System and method for detecting an analyte in a sample

Abstract

The present invention relates to a system and methods for detection of at least one analyte of interest in sample. The system comprises at least one pair or proximity probes, comprising a first and a second proximity probe wherein the nucleic acid domain of the first proximity probe is partially double-stranded. The first proximity probe comprises an anchor moiety for capture on a solid phase, wherein the anchor moiety is attached to the end of one of the strands of the partially double-stranded nucleic acid domain of the first proximity probe. The system and method of the present invention provide a new and improved way for solid phase purification in proximity based assays.

Description

[0001] SYSTEM AND METHOD FOR DETECTING AN ANALYTE IN A SAMPLE TECHNICAL FIELD OF THE INVENTION

[0002] The present invention relates to the field of detecting the presence of at least one analyte, particularly a protein analyte, in at least one sample. More specifically, the present invention relates to dual-recognition immunoassays, particularly PEA (Proximity Extension Assay) and PLA (Proximity Ligation Assay) for detecting and measuring at least one analyte in a sample and to a system for use in such assays. The methods and system find particular use in multiplex assays for detecting multiple protein analytes that require larger sample volumes for detection and that use solid-phase purification as part of the assay.

[0003] BACKGROUND OF THE INVENTION

[0004] Modern personalised medicine requires the ability to assess large panels of biomarkers, e.g. in the field of oncology. As personalised medicine becomes ever more widespread, the ability to accurately identify and quantify a large number of biomarkers in a sample is increasing in importance.

[0005] Hence, modern proteomics methods need 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 dual-recognition immunoassays, particularly proximity extension assays (PEA) and proximity ligation assays (PLA). PEA and PLA are described in WO 01 / 61037. PEA is further described in WO 03 / 044231 , WO 2004 / 094456, WO 2005 / 123963, WO 2006 / 137932 and WO 2013 / 113699.

[0006] Multiplexed PLA is also further described in i.a. Lundberg et al. Molecular & Cellular Proteomics 10:10.1074 / mcp.M110.004978, 1-10, 2011 and multiplexed PEA is further described in e.g. Assarsson et al., PLoS 1 , 2014, 9, 4, e95192; Wik et aL, 2021 , Mol Cell 30 Proteomics 20, 100168 and Siegbahn A, et al., PLoS One. 2023 Nov 14; 18(11): e0293465. Briefly, in such assays, analytes (particularly proteins, or parts thereof) are detected through the binding of a pair of binders (usually antibodies, or parts thereof) comprising oligonucleotides (particularly DNA) tails that are attached thereto. A binding event brings the oligonucleotides that are coupled to each member of the pair of binders in proximity (“proximity probes”) and allows them to interact to either extend (PEA) or be ligated (PLA) to generate a reporter nucleic acid molecule.

[0007] This reporter nucleic acid molecule can then be detected e.g. through qPCR or by sequencing the DNA reporter using Next Generation Sequencing (NGS). The presence of ananalyte of interest in a sample is hence indirectly determined through detecting the presence of the reporter nucleic acid molecule produced after a hybridization event between the oligonucleotides of the respective binders of each pair, wherein the hybridization event is either directly between the respective oligonucleotides or between the oligonucleotides and a splint oligonucleotide.

[0008] WO2017068116 relates to a method of manufacturing pairs of proximity probes, wherein each probe of the proximity probe pair comprises a universal oligonucleotide conjugate bound to the analyte binding moiety of the probe. One advantage of using universal oligonucleotide conjugates is that it reduces the number of chemical activation reactions that are required to manufacture a plurality of proximity probe pairs, thereby providing for a greater consistency to be achieved and for the reactions to be performed in large batches. Different tag oligonucleotides comprising unique domains are hybridized to the universal conjugates. As an example, at least one of the probes of each proximity probe pair comprises in its unique domain a sequence that is specific and representative to a particular target analyte.

[0009] Both homogenous and heterogenous versions of proximity-based assays are known in the art. Homogenous assays are performed entirely in solution, whereas heterogenous assays capture the probe-analyte complex onto a solid phase to allow for washing steps to wash away material that may negatively affect the performance of the assay. Solid phase protocols in proximity-based assays have conventionally immobilized the probe-analyte complex onto a solid phase. This has e.g. been achieved through use of a capture antibody or other binding agent with specificity for the analyte, being bound to a solid phase and used to immobilize the analyte prior to formation of the probe-analyte complex (see e.g.

[0010] W09700446). Another mechanism has been to include an anchor moiety, e.g. a oligo-dA strand or a biotin) on one oligonucleotide part of one of the proximity probes, or a biotin moiety directly attached to the antibody part of one of the proximity probes (see e.g W02004094456, W02021113290).

[0011] There is a constant need in the art for improving the sensitivity of dual-recognition assays, particularly when the purpose is to detect the presence of analytes that may be present in low concentrations in a sample. Different ways of improving sensitivity exist in the prior art, but there is always a need for identifying even more improved or at least alternative ways to improve the sensitivity of the assay. One known way to do so is to increase the total volume of the samples, but this can cause other problems such as precipitation during amplification (PCR) of the reporter product and may risk affecting the outcome of the data. In these situations, a solid-phase purification step is often needed.In this regard, there is also always a need to introduce more flexible, versatile solid-phase protocols for use in dual-recognition assays.

[0012] SUMMARY OF THE INVENTION

[0013] The present invention aims to solve problems relating to scale-up, effectivization and / or automation of dual-recognition immunoassays, more specifically in relation to proximitybased assays. More precisely, the present invention aims to solve or at least mitigate problems that may affect sensitivity and accuracy of the assay and that is caused by impurities affecting the output data.

[0014] More particularly, the present invention aims to solve problems that may arise when increasing the volume of the samples in the assay, which is sometimes desirable to improve sensitivity. However, increasing the sample volume is associated with other issues.

[0015] These issues are addressed by the present invention by providing an improved, or at least an alternative system and method for increasing sensitivity of an assay as further described herein. Naturally, a system and method according to the present invention also benefits the outcome of assays using standard sample volumes by in general improving sensitivity. In this regard, the present inventors have found that a proximity based method for detecting at least one analyte of interest benefits from a solid-phase purification step using a solidphase to which the reporter nucleic acid molecule may be captured through the presence of an anchor moiety incorporated in a nucleic acid domain of the proximity probe used to generate the reporter molecule.

[0016] According to the invention, a first proximity probe of a proximity probe pair comprises a first nucleic acid domain that is partially double-stranded, comprising a first hybridization oligonucleotide and a first conjugation oligonucleotide, wherein an anchor moiety is attached to one of the ends of the first hybridization oligonucleotide. The analyte-binding domain is attached to the conjugation oligonucleotide and the first hybridization oligonucleotide is hybridized to the first conjugation oligonucleotide.

