index oligonucleotide of (b) is, at its 3’-end, blocked from being extended and / or protected from being degraded by the polymerase.

[0017] Further provided is a system comprising: a) at least one pair of oligonucleotides, wherein the first member and the second member of the oligonucleotide pair have complementary nucleotide sequences at their respective 3’-ends and are able to hybridize to each other at the 3’-ends, the first and / or second member comprising an identification sequence unique to the individual member or unique to the oligonucleotide pair; b) a polymerase having 3’-exonuclease activity; characterized in that the first or the second member of the oligonucleotide pair of (a) is, at its 3’-end, blocked from being extended and / or from being degraded by the polymerase.

[0018] Additionally, the present invention provides a method for detecting at least one analyte in a sample, comprising: contacting the sample with at least one pair of proximity probes, each proximity probe pair being specific for one analyte, and comprising:

[0019] - a pair of oligonucleotides, wherein the first member and the second member of the oligonucleotide pair have complementary nucleotide sequences at their respective 3’-ends and are able to hybridize to each other at the 3’-ends, the first and / or second member comprising an identification sequence unique to the individual member or unique to the oligonucleotide pair;

[0020] - a first analyte-binding moiety specific for a first analyte, and a second analyte-binding moiety specific for said first analyte; under conditions suitable for binding of the analyte-binding moieties to the corresponding analyte and hybridization of the complementary parts of the two members of the oligonucleotide pair; contacting the sample with a polymerase having 3’-exonuclease activity to extend one member of the oligonucleotide pair to form a reporter nucleic acid molecule; contacting the sample with an index oligonucleotide, comprising a samplespecific index sequence and a member association sequence; wherein the index oligonucleotide is, at its 3’-end, blocked from being extended and / or from being degraded by a polymerase having 3’-exonuclease activity; contacting the sample with the polymerase having 3’-exonuclease activity to incorporate the sample-specific index sequence in the reporter nucleic acid molecule optionally amplifying the reporter nucleic acid molecule; quantifying the amount of reporter nucleic acid molecule; and correlating the amount of reporter nucleic acid molecule to the amount of analyte in the sample and correlating the amount of reporter nucleic acid molecule, comprising the sample-specific index sequence, to the corresponding sample.

[0021] Also provided is a method for detecting at least one analyte in a sample, comprising: contacting the sample with at least one pair of proximity probes, each proximity probe pair being specific for one analyte, and comprising:

[0022] - a pair of oligonucleotides, wherein the first member and the second member of the oligonucleotide pair have complementary nucleotide sequences at their respective 3’-ends and are able to hybridize to each other at the 3’-ends, the first and / or second member comprising an identification sequence unique to the individual member or unique to the oligonucleotide pair, wherein the first or the second member of each oligonucleotide pair is, at its 3’-end, blocked from being extended and from being degraded by a polymerase having 3’-exonuclease activity;

[0023] - a first analyte-binding moiety specific for a first analyte, and a second analytebinding moiety specific for said first analyte; under conditions suitable for binding of the analyte-binding moieties to the corresponding analyte and hybridization of the complementary parts of the two members of the oligonucleotide pair; contacting the sample with a polymerase having 3’-exonuclease activity to extend one member of the oligonucleotide pair to form a reporter nucleic acid molecule; optionally amplifying the reporter nucleic acid molecule; quantifying the amount of reporter nucleic acid molecule; and correlating the amount of reporter nucleic acid molecule to the amount of analyte in the sample.

[0024] Also provided herein is a method for detecting multiple analytes in multiple samples, comprising: contacting the samples with multiple proximity probe pairs, each proximity probe pair being specific for one analyte and comprising:

[0025] - a pair of oligonucleotides, wherein the first member and the second member of the oligonucleotide pair can interact directly or indirectly to form a doublestranded helix, the first and / or second member comprising an identification sequence unique to the individual member or unique to the oligonucleotide pair;

[0026] - a first analyte-binding moiety specific for a first analyte, and a second analyte-binding moiety specific for said first analyte; under conditions suitable for binding of the analyte-binding moieties of each proximity probe to the corresponding analyte and hybridization of the complementary parts of the two members of the oligonucleotide pair of each proximity probe and hybridization of the complementary parts of the two members of the oligonucleotide pair of each proximity probe;

[0027] - contacting each sample with a polymerase having 3’-exonuclease activity to extend one member of the oligonucleotide pair of each proximity probe to form a reporter nucleic acid molecule;

[0028] - contacting each sample with an index oligonucleotide comprising a sample-specific index sequence and a member association sequence, wherein the index oligonucleotide is, at its 3’-end, blocked from being extended and / or from being degraded by a polymerase having 3’-exonuclease activity, and wherein the step of contacting each sample with a polymerase is additionally to incorporate the sample-specific index sequence in the reporter nucleic acid molecule; or contacting each sample with an index oligonucleotide comprising a sample-specific index sequence and a member association sequence, wherein the index oligonucleotide is, at its 3’-end, blocked from being extended and / or from being degraded by a polymerase having 3’-exonuclease activity, and contacting each sample with a polymerase having 3’-exonuclease activity to incorporate the sample-specific index sequence in the reporter nucleic acid molecule; optionally amplifying each reporter nucleic acid molecule; quantifying the amount of each reporter nucleic acid molecule comprising the samplespecific index sequence; and correlating the amount of each reporter nucleic acid molecule, comprising the samplespecific index sequence, to the amount of the corresponding analyte in each corresponding sample.

[0029] Also provided herein is a method for detecting multiple analytes in multiple samples, comprising: contacting the samples with multiple proximity probe pairs, each proximity probe pair being specific for one analyte and comprising:

[0030] - a pair of oligonucleotides, wherein the first member and the second member of the oligonucleotide pair can interact directly or indirectly to form a doublestranded helix while leaving a portion of both members single-stranded, the first and / or second member comprising an identification sequence unique to the individual member or unique to the oligonucleotide pair, wherein the first or the second member of each oligonucleotide pair is, at its 3’-end, blocked from being extended and / or from being degraded by a polymerase having 3’-exonuclease activity;

[0031] - a first analyte-binding moiety specific for a first analyte, and a second analyte-binding moiety specific for said first analyte; under conditions suitable for binding of the analyte-binding moieties of each proximity probe to the corresponding analyte c;

[0032] - contacting each sample with a polymerase having 3’-exonuclease activity to extend one member of the oligonucleotide pair of each proximity probe to form a reporter nucleic acid molecule;

[0033] - contacting each sample with an index oligonucleotide comprising a sample-specific index sequence and a member association sequence, and wherein the step of contacting each sample with a polymerase is additionally to incorporate the samplespecific index sequence in the reporter nucleic acid molecule; or contacting each sample with an index oligonucleotide comprising a sample-specific index sequence and a member association sequence, and contacting each sample with a polymerase having 3’-exonuclease activity to incorporate the sample-specific index sequence in the reporter nucleic acid molecule; optionally amplifying each reporter nucleic acid molecule; quantifying the amount of each reporter nucleic acid molecule comprising the samplespecific index sequence; and correlating the amount of each reporter nucleic acid molecule, comprising the samplespecific index sequence, to the amount of the corresponding analyte in each corresponding sample.

[0034] The present invention further provides use of the herein disclosed system for detecting at least one analyte in at least one sample.

[0035] The present invention is defined by the appended independent claims. Non-limiting embodiments, such as currently preferred embodiments, will become apparent from the dependent claims, the appended drawings, and the following description. It is noted that the present invention relates to all possible combinations of features recited in the claims.

[0036] Brief description of the drawings

[0037] Fig. 1 schematically illustrates a method for detecting at least one analyte in a sample, in accordance with the present invention.

[0038] Fig. 2 schematically illustrates a method for detecting multiple analytes in multiple samples, in accordance with the present invention.

[0039] Fig. 3 schematically illustrates a method for detecting an analyte in a sample, including use of a blocked index oligonucleotide.

[0040] Fig. 4 schematically illustrates a method for detecting an analyte in a sample, including use of a blocked oligonucleotide pair member.

[0041] Fig. 5 shows the results of the experiments described in Example 1 herein.

[0042] Fig. 6 shows the results of the experiments described in Example 2 herein.

[0043] Fig. 7 shows the results of the experiments described in Example 3 herein.

[0044] Fig. 8 shows results of the experiments described in Example 4 herein.

[0045] Fig. 9 shows results of the experiments described in Example 4 herein. Definition of terms and abbreviations

[0046] All terms and abbreviations used in the present specification shall be construed to have the meaning normally given to them in the relevant art unless another meaning is clearly intended. Unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. For the sake of clarity, a few terms and abbreviations are defined below.

[0047] The terms “a” and “an” are defined as one or more unless explicitly stated otherwise herein.

[0048] Compositions “comprising” one or more recited elements may also include other elements not specifically recited. The term "comprising” also encompasses the term “consisting of”.

[0049] A “system”, which can also be described as a “kit of parts”, or simply “kit”, is a product. In particular, it is a product comprising multiple parts combined and arranged to be provided as a single entity to a user. The parts of a system, in the present technological field, are generally one or more reagents, in one or more reagent compositions, comprised in vials or other containers, optionally reaction plates, optionally instructions for use or internet hyperlinks to such instructions, and optionally a box in which the parts are packaged.