[0017] Thus, in a first aspect, the present invention relates to a system for detection of at least one analyte of interest in a sample, said system comprising at least one proximity probe pair, each proximity probe pair comprising:

[0018] a. a first proximity probe comprising a first analyte binding domain coupled to a first nucleic acid domain, and;

[0019] b. a second proximity probe comprising a second analyte binding domain coupled to a second nucleic acid domain,wherein the first and second analyte binding domains of said proximity probe pair are capable of simultaneously and specifically binding to an analyte of interest present, or suspected of being present, in the sample,

[0020] in each proximity probe pair, the first nucleic acid domain is partially double-stranded, comprising:

[0021] i) a first conjugation oligonucleotide and, ii) a first hybridization oligonucleotide

[0022] together providing a double-stranded part of the first nucleic acid domain,

[0023] wherein the first conjugation oligonucleotide is conjugated to the analyte binding domain of the first proximity probe via its 5’ end or 3’ end,

[0024] wherein the first conjugation oligonucleotide and the first hybridization oligonucleotide are configured to hybridise to each other through paired universal sequence elements present in the conjugation oligonucleotide and the hybridization oligonucleotide, wherein the first hybridization oligonucleotide and the second nucleic acid domain together are capable of directly or indirectly interacting to generate a reporter nucleic acid molecule when said first and second proximity probes are both bound to the same analyte and,

[0025] wherein the first hybridization oligonucleotide comprises, at its 5’ or 3’ end, an anchor moiety for capture onto a solid phase.

[0026] In a second aspect, the present invention relates to a method for detecting at least one analyte of interest in a sample, said method comprising the steps of:

[0027] i) Providing a system according to the first aspect,

[0028] ii) Contacting a sample with the system of step i),

[0029] iii) Allowing the system of step ii) to interact with the sample to generate matched pairs of proximity probes bound to their analyte of interest,

[0030] iv) Generating a reporter nucleic acid molecule from interacting first and second nucleic acid domains of the system of step iii) to obtain a reporter nucleic acid molecule comprising the anchor moiety,

[0031] v) Bringing the reporter nucleic acid molecule of step iv) into contact with a solid phase, wherein said solid phase comprises a capture group configured to capture the anchor moiety present in the reporter nucleic acid molecule,

[0032] vi) Allowing the reporter nucleic acid molecule to be captured onto the solid phase via the anchor moiety,

[0033] vii) Washing the solid phase one or more times,viii) Performing an amplification reaction of the reporter nucleic acid molecule captured on the solid phase wherein the amplification reaction results in the production of a reaction mixture comprising amplified reporter nucleic acid molecules free from the solid phase, ix) Detecting the at least one analyte of interest through detection of the amplified reporter nucleic acid molecules.

[0034] In a third aspect, the present invention relates to a method for detecting at least one analyte of interest in a sample, said method comprising the steps of:

[0035] i) Providing a system according to the first aspect,

[0036] ii) Contacting a sample with the system of step i)

[0037] iii) Allowing the system of step ii) to interact with the sample to generate matched pairs of proximity probes bound to their analyte of interest,

[0038] iv) Bringing the matched pairs of proximity probes of step iii) into contact with a solid phase, wherein said solid phase comprises a capture group configured to capture the anchor moiety present in the first nucleic acid domain,

[0039] v) Allowing the matched pairs of proximity probes to be captured onto the solid phase through the anchor moiety present in the first nucleic acid domain,

[0040] vi) Optionally washing the solid phase one or more times,

[0041] vii) Generating a reporter nucleic acid molecule from interacting nucleic acid domains of the system of step iii) to obtain a reporter nucleic acid molecule captured on the solid phase through the anchor moiety, or

[0042] releasing the matched pairs of proximity probes from the solid phase followed by generation of a reporter nucleic acid molecule,

[0043] viii) Optionally washing the solid phase one or more times,

[0044] ix) Performing an amplification reaction of the reporter nucleic acid molecule captured to the solid phase wherein the amplification reaction results in the production of a reaction mixture comprising amplified reporter nucleic acid molecules free from the solid phase,

[0045] x) Detecting the at least one analyte of interest through detection of the amplified reporter nucleic acid molecules.

[0046] In a fourth embodiment, the present invention relates to a control reagent comprising a first nucleic acid domain and a second nucleic acid domain coupled together to be in constant proximity, the first nucleic acid domain is partially double-stranded, comprising:

[0047] i) a first conjugation oligonucleotide and,

[0048] ii) a first hybridization oligonucleotide

[0049] together providing a double-stranded part of a first nucleic acid domain,

[0050] wherein the first conjugation oligonucleotide is coupled to the second nucleic acid domain to keep the first and second nucleic acid domains in constant proximity,wherein the first conjugation oligonucleotide and the first hybridization oligonucleotide are configured to hybridize to each other through paired universal sequence elements present in the conjugation oligonucleotide and the hybridization oligonucleotide, wherein the first and / or second nucleic acid domain of the control reagent comprise a control reagent identification sequence identifying the control reagent,

[0051] wherein the first hybridization oligonucleotide and the second nucleic acid domain together are capable of directly or indirectly interacting to generate a reporter nucleic acid molecule, and

[0052] wherein the first hybridization oligonucleotide comprises, at its 5’ or 3’ end, an anchor moiety for capture onto a solid phase.

[0053] BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 shows a number of probe designs useful in all aspects of the present invention (1 to 6) and control reagent designs useful for internal extension controls (7 and 8).

[0055] Figure 2 shows a probe design of the prior art (A) and preferred probe designs according to the present invention (B, C and D) useful in all aspects of the invention.

[0056] Figure 3 is an illustration of a method according to the second aspect of the invention, wherein the reporter nucleic acid molecule comprising an anchor moiety is generated before capture on the solid phase, and the probe-analyte complex is washed away before amplifying the generated reporter nucleic acid molecule captured on the solid phase.

[0057] Figure 4 is an illustration of an embodiment of a method according to the third aspect of the invention, where the probe-analyte complex is captured on the solid phase followed by washout of any unbound probes and analytes before generating a reporter nucleic acid molecule when the probe-analyte complex is still bound to the solid phase or wherein the probe-analyte complex is released from the solid phase and a reporter nucleic acid molecule is generated and subsequently amplified away from the solid phase.

[0058] Figure 5 shows the Ct values for the extension control and the respective analytes (Panel A) and panel B shows the signal to noise for the respective analytes, for different sample volumes.

[0059] DEFINITIONS OF TERMS AND ABBREVIATIONS

[0060] 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. For the sake of clarity, a few terms and abbreviations are defined below.

[0061] The singular “a” and “an” shall be construed as including also the plural.Compositions “comprising” one or more recited elements may also include other elements not specifically recited. The term "comprising” also encompasses the term “consisting of’. An ’’immunoassay” is a type of assay for a specific analyte to be detected, that utilizes molecules binding specifically and preferentially to the analyte in question and wherein binding events generate or result in a signal that can be detected. While the prefix “immuno-” implies, and originates from, the original use of antibodies as the specific binding molecules, immunoassays may also use other types of specific analyte binding molecules, as further exemplified herein.

[0062] “Dual recognition immunoassay” is an immunoassay wherein two simultaneous and specific binding events are required for a signal to be generated. PEA (Proximity Extension Assay) and PLA (Proximity Ligation Assay) are examples of dual recognition immunoassays. These are assays well-known in the art as further described herein. Dual recognition immunoassays differ from e.g. sandwich ELISAs that generally includes capture of the analyte by a capture antibody and subsequent addition of a second antibody (detection antibody) coupled to a signal-generating moiety. While this involves two binding events, these two events are not both required to generate a signal, as the signal is generated solely by the signal-generating moiety on the detection antibody.

[0063] “Multiplexing” of biological assays, such as proximity assays, means performing a plurality of assays in parallel and, preferably, in the same reaction space, 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. As used herein, the term “multiplex” is used to refer to an assay in which multiple or a plurality of analytes (i.e. at least two) different analytes are assayed at the same time, and more particularly in the same sample.

[0064] The term “plurality” or “multiple” as used in the present invention means more than one (that is to say, two or more), in line with its standard definition.

[0065] 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 and / or samplespecific probe composition of the invention. The analyte is thus the "target" of a method and / or sample-specific probe composition of the invention, i.e. the substance to be detected or screened for using the method and / or sample-specific probe composition of the invention. The term "detecting" or "detected” and the like 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 specific sample or not). Accordingly, if a method of the invention isperformed 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.

[0066] A “proximity probe” as referred to herein, is a probe for the detection of an analyte in a sample that is generally used in pairs, i.e. in general a first and a second proximity probe is used for detection of an analyte (but it may also comprise more than two probes in combination). A first and a second proximity probe that are used as a pair are herein referred to as a “proximity probe pair”. 'The terms “proximity probing” and “proximity assays” are also used herein.

[0067] The “analyte binding domain” or “analyte binding moiety”, which are terms used interchangeably herein, of a proximity probe of a proximity probe pair, may comprise any entity capable of binding specifically to an analyte of interest (or part thereof) and capable of being coupled to “a nucleic acid domain”. That the analyte binding domain binds “specifically” to or 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. Further, the analyte binding domain may bind to the analyte directly or indirectly. In other words, the proximity probe(s) 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.

[0068] The “nucleic acid domain” or “nucleic acid moiety”, which are terms that may be used interchangeably herein, of a proximity probe of a proximity probe pair, comprises a domain or moiety capable of generating a detectable signal when dual recognition is achieved between the nucleic acid domain of the first and second proximity probe of a proximity probe pair. The nucleic acid domain is coupled to the analyte binding domain of each proximity probe. The nucleic acid domain or moiety is sometimes referred to herein as an “oligonucleotide”.

[0069] Generally, the nucleic acid domain has a length in the range of 20-100 nucleotides but may be shorter or longer as required in the specific assay / method in which the proximity probe is intended to be used.

[0070] A “sample” is a discrete volume of material which is subjected to the method according to the invention.

[0071] A “sample identification sequence” is a nucleic acid sequence that serves to identify the sample from which a reporter nucleic acid molecule is derived.An “analyte identification sequence” is a nucleic acid sequence that serves to identify a specific analyte, analogous to a sample identification sequence identifying a specific sample. “Readout” as used herein is intended to refer to the process of detecting the presence of and / or quantifying the amount of reporter nucleic acid molecules and correlating these amounts to the amounts of the respective at least one analyte of interest originating from a specific sample. Accordingly, a readout can be seen as a step of detecting the signal in the assay, or more particularly the reporter nucleic acid molecules, in a quantitative manner. An “anchor moiety” or “anchor group” as referred to herein, is a physical structure used for capturing a proximity probe or a proximity probe-analyte complex onto a solid phase to purify proximity probe pairs bound to an analyte from unbound probes and other impurities. The anchor moiety is bound or captured directly or indirectly to the solid phase. The moiety on the solid phase capturing the anchor moiety is correspondingly termed “capture moiety”. How the anchor moiety is bound or captured to the proximity probe is described elsewhere herein. The present invention makes use of “identification sequences”. An identification sequence may e.g. be a unique sequence (sometimes termed a “barcode sequence” or simply “barcode” or “index”) that is detected in a sequence-specific manner and used to identify the origin of the molecule in which it is present. It may, for example, be 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). Recognition sites for restriction enzymes may also be used as identification sequences.

[0072] A “sample-specific probe composition” as referred to herein, comprises a proximity probe pair comprising a first proximity probe and a second proximity probe wherein the nucleic acid domain of at least one of the first or the second proximity probe comprises a sample identification sequence. The term “sample-specific probe composition” may also be referred to herein as a “sample-specific proximity probe composition” or in short to as a “probe composition”. When bringing the sample-specific probe composition into contact with a sample, a “sample-probe combination” is obtained. Hence, a “sample-probe combination” comprises the sample-specific proximity probe composition and the sample. The sampleprobe combinations are “pooled” before reporter nucleic acid molecules are generated from the interacting first and second nucleic acid domains of the proximity probes. This procedure is also referred to herein as “pooling” of the sample probe combinations. This means that different sample-specific probe compositions combined with their respective samples are mixed to allow for a more efficient analysis of the reaction products by enabling single reaction condition for the steps of the method following the pooling.DETAILED DESCRIPTION OF THE INVENTION

[0073] The present invention introduces a new, more versatile, probe configuration for use in solid phase protocols for proximity-based assays. Whereas the prior art solid phase protocols have immobilized only the probe-analyte complex, the present invention allows for immobilization of the reporter nucleic acid molecule. A potential drawback with solid-phase protocols based on immobilization of the probe-analyte complex is that the probe-analyte complex is a large complex held together in part by more or less strong non-covalent interactions. There is always a risk that wash steps performed while having such a complex immobilized on a solid-phase disrupts cognate probe-analyte complex to some extent, thereby reducing the number of cognate probe-analyte complexes generating signal, ultimately leading to a less than optimal signal output. The reporter molecule is not as complex and may be more resistant to disruption and / or being washed away when immobilized on a solid phase during a wash step. The probe design according to the present invention thus enables more robust signal generation when using a solid-phase protocol. It is also versatile in that it allows both for immobilization of the generated nucleic acid reporter molecule as well as immobilization of the probe-analyte complex, as further described herein. As also mentioned elsewhere herein, it is particularly useful for analysing larger sample volumes, which is sometimes desired but associated with complications.