[0050] The term “analyte” as used herein (in respect of all aspects of the present invention) means any substance (e.g. molecule) or entity it is desired to detect by the method of the invention. The analyte is thus the "target" of the assay method of the invention, i.e. the substance detected or screened for using the method of the invention. The analyte may accordingly be any biomolecule or chemical compound it is desired to detect, for example a peptide or protein, or a nucleic acid molecule or a small molecule, including organic and inorganic molecules. The analyte may be a cell or a microorganism, including a virus, or a fragment or product thereof. It will be seen therefore that the analyte can be any substance or entity for which a specific binding partner (e.g. an affinity binding partner) can be developed. All that is required is that the analyte is capable of simultaneously binding at least two binding partners (more particularly, the analyte-binding domains of at least two proximity probes).

[0051] The present invention makes use of “identification sequences”. An identification sequence may e.g. be a unique sequence (usually termed a “barcode sequence” or simply “barcode”) that is detected in a sequence-specific manner, for example, sequenced for identification in the readout step, or which provides a specific binding (hybridization) site for a probe or primer used in the detection, e.g., a unique primer binding site that can be used for readout using quantitative PCR (qPCR). A “sample-specific” index sequence, comprised in an index oligonucleotide, is intended to refer to a sequence, which is unique to each sample to be analysed. It is to be understood that each well in a plate, on which the detection of analytes is to take place, may be loaded with a unique sample or the same sample may be loaded into two or more wells in order to measure / produce duplicate / triplicate results, and / or where one well is used as a control for example. Accordingly, a unique index sequence is required for each well in order to distinguish between samples of all wells, including duplicates / triplicates of the same sample. The method of detecting an analyte in a well may be referred to as assaying, or an assay. In other words, a unique index sequence is required for each well or assay in order to distinguish between all wells or assays. Accordingly, a sample-specific index sequence may alternatively be referred to as a “well-specific” or “assay-specific” index sequence.

[0052] A ’’proximity probe” is a molecular probe comprising two main parts, one part (“analytebinding moiety”) providing the probe with analyte-specific binding properties and one part (“nucleic acid-moiety” or simply “oligonucleotide”) comprising a nucleic acid that in turn comprises an identification sequence that correlates with the analyte to which the probe specifically binds.

[0053] A “proximity probe pair” is intended to mean a set of at least two probes that bind to the same analyte. Currently commercially available proximity assays use sets of two probes, usually termed “forward” and “reverse” probes, or “capture” and “detection” probes.

[0054] However, proximity assays using three (US 8,268,554) or four (Tavoosidana et al., PNAS, May 9, 2011 , 108 (21) 8809-8814) proximity probes binding specifically to the analyte have been described. That two (or more) proximity probes are capable of simultaneous binding to different parts of the same analyte of interest may be indicated by indicating that the probes are “matched”, indicating that, when the matched probes are bound to the analyte of interest, their respective nucleic acid moieties are capable of interacting, directly (e.g, by hybridization to each other) or indirectly (e.g. by hybridization to a common splint oligonucleotide), to yield, form or otherwise generate a reporter nucleic acid.

[0055] A “reporter molecule” is a molecule, which includes an identification sequence unique to each analyte. The reporter molecule may be quantified and correlated with the amount of analyte present in a sample. Herein, the terms “reporter molecule” and “reporter nucleic acid molecule” are used interchangeably.

[0056] “Readout” is intended to refer to the process of quantifying the amount of reporter molecules with the respective unique identification sequences and correlating these amounts to the amounts of the respective analytes of interest in the analysed sample. Accordingly, a readout can be seen as a step of detecting the signal in the assay, or more particularly the reporter molecules, in a quantitative manner.

[0057] The term “multiple” as used in the present disclosure means more than one (that is to say, two or more), in line with its standard definition. However, the method of the first aspect of the invention requires separate multiplex reactions to be performed for multiple (i.e. at least two) aliquots of a sample. As used herein, the term “multiplex” is used to refer to an assay in which multiple (i.e. at least two) different analytes are assayed at the same time, and more particularly in the same aliquot of the sample, or in the same reaction mixture. Thus it is apparent that the minimum number of analytes to be detected according to the method of the first aspect of the present invention is four (two analytes to be detected in each of two aliquots of sample). However, it is preferred that considerably more analytes than four are detected according to the present method. Preferably at least 10, 20, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400 or 1500 or more analytes are detected according to the present method.

[0058] The term "detecting" or "detected" is used broadly herein to include any means of determining the presence or absence of an analyte (i.e. determining whether a target analyte is present in a sample of interest or not). Accordingly, if a method of the invention is performed and an attempt is made to detect a particular 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” has still been performed, because its presence or absence from the sample has been assessed. The step of “detecting” an analyte is not dependent on that detection proving successful, i.e. on the analyte actually being detected.

[0059] Detecting an analyte may further include any form of measurement of the concentration or abundance of the analyte in the sample. Either the absolute concentration of a target analyte may be determined, or a relative concentration of the analyte, for which purpose the concentration of the target analyte may be compared to the concentration of another target analyte (or other target analytes) in the sample or in other samples.

[0060] Thus "detecting" may include determining, measuring, assessing or assaying the presence or absence or amount of an analyte in any way. Quantitative and qualitative determinations, measurements or assessments are included, including semi-quantitative determinations. Such determinations, measurements or assessments may be relative, for example when two or more different analytes in a sample are being detected, or absolute. As such, the term "quantifying" when used in the context of quantifying a target analyte in a sample can refer to absolute or to relative quantification. Absolute quantification may be accomplished by inclusion of known concentration(s) of one or more control analytes and / or referencing the detected level of the target analyte with known control analytes (e.g. through generation of a standard curve). Alternatively, relative quantification can be accomplished by comparison of detected levels or amounts between 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. Methods by which quantification can be achieved in the method of the invention are discussed further below.

[0061] When the present disclosure refers to multiple, or a plurality of, nucleic acid molecules, it is intended to refer to nucleic acid molecules of multiple, or a plurality of, different nucleotide sequences, unless otherwise indicated by context. Analogously, a species of nucleic acid molecules with identical sequence is generally referred to in the singular, although nucleic acid molecules of the same species may be present in several copies. For example, “multiple oligonucleotides” in general refers to several oligonucleotides with different nucleotide sequences, whereas “an oligonucleotide” refers to one or more molecules with identical nucleotide sequence.

[0062] Detailed description

[0063] The present invention relates to products and methods for improved signal-to-noise ratio in proximity assays for detecting analytes in one or more samples.

[0064] The present inventors have surprisingly found that reducing certain extension events in a proximity assay can significantly increase the signal and reduce the noise, thereby also significantly improve the signal-to-noise ratio.

[0065] More particularly, the present invention provides a system comprising: a) at least one pair of oligonucleotides, wherein the first member and the second member of the oligonucleotide pair have complementary nucleotide sequences at their respective 3’-ends and are able to hybridize to each other at the 3’-ends while leaving a portion of both members single-stranded, the first and / or second member comprising an identification sequence unique to the individual member or unique to the oligonucleotide pair; b) optionally, at least one index oligonucleotide, comprising an index sequence and a member association sequence; and c) a polymerase having 3’-exonuclease activity; characterized in that the first or the second member of the oligonucleotide pair of (a) is, at its 3’-end, blocked from being extended and / or from being degraded by the polymerase, and / or the index oligonucleotide of (b) is, if present, at its 3’-end, blocked from being extended and / or from being degraded by the polymerase.

[0066] In a first aspect, the invention relates to a system comprising the index oligonucleotide and wherein this index oligonucleotide is blocked from being extended and / or protected from being degraded by the polymerase. As shown in Example 4, this provides significantly improved signal-to-noise as compared with a system wherein the index oligonucleotides are not modified in this way.

[0067] In a second aspect, the invention relates to a system wherein one of the members of the oligonucleotide pair is blocked from being extended and protected from being degraded. One of the oligonucleotide pair members is not blocked from being extended, so that the extension as shown step II. of Fig 7 can take place.

[0068] In certain embodiments of the invention, the above two aspects are combined so that both one of the oligonucleotide pair members and the index oligonucleotide are modified at their respective 3’-ends, in order to block extension or protect from digestion by the polymerase included in the system.

[0069] The herein disclosed system is useful to apply in a proximity assay, such as PEA.

[0070] The pair of oligonucleotides that can hybridize to each other may herein be called a hybridizable oligonucleotide pair.

[0071] In the experimental section herein, it is shown that when one of the members of such a hybridizable oligonucleotide pair is blocked at its 3’-end, it results in an increased signal-to- noise ratio when using it to detect an analyte in a sample in a proximity assay. It is further shown herein that when applying an index oligonucleotide which is blocked at its 3’-end, an increased signal-to-noise ratio is achieved. Without being bound by theory, it is hypothesized that the invention reduces the amount of amplified noise-generating incorrect / incomplete reporter molecules, thereby improving the signal-to-noise ratio.