[0074] The probe design according to the present invention makes use of a proximity probe pair, a first and a second proximity probe, wherein both members of the proximity probe pair comprise an analyte-binding domain and a nucleic acid domain. At least one of the proximity probes, the first proximity probe, is partially double-stranded. The partially double-stranded first proximity probe has a nucleic acid strand conjugated to it (a “first conjugation oligonucleotide”). A further oligonucleotide strand (a “first hybridization oligonucleotide”) is hybridized to the conjugation oligonucleotide. Such probes are known in the art and may be produced by the methods disclosed in U.S. Patent 10,781,473, incorporated by reference in its entirety. The probes according to the present invention differ from the prior art in that one hybridization oligonucleotide has an anchor moiety at one end, facilitating capture onto a solid phase. As in the prior art, one end of the hybridization oligonucleotide is capable of interacting with the nucleic acid domain of the second proximity probe to generate a reporter nucleic acid molecule.

[0075] The second proximity probe in the proximity probe pair may be designed in the same way as for the first proximity probe, i.e., with a conjugation and hybridization oligonucleotide (a second conjugation oligonucleotide and a second hybridization oligonucleotide, respectively) or have only a conjugation oligonucleotide (a second conjugation oligonucleotide) conjugatedto the analyte binding domain, both which that can interact, directly or indirectly, with the first partially double-stranded proximity probe to generate a reporter nucleic acid molecule. As is known in the art, the oligonucleotide domains of the first and / or second proximity probes in a pair comprise identification sequences specific to the analyte to which the probes bind, which identification sequences are incorporated into the reporter nucleic acid molecule in order to identify the analyte.

[0076] According to the present invention, the first hybridization oligonucleotide has a first end and a second end, wherein an anchor moiety is present at the first end and wherein the second end is configured to be able to generate a reporter nucleic acid molecule through extension or ligation. In other words, the first hybridization oligonucleotide can comprise an anchor moiety at its 3’ or 5’ end and be configured to be able to generate a reporter nucleic acid molecule, together with the second nucleic acid domain, through extension or ligation, indirectly or directly, by its 3’ or 5’ end, (vice versa). The first conjugation oligonucleotide can be conjugated to the first analyte-binding domain via its 5’ or its 3’ end depending on the probe design used for the proximity probe pair.

[0077] The second hybridization oligonucleotide, if present, can comprise an end that is configured to be able to generate a reporter nucleic acid molecule through extension or ligation. In other words, the second hybridization oligonucleotide can be configured to be able to generate a reporter nucleic acid molecule, together with the first nucleic acid domain, through extension or ligation, indirectly or directly, by its 3’ or 5’ end, vice versa. The second conjugation oligonucleotide can be conjugated to the second analyte-binding domain via its 5’ or its 3’ end depending on the probe design used for the proximity probe pair.

[0078] Thus, the reporter nucleic acid molecule may be generated using different designs of the proximity probe pair, as will be described further herein.

[0079] Hence, there is provided by the present invention a system for detection of at least one analyte of interest in a sample, wherein said system comprises at least one proximity probe pair comprising a first and a second proximity probe.

[0080] More specifically, there is in a first aspect provided a system for detection of at least one analyte of interest in a sample, said system comprising at least one proximity probe pair, each proximity probe pair comprising:

[0081] a. a first proximity probe comprising a first analyte binding domain coupled to a first nucleic acid domain, and;

[0082] b. a second proximity probe comprising a second analyte binding domain coupled to a second nucleic acid domain,wherein the first and second analyte binding domains of said proximity probe pair are capable of simultaneously and specifically binding to an analyte of interest present, or suspected of being present, in the sample,

[0083] in each proximity probe pair, the first nucleic acid domain is partially double-stranded, comprising:

[0084] i) a first conjugation oligonucleotide and, ii) a first hybridization oligonucleotide

[0085] together providing a double-stranded part of the first nucleic acid domain,

[0086] wherein the first conjugation oligonucleotide is conjugated to the analyte binding domain of the first proximity probe via the 5’ end or 3’ end of the conjugation oligonucleotide, wherein the first conjugation oligonucleotide and the first hybridization oligonucleotide are configured to hybridise to each other through paired universal sequence elements present in the conjugation oligonucleotide and the hybridization oligonucleotide, wherein the first hybridization oligonucleotide and the second nucleic acid domain together are capable of directly or indirectly interacting to generate a reporter nucleic acid molecule when said first and second proximity probes are both bound to the same analyte, and

[0087] wherein the first hybridization oligonucleotide comprises, at its 5’ or 3’ end, an anchor moiety for capture onto a solid phase.

[0088] According to the first aspect, the first proximity probe of a proximity probe pair comprises an anchor moiety that may be captured on, and directly bound to, a solid phase. When the first proximity probe is captured on the solid phase, the other proximity probe of the same proximity probe pair may, in some aspects of the invention, be described as indirectly captured to the solid phase as the probe is interacting with the other proximity probe of the proximity probe pair through its concurrent binding to the same analyte molecule, while not being directly bound to the solid phase.

[0089] In one embodiment, the first and / or second nucleic acid domain of each proximity probe pair comprise an analyte identification sequence identifying the specific analyte of interest, In one embodiment, the first conjugation oligonucleotide conjugated to the first analytebinding domain is configured to be displaced from the first hybridization oligonucleotide upon the generation of a reporter nucleic acid molecule.

[0090] In one embodiment, the first hybridization oligonucleotide of the first nucleic acid domain is configured to be extended using the second nucleic acid domain as a template, optionally wherein second nucleic acid domain is configured to be extended using the first hybridization oligonucleotide of the first nucleic acid domain as a template. In other words, a second endof the first hybridization oligonucleotide contains a hybridization region that is matched with a hybridization region on the nucleic acid domain part of the second proximity probe so that the first hybridization oligonucleotide can be extended using the nucleic acid domain of the second proximity probe as a template, to generate a reporter nucleic acid molecule, wherein the first end of the first hybridization oligonucleotide comprises an anchor moiety.