[0072] Herein, the phrase “the first member and the second member of the oligonucleotide pair have complementary nucleotide sequences at their respective 3’-ends and are able to hybridize to each other at the 3’-ends” is intended to mean that the complementary nucleic acid sequences are located at or near the 3’-end of each member of the oligonucleotide pair. Further, the phrase ”at or near the 3’-end” is intended to mean that the complementary nucleic acid sequences start at the first nucleotide of the 3’-end of the respective member, or start at a nucleotide near the 3’-end of the respective member, for example at a nucleotide located about 1-10 nucleotides upstream of the first nucleotide of the 3’-end, or that the complementary sequence of one member starts at the first nucleotide of the 3’-end of said member while the complementary sequence of the other member starts at a nucleotide located about 1-10 nucleotides upstream of the first nucleotide of the 3’-end. The phrase “upstream of the 3’-end” means in the direction towards the 5’-end of an oligonucleotide.

[0073] Herein, when the first and second member of the oligonucleotide pair hybridize to each other at the 3’-ends, while leaving a portion of both members single-stranded, it is to be understood that the portion left single-stranded is located at the 5’-end of each member, or towards the 5’-end of each member in relation to the 3’-end portion of each member that hybridizes to each other.

[0074] In an embodiment, the first or the second member of the hybridizable oligonucleotide pair is blocked from being extended and from being degraded by a polymerase having 3’- exonuclease activity, by a chemical modification of the nucleotide located at the 3’-end of said member of the oligonucleotide pair.

[0075] In an embodiment, the index oligonucleotide is blocked from being extended and from being degraded by a polymerase having 3’-exonuclease activity, by a chemical modification of the nucleotide located at the 3’-end of said index oligonucleotide.

[0076] Methods for blocking by chemical modification of a nucleotide are well known in the art and such chemically modified nucleotides are commercially available, e.g., from Integrated DNA Technologies (see ). For example, an inverted dT incorporated at the 3’-end of an oligonucleotide leads to a 3’-3’ linkage which inhibits both degradation by 3’ exonucleases and extension by DNA polymerases, 2'-OMe-RNA CE ( 3 - cyanoethyl) phosphoramidites produce synthetic oligonucleotides containing nuclease resistant 2'-O-methyl ribonucleotide linkages, and 2Z,3Z-Dideoxycytidine is used as a DNA chain-terminating nucleotide for DNA sequencing methods. Inclusion of a phosphorothioate bond renders the internucleotide linkage resistant to nuclease degradation. Inclusion of a three carbon (C3) spacer at the 3’-end prevents extension by DNA polymerases. Other modifications protecting against digestion by a 3’ to 5’ exonuclease activity include locked nucleic acids (LNA), inverted nucleic acids, 2' Fluoro bases, 3' phosphorylation, 2'-0-Methyl modifications (or other 2’-0-alkyl modification), propyne-modified bases (e.g., deoxycytosine, deoxyuridine), L-DNA nucleotides, L-RNA nucleotides, nucleotides with inverted linkages (e.g., 5’-5’ or 3’-3’), 5’ inverted bases (e.g., 5’ inverted 2’,3’-dideoxy dT), methylphosphonate backbones, a 3' amino, a 3' phosphate, a dideoxy, and trans nucleic acids.

[0077] Non-limiting examples of a chemical modification of the nucleotide located at the 3’-end of an oligonucleotide, meaning the nucleotide located furthest downstream in an oligonucleotide, are 2'-O-Methyl RNA, Dideoxycytidine (Dideoxy-C), and Inverted dT. Any one of these non-limiting examples is suitable to apply in order to block the 3’-end of the first or second member of the oligonucleotide pair, and / or to block the 3’-end of the index oligonucleotide, from being extended and from being degraded by a polymerase having 3’- exonuclease activity.

[0078] The herein disclosed system may further comprise a first analyte-binding moiety specific for a first analyte, and a second analyte-binding moiety specific for said first analyte, wherein the first member of the oligonucleotide pair is directly or indirectly attached to the first analyte-binding moiety, and the second member of the oligonucleotide pair is directly or indirectly attached to the second analyte-binding moiety, wherein the first and the second analyte-binding moieties can simultaneously bind to the first analyte. In such embodiment, the respective analyte-binding moiety and oligonucleotide member together make up a detection probe, commonly referred to as a proximity probe.

[0079] The first and second analyte-binding moieties may independently be selected from antibodies, antibody fragments, and nucleic acids, including aptamers.

[0080] In an embodiment, at least one of the two members of the oligonucleotide pair comprises an analyte-specific identification nucleotide sequence for identification of the analyte for which the first and the second analyte-binding moieties are specific.

[0081] Multiplexing of biological assays, such as proximity assays, means performing a plurality of assays in parallel and, preferably, in the same reaction container, e.g. a test tube or a well in a microtiter plate. Multiplexing thus has the potential to massively increase throughput of samples and reduce footprint of the necessary equipment.

[0082] Accordingly, the above-described system may comprise multiple pairs of oligonucleotides, wherein the first member and the second member of each oligonucleotide pair have complementary nucleotide sequences at their respective 3’-ends and are able to hybridize to each other at the 3’-ends, the first and / or second member of each oligonucleotide pair comprising an identification sequence unique to the individual member or unique to the oligonucleotide pair; and optionally may comprise multiple index oligonucleotides, each comprising an index sequence and a member association sequence; characterized in that the first or the second member of each oligonucleotide pair is, at its 3’-end, blocked from being extended and from being degraded by the polymerase, and / or each index oligonucleotide is, if present, at its 3’-end, blocked from being extended and from being degraded by the polymerase.

[0083] The present invention further provides a system comprising: a) multiple pairs of oligonucleotides, wherein the first member and the second member of each oligonucleotide pair have complementary nucleotide sequences at their respective 3’-ends and are able to hybridize to each other at the 3’-ends, the first and / or second member of each oligonucleotide pair comprising an identification sequence unique to the individual member or unique to the oligonucleotide pair; b) optionally multiple index oligonucleotides, each comprising (i) an index sequence unique to said index oligonucleotide, and (ii) a member association sequence; and c) a polymerase having 3’-exonuclease activity; characterized in that the first or the second member of each oligonucleotide pair of (a) is, at its 3’-end, blocked from being extended and / or from being degraded by the polymerase, and / or each index oligonucleotide of (b) is, if present, at its 3’-end, blocked from being extended and / or from being degraded by the polymerase.

[0084] In embodiments of the present invention, the first member and the second member of the oligonucleotide pair are able to hybridize to each other at the 3’-ends while leaving a portion of one or both member(s) single-stranded.

[0085] The identification sequence may e.g. be a unique sequence (also termed a “barcode sequence” or simply “barcode”) that is detected in a sequence-specific manner, for example, sequenced for identification in the readout step, or which provides a specific binding (hybridization) site for a probe or primer used in the detection, e.g., a unique primer binding site that can be used for readout using quantitative PCR (qPCR). Herein, the terms “identification sequence” and “ID sequence” are used interchangeably.

[0086] An analyte of interest may be any analyte it is desired to detect. In an embodiment, the analyte is a protein. However, it may be any biological or chemical entity it is desired to detect. As indicated above, proximity probes are used in the art to detect a wide variety of analytes, and these may include interactions and complexes etc. Thus, the proximity probes of a pair (or more) may each bind to the same target molecule (but at different sites, such that the individual proximity probes may each bind to their respective target binding sites at the same time (i.e. simultaneously)), or to different molecules (e.g. where the target analyte is an interaction, in which case each proximity probe binds to a different member of the interaction, or where a post-translational modification of a given protein is being detected). Indeed, the target analyte may be the co-localization of two molecules in close proximity.

[0087] As is known in the art, the analyte-specific binding domain of a detection probe may be any entity capable of binding specifically to a target analyte (or part thereof) and being coupled to a nucleic acid moiety. That the binding domain is “specific” to a certain analyte means, as is known to the skilled person, that it recognizes the analyte with low cross-reactivity (off-target binding) with other potentially present analytes, within the relevant application and experimental context. A framework for determining specificity for binders have been established by an International Working Group for Antibody Validation (llhlen et al., Nat Methods, 2016 Oct; 13(10), 823-827, incorporated herein by reference).

[0088] Typically, the first and second (and optionally third, fourth or further if more than two probes are included) analyte-specific binding domain may independently be a protein, for example, an antibody, or an antigen-binding part thereof, including, but not limited to, monoclonal, recombinant monoclonal, and polyclonal antibodies and antigen-binding antibody derivatives and fragments. However, the analyte-specific binding domain may be of any nature, including lectins, soluble cell surface receptors, combinatorially derived proteins from phage display or ribosome display, peptides, carbohydrates, molecularly imprinted polymers (MIPs), nucleic acids, such as an aptamer or a nucleic acid molecule comprising the complementary sequence for a target nucleic acid, or combinations thereof.

[0089] Reagents useful as analyte-specific binding domains are commercially available from a number of manufacturers that offer off-the-shelf reagents or develop new binding reagents for specific analytes and specific needs. Such manufacturers include, among others, Thermo Fisher Scientific (Boston, MA, USA), Abeam (Cambridge, United Kingdom), Bio-Techne (Minneapolis, MN, USA), Proteogenix (Schiltigheim, France), Sino Biological (Beijing, China), Agrisera (Vannas, Sweden), Novaptech (Pessac, France), Aptamer Group (York, United Kingdom). Reagents useful as analyte-specific binding domains may also be developed independently of commercial suppliers, according to protocols well-known to the skilled person. Such protocols are e.g. described in “Monoclonal Antibody Production” (National Academy Press, Washington, DC, USA, 1999), Carey-Hanly et al. (ILAR Journal, 1995, 37(3): 93-118), llgu and Nilsen-Hamilton (Analyst. 2016, 141(5): 1551-1568). Reagents may also comprise antibody derivates or fragments, such as Fab, Fab', F(ab')2, Fv fragments; diabodies; single-domain antibodies (sdAb, Desmyter et al., 1996, Nat. Structure Biol. 3: 803-811), nanobodies, single-chain Fv (scFv, Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85: 5879-5883), divalent scFV (di-scFvs), tandem scFvs, triabodies, diabodies, single-chain diabodies (scDb), bi-specific T-cell engagers (BiTEs, Kufer et al., 2004, Trends Biotechnol. 22: 238-244), and Dual Affinity Retargeting molecules (DARTs, diabodies additionally stabilized through a C-terminal disulfide bridge). The specificity of analytespecific reagents with regard to the intended detection assay may be evaluated using the framework proposed by the International Working Group for Antibody Validation, cited above.

[0090] Further, the analyte-specific binding domain may bind to the analyte directly or indirectly. In other words, the detection probe may be a primary reagent which binds directly to the analyte, or a secondary reagent which binds indirectly, by virtue of binding to an intermediate molecule (a primary reagent) which is itself bound directly to the analyte.

[0091] As described further above, in addition to the analyte-specific binding domain, a detection probe as used in the present invention also comprises a nucleic acid moiety, also referred to herein as an oligonucleotide. The oligonucleotide must be long enough to comprise the necessary functional elements used in the detection assay for which the detection probe is intended to be used. That is, at least a sequence capable of generating an identification sequence in the reporter molecule. This is typically 5-20 nucleotides, such as 5-10, 5-15, IQ- 15 or 15-20 nucleotides. The oligonucleotide may also contain sequences related to primer sites and / or sequencing adaptors for readout, as known in the art. Generally, the oligonucleotide has a length in the range of 20-100 nucleotides but may be shorter or longer as required in the specific detection assay in which the detection probe is intended to be used.

[0092] Conjugation of a nucleic acid moiety to an antibody can be performed in several ways known to the skilled person, e.g. as reviewed by Dugal-Tessier et al. (J. Clin. Med.2021 , 10, 838). Commercial kits for preparing antibody-oligonucleotide conjugates are also readily available from a number of suppliers. The oligonucleotides may be coupled to the analyte binding domains by any means known in the art, and which may be desired or convenient and may be direct, or indirect, e.g. via a linking group. For example, the domains may be associated with one another by covalent linkage (e.g. chemical cross-linking) or by non-covalent association e.g. via streptavidin-biotin based coupling (biotin being provided on one domain, particularly the oligonucleotide domain, and streptavidin on the other). The oligonucleotide and analyte binding domain are joined together either directly through a bond or indirectly through a linking group. Where linking groups are employed, such groups may be chosen to provide for covalent attachment of the nucleic acid moiety and analyte binding domain through the linking group. The linking group, when present, is in many embodiments biologically inert. In representative embodiments, the linking group is generally at least about 50 Daltons, usually at least about 100 Daltons and may be as large as 1000 Daltons or larger, for example, up to 1000000 Daltons if the linking group contains a spacer, but generally will not exceed about 500 Daltons and usually will not exceed about 300 Daltons. Generally, such linkers will comprise a spacer group terminated at either end with a reactive functionality capable of covalently bonding to the nucleic acid domain or analyte binding domain. Spacer groups of interest may include aliphatic and unsaturated hydrocarbon chains, spacers containing heteroatoms such as oxygen (ethers such as polyethylene glycol) or nitrogen (polyamines), peptides, carbohydrates, cyclic or acyclic systems that may possibly contain heteroatoms. Spacer groups may also be comprised of ligands that bind to metals such that the presence of a metal ion coordinates two or more ligands to form a complex. Specific spacer elements include: 1 ,4-diaminohexane, xylylenediamine, terephthalic acid, 3,6-dioxaoctanedioic acid, ethylenediamine-N,N-diacetic acid, 1,1'-ethylenebis(5-oxo-3-pyrrolidinecarboxylic acid), 4,4'-ethylenedipiperidine.

[0093] Potential reactive functionalities include nucleophilic functional groups (amines, alcohols, thiols, hydrazides), electrophilic functional groups (aldehydes, esters, vinyl ketones, epoxides, isocyanates, maleimides), functional groups capable of cycloaddition reactions, forming disulfide bonds, or binding to metals. Specific examples include primary and secondary amines, hydroxamic acids, N-hydroxysuccinimidyl esters, N-hydroxysuccinimidyl carbonates, oxycarbonylimidazoles, nitrophenylesters, trifluoroethyl esters, glycidyl ethers, vinylsulfones, and maleimides.

[0094] Specific linker groups that may find use in the presently disclosed proximity probes include heterofunctional compounds, such as azidobenzoyl hydrazide, N-[4-(p- azidosalicylamino)butyl]-3'-[2'-pyridyldithio]propionamide, bis-sulfosuccinimidyl suberate, dimethyladipimidate, disuccinimidyltartrate, N- maleimidobutyryloxysuccinimide ester, N- hydroxy sulfosuccinimidyl-4- azidobenzoate, N-succinimidyl [4-azidophenyl]-1 ,3'- dithiopropionate, N-succinimidyl [4-iodoacetyl]aminobenzoate, glutaraldehyde, and succinimidyl-4-[N- maleimidomethyl]cyclohexane-1 -carboxylate, 3-(2-pyridyldithio)propionic acid N- hydroxysuccinimide ester (SPDP), 4-(Nmaleimidomethyl)-cyclohexane-1 -carboxylic acid N-hydroxysuccinimide ester (SMCC), and the like. The nucleic acid domain of the detection probes may be made up of ribonucleotides and / or deoxyribonucleotides as well as synthetic nucleotide residues that are capable of participating in Watson-Crick type or analogous base pair interactions. Thus, the nucleic acid domain may be DNA or RNA or a combination or any modification thereof e.g. PNA or other derivatives containing non-nucleotide backbones.

[0095] In one embodiment, the detection probes are manufactured by coupling a universal oligonucleotide to the analyte-specific binding domain and subsequently hybridizing a tag oligonucleotide to the universal oligonucleotide, wherein the tag oligonucleotide comprises a sequence capable of generating the identification sequence in a reporter molecule, a sequence complementary to the universal oligonucleotide to facilitate hybridization, and any other functional sequences necessary to perform the detection assay for which the detection probes are intended. Methods for manufacturing of such detection probes are described i.a. in US10781473B2.

[0096] In an embodiment of the present invention, one or both members of the hybridizable oligonucleotide pair is attached at its 5’-end to its respective analyte-binding moiety. In another embodiment, one or both members of the hybridizable oligonucleotide pair is hybridized to a partially complementary oligonucleotide, which is attached (at its 5’-end or 3’- end) to its respective analyte-binding moiety, as described in US10781473B2.

[0097] The present invention does not achieve blocking of the 3’-end of the first or second member of the hybridizable oligonucleotide pair by conjugation of the 3’-end to an analyte-binding moiety.

[0098] Further, the present invention does not achieve blocking of the 3’-end of the first or second member of the hybridizable oligonucleotide pair by use of one or more so-called blocking oligonucleotides, which first bind to the first and / or second member of the oligonucleotide pair to create a double-stranded oligonucleotide and then are displaced.

[0099] Accordingly, the 3’-end of the first or second member of the oligonucleotide pair is not blocked by a separate entity, such as a nucleic acid sequence or an amino acid sequence, which binds to or is conjugated to the first or second member and which thereby blocks the 3’-end of the first or second member from being extended and from being degraded by a polymerase having 3’-exonuclease activity.

[0100] Additionally, the 3’-end of the index oligonucleotide is not blocked by a separate entity, such as a nucleic acid sequence or an amino acid sequence, which binds to or is conjugated to the index oligonucleotide and which thereby blocks the 3’-end of the first or second member from being extended and from being degraded by a polymerase having 3’-exonuclease activity.

[0101] In other words, in the context of the presently disclosed system, the first and second member of the hybridizable oligonucleotide pair, which are able to hybridize to each other at the 3’-ends, both have their 3’-end free before they hybridize to each other, meaning that none of the two 3’-ends is bound or conjugated to any third moiety (such as an antibody or a third oligonucleotide) before they hybridize to each other. In other words, the 3’-end of the first member is free or non-conjugated, and the 3’-end of the second member is free or nonconjugated.

[0102] Where proximity probes are used as the detection probes, each nucleic acid moiety of a proximity probe of a matched pair (or more) may comprise an identification sequence, or a partial identification sequence. The reporter molecule which is generated may comprise an identification sequence from each of the matched proximity probes. In other words, the identification sequence of the reporter molecule may be a combination, or composite, of the identification sequences of the individual nucleic acid moieties of matched proximity probes. The identification sequences of individual matched proximity probes may be the same or different. According to a non-limiting embodiment, each identification sequence in matched proximity probes is indicative of, or corresponds to, the analyte of interest. However, it is not required for a particular identification sequence of an individual proximity probe to be indicative of an analyte of interest - it is the identification sequence of the reporter nucleic acid molecule which is indicative of the analyte of interest. As indicated above, the identification sequence of the reporter nucleic acid molecule may be a combination or composite. Alternatively, the identification sequence of the reporter nucleic acid molecule may be derived from the nucleic acid moiety of a single proximity probe (although it will be understood that interaction of the nucleic acid moieties of matched proximity probes will be required for the reporter nucleic acid molecule to form).