[0091] In one embodiment, also the second nucleic acid domain of the second proximity probe of the proximity probe pair is partially double-stranded, said second partially double-stranded nucleic acid domain comprising: i) a second conjugation oligonucleotide and, ii) a second hybridization oligonucleotide, wherein the second conjugation oligonucleotide and the second hybridization oligonucleotide are configured to hybridise to each other through paired universal sequence elements present in the conjugation oligonucleotide and the hybridization oligonucleotide.

[0092] The universal sequence elements are sequence elements used to pair the hybridization and conjugation oligonucleotide within the respective proximity probe pair, but which sequence elements are not unique (in sequence) for the respective proximity probe pair, therefore referred to as “universal”.

[0093] Examples of universal oligonucleotides comprising such universal sequence elements, probes comprising them and methods for making them are further described in US10781473, incorporated by reference herein.

[0094] The paired universal sequence elements of the conjugation oligonucleotide and the hybridization oligonucleotide of the first and optionally the second partially double-stranded nucleic acid domains may be positioned internally within or at the very ends of the conjugation and the hybridization oligonucleotide. When the paired universal sequence elements are located internally, the hybridization oligonucleotides have both a 3’-overhang and a 5’-overhang. The sequences of the hybridization oligonucleotides will template the sequences of the generated reporter molecules. In such embodiments it is particularly convenient to add sequence elements that are required at the ends of reporter molecules at the 5’-ends of the hybridization oligos, e.g. sequencing adapters such as P5 and P7. The 5’-overhang in this version may also contain an identification sequence, e.g. an analyte or sample identification sequence. The 3’-overhang in this version may also contain an identification sequence, e.g. an analyte or sample identification sequence. In one embodiment, the 5’-overhang contains a sample identification sequence and the 3’-overhang contains an analyte identification sequence. In one embodiment, the 5’-overhang contains an analyte identification sequence and the 3’-overhang contains a sample identification sequence.In one embodiment, the second conjugation oligonucleotide conjugated to the second analyte-binding domain is configured to be displaced from the second hybridization oligonucleotide upon the generation of a reporter nucleic acid molecule.

[0095] In one embodiment, the first and the second hybridization oligonucleotides are configured to be extended using each other as templates, optionally wherein the second hybridization oligonucleotide is blocked in its 3'end to prevent extension of said second hybridization oligonucleotide.

[0096] In one embodiment, the second end of the first hybridization oligonucleotide contains a hybridization region that is matched with a hybridization region on a splint oligonucleotide, which splint oligonucleotide has a further hybridization region capable of hybridizing to a hybridization region in the second proximity probe, so that the first hybridization oligonucleotide can be ligated to the nucleic acid domain of the second proximity probe, or to a strand of the splint oligonucleotide, to generate a nucleic acid reporter molecule.

[0097] In other words, the first hybridization oligonucleotide can be ligated end-to-end with the second hybridization oligonucleotide through the splint oligonucleotide. Alternatively, the nucleic acid domains of the proximity probe pair may hybridise to the splint oligonucleotide such that there is a gap between the 3’ terminus of one nucleic acid domain and the 5’ terminus of the other nucleic acid domain. In this embodiment, the duplex formed between the splint oligonucleotide and two probe nucleic acid domains comprises a length of singlestranded nucleic acid from the splint oligonucleotide, separating the two parts of the duplex. The single-stranded gap may be any number of nucleotides in length. In this embodiment, a gap-filling extension reaction is performed to fill the gap between the ends of the two probe nucleic acid domains (i.e. the probe nucleic acid domain comprising the free 3’ end is extended, in order to fill the gap). Following gap-filling, the nucleic acid domains of the two splint oligonucleotides are ligated to each other using a ligase enzyme. Ligation of nucleic acid domains to each other following gap-filling is referred to herein as “indirect ligation” of the nucleic acid domains to each other. The gap-filling extension reaction is performed using a polymerase enzyme lacking strand displacement activity, such that extension ends when the gap is filled, rather than displacing the hybridised nucleic acid domain downstream of the free 3’ end. Non-displacing polymerases include the T4 DNA polymerase. Other such polymerases are known in the art.

[0098] Alternatively, the ligation may be through a molecular bridge that may be partially doublestranded, said bridge linking the first hybridization oligonucleotide to the second hybridization oligonucleotide to generate a reporter nucleic acid molecule through ligation.An example of such a splint oligonucleotide that may be used in the context of the present invention is described in Hammond et a., 2012, PLoS ONE 7(7): e40405, incorporated by reference herein.

[0099] Hence, in one embodiment, the first and the second hybridization oligonucleotide are configured to hybridize to a splint oligonucleotide, wherein said splint oligonucleotide comprises paired hybridization sites for the first and second hybridization oligonucleotide, optionally wherein the splint oligonucleotide is partially double-stranded.

[0100] The hybridisation between the first and second hybridization oligonucleotide may be described herein as to occur between paired hybridisation sites present in the respective hybridization oligonucleotides, or present in the splint oligonucleotide.

[0101] In one embodiment, the paired hybridization sites of the respective hybridization oligonucleotides of the first and the second partially double-stranded nucleic acid domain are positioned at the 3’ end of each hybridization oligonucleotide.

[0102] In one embodiment, the system further comprises a polymerase (for Proximity Extension) or a ligase (for Proximity Ligation), in other words the system further comprises means for extension or ligation to generate a reporter nucleic acid molecule Polymerases useful for extending the two members of the oligonucleotide pair, and / or the sample index oligonucleotide, include DNA polymerases with and without 3’-exonuclease activity.

[0103] Polymerases without 3’-exonuclease activity include Taq polymerase, Bst polymerase, Tth and various variants of wild-type polymerases that have been engineered to lack 3’-exonuclease activity. It is known in the art of PEA to use DNA polymerases having exonuclease activity, such as T4 and T7 DNA polymerase, Phi29, DNA polymerase I, Pfu DNA polymerase, Pwo DNA polymerase, cf. US9,777,315. The polymerase may have strand displacement activity. Polymerases with strand displacement activity include DNA Polymerase I, Bst (including Bst 2.0 and 3.0), Phi29 DNA polymerase. Ligases useful in the system include T4 DNA ligase, SplintR ligase, Taq DNA ligase, Pfu DNA ligase.

[0104] The system may also comprise a internal control reagent, which is also described as a separate fourth aspect of the present invention), wherein said control reagent comprises an anchor moiety and may thereby be captured on a solid phase in the same manner as the first proximity probe of the present invention. Examples of such control reagents, incorporating an anchor moiety, are shown e.g. in Figure 1 (version 7 and 8).