[0103] In an embodiment, the analyte-specific binding domain is not a nucleic acid which binds by hybridization. In another embodiment, the analyte-specific binding domain is not a nucleic acid. In another embodiment, the detection probe is not composed wholly of nucleic acid. In another embodiment, the detection probe is not a gene-specific probe.

[0104] As illustrated in Fig. 1, the present invention additionally provides a method 100 for detecting at least one analyte in a sample, comprising: contacting 110 the sample with at least one pair of proximity probes, each proximity probe pair being specific for one analyte, and comprising: - a pair of oligonucleotides, wherein the first member and the second member of the oligonucleotide pair have complementary nucleotide sequences at their respective 3’-ends and are able to hybridize to each other at the 3’-ends, the first and / or second member comprising an identification sequence unique to the individual member or unique to the oligonucleotide pair, wherein the first or the second member of each oligonucleotide pair is optionally, at its 3’-end, blocked from being extended and protected from being degraded by a polymerase having 3’-exonuclease activity;

[0105] - a first analyte-binding moiety specific for a first analyte, and a second analytebinding moiety specific for said first analyte; under conditions suitable for binding of the analyte-binding moieties to the corresponding analyte and hybridization of the complementary parts of the two members of the oligonucleotide pair; contacting 120 the sample with a polymerase having 3’-exonuclease activity to extend one member of the oligonucleotide pair to form a reporter nucleic acid molecule; optionally amplifying 130 the reporter nucleic acid molecule; quantifying 140 the amount of reporter nucleic acid molecule; and correlating 150 the amount of reporter nucleic acid molecule to the amount of analyte in the sample.

[0106] In the method 100 for detecting at least one analyte, each proximity probe pair has a unique part or feature and is specific for only one type of analyte. The method includes contacting the sample with as many proximity probe pairs as there are analytes desirable to detect, or in other words, one proximity probe pair for each analyte to be detected.

[0107] The method 100 may further comprise: contacting 115; 125 the sample with an index oligonucleotide, comprising a samplespecific index sequence and a member association sequence; optionally wherein the index oligonucleotide is, at its 3’-end, blocked from being extended and protected from being degraded by a polymerase having 3’-exonuclease activity; contacting 120; 127 the sample with a polymerase having 3’-exonuclease activity to incorporate the sample-specific index sequence in the reporter nucleic acid molecule; and wherein the step of correlating 150 the amount of reporter nucleic acid molecule to the amount of analyte in the sample further comprises correlating the amount of reporter nucleic acid molecule, comprising the sample-specific index sequence, to the corresponding sample.

[0108] In an embodiment of the method 100, the step of contacting the sample with an index oligonucleotide is performed as a step 115, i.e. , after the step of contacting 110 the sample with at least one pair of proximity probes and before the step of contacting 120 the sample with a polymerase, in which case the step of contacting 120 the sample with a polymerase is performed to extend one member of the oligonucleotide pair to form a reporter nucleic acid molecule and to incorporate the index sequence in the reporter nucleic acid molecule. In an alternative embodiment of the method 100, the step of contacting the sample with an index oligonucleotide is performed as a step 125, i.e. , after the step of contacting 120 the sample with a polymerase and before the optional step of amplifying 130 the reporter nucleic acid molecule, if present, and before the step of quantifying 140 the amount of reporter nucleic acid molecule, in which case an additional step of contacting 127 the sample with a polymerase is performed to incorporate the index sequence in the reporter nucleic acid molecule. In other words, according to this alternative embodiment, the method 100 includes adding a polymerase to the sample twice, in both step 120 and step 127.

[0109] In embodiments including contacting the sample with an index oligonucleotide and incorporating the sample-specific index sequence in the reporter nucleic acid molecule, it follows that in the step of quantifying 140, the reporter nucleic acid molecule comprises the sample-specific index sequence, which means that in the step of correlating 150, the amount of reporter nucleic acid molecule can be correlated not only to the amount of analyte but also to the corresponding sample.

[0110] In an embodiment, only the index sequence (i.e., the sample-specific or assay-specific part of the index oligonucleotide) is incorporated in the reporter nucleic acid molecule, not the entire index oligonucleotide. In an alternative embodiment, the entire index oligonucleotide is incorporated in the reporter nucleic acid molecule.

[0111] As illustrated in Fig. 2, further provided is a method 200 for detecting multiple analytes in multiple samples, comprising: contacting 210 the samples with multiple proximity probe pairs, each proximity probe pair being specific for one analyte and comprising:

[0112] - a pair of oligonucleotides, wherein the first member and the second member of the oligonucleotide pair have complementary nucleotide sequences at their respective 3’-ends and are able to hybridize to each other at the 3’-ends, the first and / or second member comprising an identification sequence unique to the individual member or unique to the oligonucleotide pair, wherein the first or the second member of each oligonucleotide pair is optionally, at its 3’-end, blocked from being extended and protected from being degraded by a polymerase having 3’-exonuclease activity;

[0113] - a first analyte-binding moiety specific for a first analyte, and a second analytebinding moiety specific for said first analyte; under conditions suitable for binding of the analyte-binding moieties of each proximity probe to the corresponding analyte and hybridization of the complementary parts of the two members of the oligonucleotide pair of each proximity probe; contacting 220 each sample with a polymerase having 3’-exonuclease activity to extend one member of the oligonucleotide pair of each proximity probe to form a reporter nucleic acid molecule; contacting 215 each sample with an index oligonucleotide comprising a sample-specific index sequence and a member association sequence, optionally wherein the index oligonucleotide is, at its 3’-end, blocked from being extended and protected from being degraded by a polymerase having 3’-exonuclease activity, and wherein the step of contacting 220 each sample with a polymerase is additionally to incorporate the samplespecific index sequence in the reporter nucleic acid molecule; or contacting 225 each sample with an index oligonucleotide comprising a sample-specific index sequence and a member association sequence, optionally wherein the index oligonucleotide is, at its 3’-end, blocked from being extended and protected from being degraded by a polymerase having 3’-exonuclease activity, and contacting 227 each sample with a polymerase having 3’-exonuclease activity to incorporate the samplespecific index sequence in the reporter nucleic acid molecule; optionally amplifying 230 each reporter nucleic acid molecule; quantifying 240 the amount of each reporter nucleic acid molecule; and correlating 250 the amount of each reporter nucleic acid molecule to the amount of the corresponding analyte in each sample.

[0114] In an embodiment of the method 200, the step of contacting each sample with an index oligonucleotide is performed as a step 215, i.e., after the step of contacting 210 the sample with multiple proximity probe pairs and before the step of contacting 220 each sample with a polymerase, in which case the step of contacting 220 the sample with a polymerase is performed to extend one member of the oligonucleotide pair to form a reporter nucleic acid molecule and to incorporate the index sequence in the reporter nucleic acid molecule. In an alternative embodiment of the method 200, the step of contacting each sample with an index oligonucleotide is performed as a step 225, i.e., after the step of contacting 220 each sample with a polymerase and before the optional step of amplifying 230 each reporter nucleic acid molecule, if present, and before the step of quantifying 240 the amount of reporter nucleic acid molecule, in which case an additional step of contacting 227 the sample with a polymerase is performed to incorporate the index sequence in the reporter nucleic acid molecule. In other words, according to this alternative embodiment, the method 200 includes adding a polymerase to each sample twice, in both step 220 and step 227. Fig. 3 illustrates an embodiment of the present invention wherein the index oligonucleotide is blocked from being extended by the polymerase used in the extension step of a PEA method.

[0115] Part I. of Fig. 3 discloses a first member of an oligonucleotide pair, which first member comprises a first sequencing adapter (here indicated as the standard P5 sequence used for high throughput sequencing on instruments available from Illumina), a forward barcode (FBC) and a hybridization site (Hyb). The second member comprises the reverse complement of the Hyb-sequence (Hyb’) to allow hybridization between the first and second members. The second member also comprises a reverse barcode (RBC) and a Common sequence (Com). In embodiments utilizing a plurality of different oligonucleotide pairs, the Common sequence of the second member is generic, or common, to a plurality of second members. This may allow for hybridization of the second members to a generic oligonucleotide coupled to an analyte binding moiety, such as an antibody. Methods for generating such probes comprising analyte binding moieties and nucleic acid moieties are further described in US patent 10,781 ,473.

[0116] As shown in Fig. 3 part I., when the hybridization sites are annealed and the members are subjected to an extension reaction (“Extension 1”), both members will be extended, as is shown in Fig. 3 part II.

[0117] After the Extension 1 , an index oligonucleotide with a blocked 3’-end is added. The index oligonucleotide comprises a member association sequence (MAS) identical, or substantially identical, to the Common sequence (Com) of the second member mentioned above and thus able to hybridize to the reverse complement (Com’) in the extended first member, an index sequence (index barcode, IBC) and a second sequence adapter sequence (here indicated as the standard P7 sequence used for high throughput sequencing on instruments available from Illumina). The reaction mixture is heated (e.g. to 95 °C) to denature the extended members and improve index oligonucleotide distribution in solution (Part III.).

[0118] A second extension (“Extension 2”) follows, triggered by annealing of the index oligonucleotide (added in excess) to the extended first member. Exemplary conditions are 50 °C for at least 10 minutes (Part IV.).

[0119] Finally, amplification of reporter molecules starts as P5 and P7 primers are added to the reaction. An exemplary run is (95 °C, 30 sec / 54 °C, 60 sec / 60 °C, 60 sec)*25 + 72 °C 120 sec and hold at 10 °C. (Part V.) Fig. 4 illustrates an embodiment of the present invention wherein one member of the oligonucleotide pair is blocked from being extended by the polymerase used in the extension step of a PEA method, and also including incorporation of an index oligonucleotide.

[0120] Part I. of Fig. 4 discloses a first member of an oligonucleotide pair, which first member comprises a first sequencing adapter (here indicated as the standard P5 sequence used for high throughput sequencing on instruments available from Illumina), a forward barcode (FBC) and a hybridization site (Hyb). The second member comprises the reverse complement of the Hyb-sequence (Hyb’) to allow hybridization between the first and second members. The second member also comprises a reverse barcode (RBC) and a Common sequence (Com). In embodiments utilizing a plurality of different oligonucleotide pairs, the Common sequence of the second member is generic, or common, to a plurality of second members. This may allow for hybridization of the second members to a generic oligonucleotide coupled to an analyte binding moiety, such as an antibody. Methods for generating such probes comprising analyte binding moieties and nucleic acid moieties are further described in US10781473B2.

[0121] As shown in Fig. 4 part I., the 3’-end of the second member is blocked from being extended by a DNA polymerase. When the hybridization sites are annealed and the members are subjected to an extension reaction (“Extension 1”), only the first member will be extended, as is shown in Fig. 4 part II.

[0122] After the Extension 1 , an index oligonucleotide is added. The index oligonucleotide comprises a member association sequence (MAS) identical, or substantially identical, to the Common sequence (Com) of the second member mentioned above and thus able to hybridize to the reverse complement (Com’) in the extended first member, an index sequence (index barcode, IBC) and a second sequence adapter sequence (here indicated as the standard P7 sequence used for high throughput sequencing on instruments available from Illumina). The reaction mixture is heated (e.g. to 95 °C) to denature the extended members and improve index oligonucleotide distribution in solution (Part III.).

[0123] A second extension (“Extension 2”) follows, triggered by annealing of the index oligonucleotide (added in excess) to the extended first member. Exemplary conditions are 50 °C for at least 10 minutes (Part IV.).

[0124] Finally, amplification of reporter molecules starts as P5 and P7 primers are added to the reaction. An exemplary run is (95 °C, 30 sec / 54 °C, 60 sec / 60 °C, 60 sec)*25 + 72 °C 120 sec and hold at 10 °C. (Part V.) The present invention further provides use of any one of the herein disclosed systems for detecting at least one analyte in at least one sample, such as for detecting multiple analytes in at least one sample, such as for detecting multiple analytes in multiple samples.

[0125] As is known in the art, different detection modalities for the readout are possible. These include sequencing. Thus, for example, an identification sequence may be a barcode which is sequenced. Any method of sequencing may be used, including sequencing-by-synthesis and sequencing-by-hybridization methods. Thus, depending on the nature of the ID sequence, any suitable method may be used to identify the ID sequence, and this may involve the use of hybridization probes and / or primers. For example, the detection (readout) step may involve amplifying the reporter nucleic acid molecule using one or more primers, at least one of which binds to the ID sequence. Alternatively, the detection method may involve amplifying the reporter and detecting the amplicons by means of specific hybridization probes which bind to the ID sequence (or to a complement thereof). In sequencing-by- hybridization, barcodes can be decoded using labelled hybridization probes, including in combinatorial fashion.

[0126] Sequencing advantageously allows a high level of multiplexing and is a convenient method of detection. As noted above, any form of sequencing may be used, including any method of sequencing-by-synthesis, for example, pyrosequencing, reversible dye terminator sequencing and ion torrent sequencing. Particularly, high throughput methods of sequencing are used, and especially massively parallel DNA sequencing. Massively parallel DNA sequencing using the reversible dye terminator method may be performed, for instance, using an Illumina® NovaSeq™ system.

[0127] In another embodiment, the ID sequences are primer binding sites, e.g. for a PCR primer, although other amplification methods may be used.

[0128] In still further embodiments, the ID sequences may be restriction sites (i.e. a nucleotide sequence recognized by a restriction enzyme). In this embodiment, the nucleic acid domain of a proximity probe may comprise a different restriction site (such that it is recognized and cleaved by a different restriction enzyme). Different combinations of restriction enzymes may thus be applied to differentiate different reporter nucleic acids.

[0129] Amplification methods based on PCR are convenient and conveniently the readout may involve quantitative PCR (qPCR) or real-time PCR. The amplicons may be detected using any convenient protocol, including the use of dyes and stains, or labels, e.g. intercalating dyes, or labelled probes which bind to the amplicons. These include molecular beacons and such like, e.g. probes with FRET labels etc.

[0130] For instance, when readout is performed by qPCR, it is preferable to be able to provide a limited set of qPCR primers that work for all panels of assays, regardless of the assay content of the various panels. It is also preferable to keep the number of qPCR primers low to reduce cost, and to minimize risk of mismatched binding and other biological artefacts. With the present invention, it is possible to select a set of detection probes where all detection probes generate reporter molecules with unique identification sequences, while at the same time all those identification sequences also correspond to a limited set of qPCR primer binding sites, such that a corresponding limited set of qPCR primers can be used for read-out of the panel. In this way, the same set of qPCR primers can be used for readout of any panel compiled from the library according to the invention.

[0131] Thus, in one embodiment, the unique identification sequences in the library can be made to correspond to a set of qPCR primers that can be used for readout of any panel compiled from the library according to the invention.

[0132] In one embodiment, the identification sequences are binding sites for qPCR primers.

[0133] In one embodiment, the identification sequences are barcode sequences, used for detection by Next Generation Sequencing (NGS).

[0134] In an embodiment where detection of analytes is to be performed by use of Next Generation Sequencing (NGS), the above-disclosed system comprises an index oligonucleotide. In a corresponding embodiment, the above-disclosed method 100 or 200 comprises contacting (115; 125; 215; 225) the sample(s) with an index oligonucleotide.

[0135] In an embodiment where detection is performed by use of qPCR, the above-disclosed system does not comprise an index oligonucleotide. In a corresponding embodiment, the above-disclosed method 100 or 200 does not comprise contacting the sample(s) with an index oligonucleotide.

[0136] The invention will be further described in the following illustrative example(s). The example(s) are merely for facilitating understanding of the invention and shall not be construed as limiting the scope of the invention, which is that of the appended claims.

[0137] All prior publications referenced in the present specification are incorporated by reference in their entirety. Examples

[0138] Example 1 : One member of oligonucleotide pair blocked at its 3’ end

[0139] The experimental protocol for Next Generation Sequencing (NGS) as described in Wik et al. was followed, except the number of assays was lower.

[0140] Fig. 5 shows NGS signals from measurements (quadruplicates) of three different proteins in undiluted plasma and in buffer (negative control), respectively, using two variants of one of the two Proximity Extension Assay (PEA) probes (where the probe comprises one of the members of the oligonucleotide pair that have complementary nucleotide sequences at their respective 3’-ends and are able to hybridize to each other at the 3’-ends while leaving a portion of both members single-stranded). One variant of the DNA oligonucleotide had its 3’ end chemically modified such that it was blocked from being extended and from being degraded by the polymerase, and the other variant had its 3’ end unmodified (non-blocked). The chemical modification tested was Inverted dT.

[0141] As seen in Fig. 3, the blocked variant generally increased the signal-to-noise ratio.

[0142] Example 2: One member of oligonucleotide pair blocked at its 3’ end

[0143] The experimental protocol for Next Generation Sequencing (NGS) as described in Wik et al. was followed, except the number of assays was lower.

[0144] Fig. 6 shows NGS signals from measurement of a single protein in plasma (top dot) and in buffer (bottom dot) using four different variants of one of the two PEA probes, where the 3’ end of the DNA oligonucleotide was either blocked with one out of three different chemical modifications or non-blocked. The three different chemical modifications tested were Dideoxycytidine (3ddC), Inverted dT (InvdT), and 2'-O-Methyl RNA (mUmAmU).

[0145] As seen in Fig. 4, the different variants of blocked oligonucleotide generally increased the signal-to-noise ratio (the longer the length of the line, the more increased signal-to-noise ratio).

[0146] Example 3: One member of oligonucleotide pair blocked at its 3’ end

[0147] The experimental procedure involving detection by qPCR as described in Assarsson et al. was followed, except the number of assays was lower.

[0148] Fig. 7 shows qPCR S / N data (delta Ct) from measurement of a dilution series of LTBR protein and buffer (i.e., LTBR Ag concentration 0 pg / ml, negative control) using two different variants of one of the PEA probes, where the 3’ end of the DNA oligonucleotide was either chemically modified (i.e. , blocked from being extended and from being degraded by the polymerase) or non-blocked. The chemical modification tested was Inverted dT.

[0149] As seen in Fig. 5, the blocked oligonucleotide generally increased the signal-to-noise ratio.