[0105] As mentioned herein, the anchor moiety is incorporated in the first proximity probe by coupling it to the first hybridization oligonucleotide of the first nucleic acid domain. Examples of suitable anchor and capture moieties include but are not limited to biotin andstreptavidin / avidin / neutravidin (using e.g. Dynabeads from Thermo Fisher Scientific) and complementary nucleic acid molecules, e.g. a poly-A strand as anchor moiety for capture on an oligo-dT bead. Other examples of binding pairs that may make up an anchor-capture pair include, but are not limited to, an antigen and an antibody against the antigen (including its fragments and derivatives), a ligand and its receptor, lectin and carbohydrates (such as fucose and fucose binding peptides as described in US8178319, incorporated herein by reference).

[0106] The first and / or second analyte-binding domain of each proximity probe may be or may comprises a binding moiety 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 the complementary sequence for a target nucleic acid, or combinations thereof. Other examples of analyte-binding domains are mentioned elsewhere herein.

[0107] A system of the first aspect of the invention may particularly be implemented in a method as described herein, but is not limited thereto.

[0108] Hence, in a second aspect, the present invention relates to a method for detecting at least one analyte of interest in a sample, said method comprising the steps of:

[0109] i) Providing a system according to the first aspect,

[0110] ii) Contacting a sample with the system of step i),

[0111] iii) Allowing the system of step ii) to interact with the sample to generate matched pairs of proximity probes bound to their analyte of interest,

[0112] iv) Generating a reporter nucleic acid molecule from interacting first and second nucleic acid domains of the system of step ii) to obtain a reporter nucleic acid molecule comprising the anchor moiety,

[0113] v) Bringing the reporter nucleic acid molecule into contact with a solid phase, wherein said solid phase comprises a capture group configured to capture the anchor moiety present in the reporter nucleic acid molecule,

[0114] vi) Allowing the reporter nucleic acid molecule to be captured onto the solid phase via the anchor moiety,

[0115] vii) Washing the solid phase one or more times,

[0116] viii) Performing an amplification reaction of the reporter nucleic acid molecule captured on the solid phase wherein the amplification reaction results in the production of a reaction mixture comprising amplified reporter nucleic acid molecules free from the solid phase, andix) Detecting the at least one analyte of interest through detection of the amplified reporter nucleic acid molecules.

[0117] In a third aspect, the present invention relates to a method for detecting at least one analyte of interest in a sample, said method comprising the steps of:

[0118] i) Providing a system according to the first aspect,

[0119] ii) Contacting a sample with the system of step i)

[0120] iii) Allowing the system of step ii) to interact with the sample to generate matched pairs of proximity probes bound to their analyte of interest,

[0121] iv) Bringing the matched pairs of proximity probes of step into contact with a solid phase, wherein said solid phase comprises a capture group configured to capture the anchor moiety present in the first nucleic acid domain,

[0122] v) Allowing the matched pairs of proximity probes to be captured onto the solid phase through the anchor moiety present in the first nucleic acid domain,

[0123] vi) Optionally washing the solid phase one or more times,

[0124] vii) Generating a reporter nucleic acid molecule from interacting nucleic acid domains of the system of step iii) to obtain a reporter nucleic acid molecule captured on the solid phase through the anchor moiety,

[0125] viii) Optionally washing the solid phase one or more times,

[0126] ix) Performing an amplification reaction of the reporter nucleic acid molecule captured to the solid phase wherein the amplification reaction results in the production of a reaction mixture comprising amplified reporter nucleic acid molecules free from the solid phase, and x) Detecting the at least one analyte of interest through detection of the amplified reporter nucleic acid molecules.

[0127] In one embodiment, the reporter nucleic acid molecules of step iv) of a method of the second and third aspect, respectively, are generated through an extension reaction of the interacting nucleic acid domains.

[0128] In one embodiment, the reporter nucleic acid molecules of step iv) of the method of the second aspect and / or step vii) of the method of the third aspect, respectively, are generated through a ligation reaction of the interacting nucleic acid domains.

[0129] In one embodiment, a plurality of analytes is detected and the generated reporter molecules obtained comprises analyte identification sequences.

[0130] In one embodiment, a plurality of samples is analysed and the method is adapted to incorporate sample identification sequences into the reporter molecules generated from each respective sample. A sample identification sequence may be present in one or both of the proximity probes already at the start of the method, as described in co-pendingPCT / SE2026 / 010040, or it may be introduced after generation of the reporter nucleic acid molecule, as described in US2023159983A1 and Wik et al. 2021, Mol Cell Proteomics 20, 100168.

[0131] In one embodiment, steps i, ii, and iii of the the method are performed separately for each of a plurality of samples, using proximity probes comprising sample identification sequences. The separate incubation reactions are then pooled after performing step iii) of the method of the first and / or the second aspect, and the pooled samples are subsequently brought into contact with the solid phase in step iv.

[0132] Herein, when sample-probe combinations are envisaged, the sample-probe combinations are “pooled” before reporter nucleic acid molecules are generated from the interacting first and second nucleic acid domains of the proximity probes. This procedure is also referred to herein as “pooling” of the sample probe combinations. This means that different samplespecific probe compositions combined with their respective samples are mixed to allow for a more efficient analysis of the reaction products by enabling single reaction condition for the steps of the method following the pooling.

[0133] In one embodiment, said amplification reaction is performed by Polymerase Chain Reaction (PCR) or by an isothermal amplification reaction, as described elsewhere here.

[0134] As set out herein, the system and method of the present invention may be performed using dual recognition assays (also referred to herein as proximity assays), such as proximity extension assays (PEA) or proximity ligation assays (PLA).

[0135] 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, sometimes two or three) proximity probes, which when brought into proximity by binding to the analyte of interest (hence "proximity probes") allow a signal to be generated.

[0136] Each proximity probe of a proximity probe pair comprises a nucleic acid domain (or moiety) coupled to an analyte binding domain (or moiety) of the probe, and generation of the signal involves an interaction between the nucleic acid domain of the first proximity probe and the nucleic acid domain of the second proximity probe of the proximity probe pair.