[0150] Example 4: Index oligonucleotide blocked at its 3’ end

[0151] The protocol described in Wik et al. (Wik et al., Mol. Cell. Proteomics, 2021, 20, 100168) was run using four types of index oligonucleotides: unblocked (as per prior art), blocked to prevent digestion by 3’-exonuclease, blocked to prevent extension by DNA polymerase, and blocked to prevent both degradation by 3’-exonuclease and extension by DNA polymerase.

[0152] Samples and control: One pooled plasma sample (plate control, “PC”) and one individual plasma sample (“PS”) were used, as well as a negative control (buffer, “NC”).

[0153] Assays: Four different blocks of proximity extension assays were used, indicated as Block 1- 4, or B1, B2, B3, and B4, herein. (HT3=B1, HT4=B2, MX1=B3, MX4=B4). The blocks differed from the blocks described in Wik et al. only in the specific assays included.

[0154] Briefly, the respective samples were incubated with the PEA probes of the respective block of assays, at +4°C overnight. Amplicons were generated and preamplified from proximal binding probe pairs in a first PCR (PCR1). The three different samples (PC, PS, and NC) were then combined with sample index oligonucleotides with index sequences unique to the different samples, and in both an extension blocked configuration (“ix”), a digestion protected configuration (“iB”), a both extension blocked and digestion protected configuration (“ixB”) and an unblocked / unprotected configuration (“i”).

[0155] As taught in Wik et al., the index oligonucleotides generally comprise a hybridization region (herein called member association sequence, MAS) allowing the index oligonucleotide to anneal to the amplicons from PCR1, the sample unique index sequence, and a PCR primer region useful in a second PCR (PCR2). As in Wik et al., the second primer region was the P7 sequence, incorporated to facilitate sequencing of the amplified reporter molecules from PCR2 on sequencing instruments from Illumina. The sample index sequences were incorporated via the PCR2 program starting with a 95°C incubation for 3 min followed by ten cycles of amplification (95°C, 30s; 68°C, 1 min).

[0156] The PEA reactions were performed in a 96-well microtiter plate with the following plate layout:

[0157] Extension block: C3 spacer (modification code / 3SpC3 / )

[0158] Digestion protection: Phosphorothioate bond (modification code A* / T* / C* / G*) Extension block + digestion protection: C3 spacer and phosphorothioate bond.

[0159] The PCR products from PCR2 were analyzed on a TapeStation system (Agilent Technologies). Images of the electrophoresis gels produced by the TapeStation instrument are shown in Fig. 8 (A: Olink® Explore HT blocks; B: custom product blocks). The desired library products of PCR2 are indicated with arrows. It can be clearly seen that when the index oligonucleotide is blocked from being extended or protected from being degraded, the PCR product contain significantly more of the desired product and less of longer and shorter products as compared to when the index oligonucleotide is not blocked or protected.

[0160] Fig. 9 shows the DNA concentration (pg / pL) in the desired band for certain combinations of oligonucleotide modifications (non-modified, extension protected) for certain samples using certain blocks of assays. It can be seen that the concentration is significantly higher when an extension protected index oligonucleotide is used, as compared to a non-modified, except for the negative control of block B4. However, it must be noted that the extension blocked index oligonucleotide has in effect only gone through 9 amplification cycles, as compared to 10 amplification cycles for the unmodified index oligonucleotide, since the first cycle only amounts to an extension of the pre-amplified DNA molecules from PCR1 and not the index oligonucleotide. A corresponding tenth amplification cycle in the PCR2 step would thus result in significantly improved yield for extension protected index oligonucleotides.

[0161] References

[0162] US 7,306,904

[0163] US 8,013,134,

[0164] US 8,580,504

[0165] US 9,902,993

[0166] US 2023 / 0159983

[0167] US 2023 / 0323424

[0168] US2022 / 0162589;

[0169] US10781473B2

[0170] WO 01 / 61037

[0171] WO 03 / 044231

[0172] WO 2004 / 094456

[0173] WO 2005 / 123963

[0174] WO 2006 / 137932

[0175] WO 2013 / 113699

[0176] WO 2021 / 191442

[0177] WO 2021 / 191448

[0178] WO 2021 / 191449

[0179] WO 2022 / 191450

[0180] WO 2022 / 112300

[0181] Lundberg et al., Mol. Cell. Proteomics 2011 , 10(4), (DOI: 10.1074 / mcp.M110.004978)

[0182] Wik et al., Mol. Cell. Proteomics, 2021 , 20, 100168

[0183] Assarsson et al., PLoS 1 , 2014, 9(4), e95192

[0184] Enroth et al., Communications Biology 2019, 2(1): 1-12 (DOI: 10.1038 / s42003-019-0464-9) WO 2014 / 144371

[0185] Uhlen et al., Nat Methods, 2016, 13(10), 823-827

[0186] “Monoclonal Antibody Production”, National Academy Press, Washington, DC, USA, 1999

[0187] Carey-Hanly et al., ILAR Journal, 1995, 37(3): 93-118 llgu and Nilsen-Hamilton, Analyst., 2016, 141(5): 1551-1568

[0188] Desmyter et al., Nat. Structure Biol., 1996, 3: 803-811

[0189] Huston et al., Proc. Natl. Acad. Sci. USA, 1988, 85: 5879-5883

[0190] Kufer et al., Trends Biotechnol., 2004, 22: 238-244

[0191] Dugal-Tessier et al. (J. Clin. Med.2021, 10, 838)

[0192] Tavoosidana et al., PNAS, May 9, 2011 , 108 (21) 8809-8814

Claims

AMENDED CLAIMS received by the International Bureau on 20 November 2025 (20.11 .2025)1. A system comprising: a) at least one pair of oligonucleotides, wherein the first member and the second5 member of the oligonucleotide pair have complementary nucleotide sequences at their respective 3’-ends and are able to hybridize to each other at the 3’-ends, the first and / or second member comprising an identification sequence unique to the individual member or unique to the oligonucleotide pair; b) at least one index oligonucleotide, comprising an index sequence and a member10 association sequence; and c) a polymerase having 3’-exonuclease activity; characterized in that the index oligonucleotide of (b) is, at its 3’-end, blocked from being extended and / or protected from being degraded by the polymerase.

152. The system according to claim 1 , wherein the index oligonucleotide of (b) is, at its 3’- end, blocked from being extended by the polymerase.

3. The system according to claim 1 , wherein the index oligonucleotide of (b) is, at its 3’-20 end, protected from being degraded by the polymerase.

4. The system according to claim 1 , wherein the index oligonucleotide of (b) is, at its 3’- end, blocked from being extended and protected from being degraded by the polymerase.25 5. The system according to any one of claims1-4, wherein the first member and the second member of the oligonucleotide pair are able to hybridize to each other at the 3’-ends while leaving a portion of one or both member(s) single-stranded.

6. The system according to any one of claims 1-5, wherein the first or the second30 member of the oligonucleotide pair of (a) is, at its 3’-end, blocked from being extended and from being degraded by the polymerase.42AMENDED SHEET (ARTICLE 19)7. A system comprising: a) at least one pair of oligonucleotides, wherein the first member and the second member of the oligonucleotide pair have complementary nucleotide sequences5 at their respective 3’-ends and are able to hybridize to each other at the 3’-ends, the first and / or second member comprising an identification sequence unique to the individual member or unique to the oligonucleotide pair; b) a polymerase having 3’-exonuclease activity; characterized in that10 the first or the second member of the oligonucleotide pair of (a) is, at its 3’-end, blocked from being extended and from being degraded by the polymerase.

8. The system according to claim 7, wherein the first member and the second member of the oligonucleotide pair are able to hybridize to each other at the 3’-ends while leaving a15 portion of one or both member(s) single-stranded.

9. The system according to any one of claims 1 to 8, further comprising a first analytebinding moiety specific for a first analyte, and a second analyte-binding moiety specific for said first analyte, wherein the first member of the oligonucleotide pair is directly or indirectly20 attached to the first analyte-binding moiety, and the second member of the oligonucleotide pair is directly or indirectly attached to the second analyte-binding moiety, wherein the first and the second analyte-binding moieties can simultaneously bind to the first analyte.

10. The system according to claim 9, wherein the first and second analyte-binding25 moieties are independently selected from the group consisting of monoclonal, recombinant monoclonal and polyclonal antibodies and antigen-binding antibody derivatives and fragments, lectins, soluble cell surface receptors, combinatorially derived proteins from phage display or ribosome display, peptides, carbohydrates, molecularly imprinted polymers (MIPs), nucleic acids, such as an aptamer or nucleic acid molecules comprising the30 complementary sequence for a target nucleic acid, or combinations thereof.

11. The system according to any one of claims 9 or 10, wherein at least one of the identification sequences unique to the individual member or unique to the oligonucleotide43AMENDED SHEET (ARTICLE 19)pair is also an analyte-specific identification nucleotide sequence for identification of the analyte for which the analyte-binding domain is specific.

12. The system according to any one of the preceding claims, comprising multiple pairs5 of oligonucleotides.

13. The system according to any one of the preceding claims, comprising multiple index oligonucleotides.10 14. A method (100) for detecting at least one analyte in a sample, comprising: contacting (110) the sample with at least one pair of proximity probes, each proximity probe pair being specific for one analyte, and comprising:- a pair of oligonucleotides, wherein the first member and the second member of the oligonucleotide pair have complementary nucleotide sequences at their15 respective 3’-ends and are able to hybridize to each other at the 3’-ends, the first and / or second member comprising an identification sequence unique to the individual member or unique to the oligonucleotide pair;- a first analyte-binding moiety specific for a first analyte, and a second analyte-binding moiety specific for said first analyte;20 under conditions suitable for binding of the analyte-binding moieties to the corresponding analyte and hybridization of the complementary parts of the two members of the oligonucleotide pair; contacting (120) the sample with a polymerase having 3’-exonuclease activity to extend one member of the oligonucleotide pair to form a reporter nucleic acid molecule;25 contacting (115; 125) the sample with an index oligonucleotide, comprising a samplespecific index sequence and a member association sequence; wherein the index oligonucleotide is, at its 3’-end, blocked from being extended and / or from being degraded by a polymerase having 3’-exonuclease activity; contacting (120; 127) the sample with the polymerase having 3’-exonuclease activity to30 incorporate the sample-specific index sequence in the reporter nucleic acid molecule optionally amplifying (130) the reporter nucleic acid molecule; quantifying (140) the amount of reporter nucleic acid molecule; and44AMENDED SHEET (ARTICLE 19)correlating (150) the amount of reporter nucleic acid molecule to the amount of analyte in the sample and correlating the amount of reporter nucleic acid molecule, comprising the sample-specific index sequence, to the corresponding sample.5 15. The method according to claim 14, wherein the index oligonucleotide of (b) is, at its 3’-end, blocked from being extended by the polymerase.

16. The method according to claim 14, wherein the index oligonucleotide of (b) is, at its 3 ’-end, protected from being degraded by the polymerase.1017. The method according to claim 14, wherein the index oligonucleotide of (b) is, at its 3’-end, blocked from being extended and protected from being degraded by the polymerase.

18. The method according to any one of claims 14-17, wherein the first member and the15 second member of the oligonucleotide pair are able to hybridize to each other at the 3’-ends while leaving a portion of one or both member(s) single-stranded.

19. The method according to any one of claims 14-18, wherein the first or the second member of each oligonucleotide pair is, at its 3’-end, blocked from being extended and from20 being degraded by a polymerase having 3’-exonuclease activity.

20. A method (100) for detecting at least one analyte in a sample, comprising: contacting (110) the sample with at least one pair of proximity probes, each proximity probe pair being specific for one analyte, and comprising:25 - a pair of oligonucleotides, wherein the first member and the second member of the oligonucleotide pair have complementary nucleotide sequences at their respective 3’-ends and are able to hybridize to each other at the 3’-ends, the first and / or second member comprising an identification sequence unique to the individual member or unique to the oligonucleotide pair, wherein the first or the second member30 of each oligonucleotide pair is, at its 3’-end, blocked from being extended and from being degraded by a polymerase having 3’-exonuclease activity;45AMENDED SHEET (ARTICLE 19)- a first analyte-binding moiety specific for a first analyte, and a second analytebinding moiety specific for said first analyte; under conditions suitable for binding of the analyte-binding moieties to the corresponding analyte and hybridization of the complementary parts of the two5 members of the oligonucleotide pair; contacting (120) the sample with a polymerase having 3’-exonuclease activity to extend one member of the oligonucleotide pair to form a reporter nucleic acid molecule; optionally amplifying (130) the reporter nucleic acid molecule; quantifying (140) the amount of reporter nucleic acid molecule; and10 correlating (150) the amount of reporter nucleic acid molecule to the amount of analyte in the sample.

21. The method according to claim 20, wherein the first member and the second member of the oligonucleotide pair have complementary nucleotide sequences at their respective 3’-15 ends and are able to hybridize to each other at the 3’-ends while leaving a portion of both members single-stranded.

22. A method (200) for detecting multiple analytes in multiple samples, comprising: contacting (210) the samples with multiple proximity probe pairs,20 each proximity probe pair being specific for one analyte and comprising:- a pair of oligonucleotides, wherein the first member and the second member of the oligonucleotide pair can interact directly or indirectly to form a doublestranded helix, the first and / or second member comprising an identification sequence unique to the individual member or unique to the oligonucleotide pair;25 - a first analyte-binding moiety specific for a first analyte, and a second analyte-binding moiety specific for said first analyte; under conditions suitable for binding of the analyte-binding moieties of each proximity probe to the corresponding analyte and hybridization of the complementary parts of the two members of the oligonucleotide pair of each proximity probe and hybridization of the30 complementary parts of the two members of the oligonucleotide pair of each proximity probe; contacting (220) each sample with a polymerase having 3’-exonuclease activity to extend one member of the oligonucleotide pair of each proximity probe to form a reporter nucleic acid molecule;35 contacting (215) each sample with an index oligonucleotide comprising a samplespecific index sequence and a member association sequence, wherein the index46AMENDED SHEET (ARTICLE 19)oligonucleotide is, at its 3’-end, blocked from being extended and / or from being degraded by a polymerase having 3’-exonuclease activity, and wherein the step of contacting (220) each sample with a polymerase is additionally to incorporate the sample-specific index sequence in the reporter nucleic acid molecule; or5 contacting (225) each sample with an index oligonucleotide comprising a samplespecific index sequence and a member association sequence, wherein the index oligonucleotide is, at its 3’-end, blocked from being extended and / or from being degraded by a polymerase having 3’-exonuclease activity, and contacting (227) each sample with a polymerase having 3’-exonuclease activity to incorporate the sample¬10 specific index sequence in the reporter nucleic acid molecule; optionally amplifying (230) each reporter nucleic acid molecule; quantifying (240) the amount of each reporter nucleic acid molecule comprising the sample-specific index sequence; and correlating (250) the amount of each reporter nucleic acid molecule, comprising the15 sample-specific index sequence, to the amount of the corresponding analyte in each corresponding sample.

23. The method according to claim 22, wherein the index oligonucleotide of (b) is, at its 3’-end, blocked from being extended by the polymerase.2024. The method according to claim 22, wherein the index oligonucleotide of (b) is, at its 3 ’-end, protected from being degraded by the polymerase.

25. The method according to claim 22, wherein the index oligonucleotide of (b) is, at its25 3’-end, blocked from being extended and protected from being degraded by the polymerase.

26. A method (200) for detecting multiple analytes in multiple samples, comprising: contacting (210) the samples with multiple proximity probe pairs, each proximity probe pair being specific for one analyte and comprising:30 - a pair of oligonucleotides, wherein the first member and the second member of the oligonucleotide pair can interact directly or indirectly to form a doublestranded helix while leaving a portion of both members single-stranded, the first and / or second member comprising an identification sequence unique to the individual member or unique to the oligonucleotide pair, wherein the first or the47AMENDED SHEET (ARTICLE 19)second member of each oligonucleotide pair is, at its 3’-end, blocked from being extended and from being degraded by a polymerase having 3’-exonuclease activity;- a first analyte-binding moiety specific for a first analyte, and a second analyte-binding moiety specific for said first analyte;5 under conditions suitable for binding of the analyte-binding moieties of each proximity probe to the corresponding analyte c; contacting (220) each sample with a polymerase having 3’-exonuclease activity to extend one member of the oligonucleotide pair of each proximity probe to form a reporter nucleic acid molecule;10 contacting (215) each sample with an index oligonucleotide comprising a samplespecific index sequence and a member association sequence, and wherein the step of contacting (220) each sample with a polymerase is additionally to incorporate the sample-specific index sequence in the reporter nucleic acid molecule; or contacting (225) each sample with an index oligonucleotide comprising a sample¬15 specific index sequence and a member association sequence, and contacting (227) each sample with a polymerase having 3’-exonuclease activity to incorporate the sample-specific index sequence in the reporter nucleic acid molecule; optionally amplifying (230) each reporter nucleic acid molecule; quantifying (240) the amount of each reporter nucleic acid molecule comprising the20 sample-specific index sequence; and correlating (250) the amount of each reporter nucleic acid molecule, comprising the sample-specific index sequence, to the amount of the corresponding analyte in each corresponding sample.25 27. The method according to any one of claims 22-26, wherein the first member and the second member of the oligonucleotide pair have complementary nucleotide sequences at their respective 3’-ends and are able to hybridize to each other at the 3’-ends while leaving a portion of both members single-stranded.30 28. The method according to any one of claims 22 to 27, wherein quantification of the amount of reporter nucleic acid molecules is performed by nucleic acid sequencing.48AMENDED SHEET (ARTICLE 19)29. The method according to any one of claims 22 to 27, wherein quantification of the amount of reporter nucleic acid molecules is performed by nucleic acid amplification.

30. The method according to any one of claims 14-29, wherein the first and second5 analyte-binding moieties are independently selected from the group consisting of monoclonal, recombinant monoclonal and polyclonal antibodies and antigen-binding antibody derivatives and fragments, lectins, soluble cell surface receptors, combinatorially derived proteins from phage display or ribosome display, peptides, carbohydrates, molecularly imprinted polymers (MIPs), nucleic acids, such as an aptamer or nucleic acid10 molecules comprising the complementary sequence for a target nucleic acid, or combinations thereof.

31. Use of a system according to any one of claims 1-13 for detecting at least one analyte in at least one sample.1549AMENDED SHEET (ARTICLE 19)

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