[0137] Thus, signal generation in the form of the generation of a reporter nucleic acid molecule is dependent on an interaction between the respective nucleic acid domains of the respective proximity probes of each proximity probe pair, more particularly between the nucleic acid or other functional moieties / domains carried by them. This only occurs when the necessary proximity probes of the same proximity probe pair have bound to the analyte of interest, thereby lending improved specificity to the detection system.In PEA, nucleic acid domains linked to the analyte binding domains of the first and second proximity probe of a proximity probe pair hybridise to one another when the proximity probes are in close proximity (i.e. when bound to a target) and are then extended using a nucleic acid polymerase. The extension product forms a reporter nucleic acid molecule, the detection of which demonstrates the presence of at least one analyte of interest, i.e. wherein the analyte is the analyte bound by each proximity probe of the relevant proximity probe pair. In PLA, nucleic acid domains linked to the analyte binding domains of a proximity probe pair come into proximity when the probes of the proximity probe pair bind their target analyte, and may at that stage 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. Such oligonucleotides are also referred to as splint oligonucleotides. The ligation product is then amplified, acting as a reporter nucleic acid molecule.

[0138] While a number of dual-recognition immunoassays may be used in the present invention, as discussed herein, presently preferred dual-recognition immunoassays are PEA and PLA. PEA and PLA are also described in WO 01 / 61037US 7,306,904 and further information regarding PLA may be found in Lundberg et al., Molecular & Cellular Proteomics 10:10.1074 / mcp.M 110.004978, 1 -10, 2011. PEA is further described in WO 03 / 044231 (US2005003361A1), WO 2004 / 094456 (US2005009050A1), WO 2005 / 123963 (US2009162840A1), WO 2006 / 137932 (US2008131883A1), WO 2013 / 113699 (US2015044674A1), WO 2021 / 191442 (US2023159983A1), WO 2021 / 191448 (US2023159983A1), WO 2021 / 191449 (US2023159983A1), WO2021 / 191450 (US2023107654A1), and WO 2022 / 112300 (US2022162589A1); Lundberg et al. Nucleic Acids Research, 2011, Vol. 39, No. 15 e102; Wik et al., 2021, Mol Cell Proteomics 20, 100168, and Siegbahn A, et al., PLoS One. 2023 Nov 14; 18(11): e0293465, all incorporated herein by reference in their entirety.

[0139] In addition, a methodology to develop and run a panel detecting 94 unique target proteins is provided in Assarsson et al. PLoS One, 2014, 9(4), e95192. Similarly, the development of a multiplexed PLA assay is described in Lundberg et al., Molecular & Cellular Proteomics 10:10.1074 / mcp.M110.004978, 1-10, 2011, all incorporated herein by reference in their entirety.

[0140] One or more analyte(s) of interest is detected by a system of the present invention. An analyte may be any biomolecule or chemical compound, for example a peptide or protein, a nucleic acid molecule, or a small molecule, including organic and inorganic molecules.An analyte can be any substance or entity for which a specific binding partner (analytebinding domain) 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 as disclosed herein). As is well known, proximity probe-based assays have found particular utility in the detection of proteins or polypeptides. Analytes of particular interest thus include proteinaceous molecules such as peptides, polypeptides, proteins or prions or any molecule which includes a protein or polypeptide component, etc., or fragments thereof. In a particularly preferred embodiment of the invention, the analyte is a wholly or partially proteinaceous molecule, most particularly a protein. Thus, preferably, the analyte is or comprises a protein or a polypeptide.

[0141] The analyte may be a single molecule or a complex that contains two or more molecular subunits, which may or may not be covalently bound to one another, and which may be the same or different. Thus, in addition to cells or microorganisms, such a complex analyte may also be a protein complex, or a biomolecular complex comprising a protein and one or more other types of biomolecule. Such a complex may thus be a homo- or hetero-multimer.

[0142] Aggregates of molecules e.g. proteins may also be target analytes, for example aggregates of the same protein or different proteins.

[0143] The analyte may also be a complex between proteins or peptides and nucleic acid molecules such as DNAor RNA. Of particular interest may be the interactions between proteins and nucleic acids, e.g. regulatory factors, such as transcription factors, and DNAor RNA. Thus, the analyte may be a protein-nucleic acid complex (e.g. a protein-DNA complex or a protein-RNA complex).

[0144] The analyte may also be a small molecule or a lipid.

[0145] Detecting an analyte may include any form of measurement of the concentration or abundance of the analyte in the sample. Either the absolute or the relative concentration of a target analyte may be determined. To determine the relative concentration of an analyte, the concentration of the target analyte may be compared to the concentration of one or more other target analyte(s) in the sample or in other samples. A relative concentration of an analyte may also 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). Methods by which quantification can be achieved in the method of the invention are discussed further below.

[0146] The system or method of the present invention may be used for detecting one or more analytes in multiple samples. The analytes may be of the same type (e.g. all the analytes may be proteins, or protein complexes), or of different types (e.g. some analytes may beproteins, others protein complexes, others lipids, others protein-DNAor protein-RNA complexes, etc., or any combination of such types of analytes).

[0147] As noted above, a target analyte may be a single entity, in particular an individual protein. If so, both proximity probes in the proximity probe pair bind the analyte (e.g. protein), but at different epitopes. The epitopes are non-overlapping, so that the binding of one proximity probe in the proximity probe pair to its epitope does not interfere with or block binding of the other proximity probe in the proximity probe pair to its epitope. Alternatively, as noted above the target analyte may be a complex, e.g. a protein complex, in which case one proximity probe in the proximity probe pair binds one member of the complex and the other probe in the pair binds the other member of the complex at sites different to the interaction sites of the different parts of the complex (e.g.. a site different the sites in the respective proteins of a protein complex where the proteins interact with each other to form the complex).

[0148] The first and second proximity probes of a proximity probe pair work together by binding to the same analyte in close proximity to be able to generate a detection signal through interaction between the first and second proximity probe. Generation of a signal from the interacting first and second proximity probe means that a specific analyte of interest has been detected in the sample.

[0149] Any sample of interest may be assayed according to the invention. The sample may contain an analyte(s) of interest or may be suspected to contain an analyte(s) of interest and the method of the present invention enables determining the presence and / or concentration of a specific analyte in a specific sample.

[0150] The sample may for example be a biological or clinical sample, e.g. any cell or tissue sample of or from an organism, or any body fluid or preparation derived therefrom, as well as samples such as cell cultures, cell preparations, cell lysates etc.

[0151] A biological or clinical sample may e.g. contain a viral or cellular material, including prokaryotic or eukaryotic cells, viruses, bacteriophages, mycoplasmas, protoplasts and organelles. Such biological material may thus comprise any type of mammalian and / or nonmammalian animal cell, plant cells, algae including blue-green algae, fungi, bacteria, protozoa etc. Environmental samples, e.g. soil and water samples, or food samples may also be analysed according to the invention. The samples may be freshly prepared or they may be prior-treated in any convenient way e.g. for storage. Further non-limiting examples of samples are food and allied products.

[0152] In particular, the sample may be a clinical sample, for instance whole blood and blood-derived products such as plasma, serum, buffy coat and blood cells, urine, faeces,cerebrospinal fluid or any other body fluid (e.g. respiratory secretions, saliva, milk etc.), tissues and biopsies. It is particularly preferred that the sample is a plasma or serum sample. Thus, the method or products of the invention may be used in the detection of biomarkers, for instance, or to assay a sample for pathogen-derived analytes.

[0153] The sample may in particular be derived from a human, though the method and products of the invention may equally be applied to samples derived from non-human animals (i.e. veterinary samples), plants, fungi, bacteria or cell cultures. The sample may be pre-treated in any convenient or desired way to prepare it for use in the method of the invention, for example by cell lysis or removal, etc.

[0154] By the present method any number of samples may be analysed simultaneously. Typically, from about 2 to about 200 samples may be analysed simultaneously, such as from about 10 to about 200 samples, such as from about 20 to about 150 samples, such as from about 80 to about 120 samples, such as about 90 to about 100 samples.

[0155] When it is intended to detect a plurality of analytes of interest, the first and / or second nucleic acid domain of each proximity probe pair comprise an analyte identification sequence identifying the specific analyte of interest. Sometimes, both of the first and second nucleic acid domains of each proximity probe pair comprise an analyte identification sequence identifying the specific analyte of interest.

[0156] A plurality of analytes may comprise at least 10, 20, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 2000, 3000, 4000, 5000, 5500, 6000, 7000 or more analytes, but is not limited to such a number.

[0157] Following generation of the reporter nucleic acid molecule of step, the reporter nucleic acid molecule is preferably amplified for ease of detection. Amplification of the reporter nucleic acid molecule may be performed by PCR, or by an isothermal amplification reaction, such as loop-mediated isothermal amplification (LAMP) or Recombinase Polymerase Amplification (RPA) but is not limited thereto.

[0158] Detection of the at least one analyte of interest through detection of the generated reporter nucleic acid molecule may be performed in various ways, such as by sequencing. By sequencing all reporter nucleic acid molecules generated, all the different reporter nucleic acid molecules generated may be identified by their analyte and optionally their sample, identification sequences. Nucleic acid sequencing is the preferred method of reporter nucleic acid detection / analysis when sample identification sequences are incorporated. If only two identification sequences are incorporated into a reporter molecule, quantitative PCR (qPCR) may be used for quantification of the reporter molecules, as known in the art.Preferably, a form of high throughput DNA sequencing is used to detect the reporter nucleic acid molecules. Examples of sequencing by synthesis techniques include pyrosequencing, reversible dye terminator sequencing and ion torrent sequencing, any of which may be utilised in the present method.

[0159] Preferably the reporter nucleic acid molecules are sequenced using massively parallel DNA sequencing. Massive parallel sequencing or massively parallel sequencing are any of several high-throughput approaches to DNA sequencing that uses the concept of massively parallel processing. This is also called next-generation sequencing (NGS) or second-generation sequencing. The use of NGS in PEA is further described in Wik et al, 2021.

[0160] Massively parallel DNA sequencing may in particular be applied to sequencing by synthesis (e.g. reversible dye terminator sequencing, pyrosequencing or ion torrent sequencing, as mentioned above). Massively parallel DNA sequencing using the reversible dye terminator method is a preferred sequencing method. Massively parallel DNA sequencing using the reversible dye terminator method may be performed, for instance, using an Illumina® NovaSeq™ system.

[0161] As is known in the art, massively parallel DNA sequencing is a technique in which multiple (e.g. thousands or millions or more) DNA strands are sequenced in parallel, i.e. at the same time. Massively parallel DNA sequencing requires target DNA molecules to be immobilised to a solid surface, e.g. to the surface of a flow cell or to a bead. Each immobilised DNA molecule is then individually sequenced. Generally, massively parallel DNA sequencing employing reversible dye terminator sequencing utilises a flow cell as the immobilisation surface, and massively parallel DNA sequencing employing pyrosequencing or ion torrent sequencing utilises a bead as the immobilisation surface.

[0162] As is known to the skilled person, immobilisation of DNA molecules to a surface in the context of massively parallel sequencing is generally achieved by the attachment of one or more sequencing adapters to the ends of the molecules. The methods herein may thus include the addition of one or more adapters for sequencing (sequencing adapters) to the reporter nucleic acid molecules.

[0163] Herein, the Illumina P5 and P7 sequencing adapters are a preferred pair of sequencing adapters. Sometimes, the P5 sequencing adapter is added to the reporter nucleic acid molecule in a first PCR amplification and the P7 sequencing adapter is added to the reporter nucleic acid molecule in a first or second second PCR amplification. Sometimes, the P7 sequencing adapter is added to the reporter nucleic acid molecule in the first PCR amplification and the P5 sequencing adapter is added to the reporter nucleic acid molecule inthe second PCR amplification. Preferably, the P5 and P7 sequences are present in the nucleic acid domains already from the start of the method.

[0164] Further details of sequencing as a manner of detecting and analysing the generated reporter nucleic acid molecules and as applicable to the present disclosure may be found in US20230159983, incorporated herein by reference.

[0165] Other suitable methods for detecting the reporter nucleic acid molecule include PCR-based methods. For instance, quantitative PCR utilising “TaqMan” probes may be performed. In this instance, the reporter nucleic acid molecules is amplified, and a probe complementary to each sequence is provided, with each different probe being conjugated to a different, distinguishable fluorophore. The presence or absence of each sequence can then be determined based on whether the particular sequence is amplified. However, it is apparent that PCR-based methods such as described above may only be suitable for analysis of relatively small numbers of different sequences at the same time, although combinatorial methods using probes for decoding identifying sequences are known and may be used to extend multiplexing capacity to a degree. Nucleic acid sequencing enables higher levels of multiplex reaction than detection using PCR, hence sequencing is the preferred method for reporter nucleic acid molecule detection.

[0166] Probe configurations

[0167] As described, the present invention builds on the principle of dual-recognition, which includes allowing the analyte-binding domains of each proximity probe of a proximity probe pair to bind simultaneously to an analyte of interest, wherein the analyte-binding domains have nucleic acid domains that interact to form a reporter nucleic acid molecule only when both analyte-binding domains are simultaneously bound to the analyte.

[0168] In general, in a proximity assay, upon binding of a pair of proximity probes to their target analyte the nucleic acid domains of the two probes come into proximity of each other and interact (i.e. directly or indirectly hybridise to one another). The interaction between the two nucleic acid domains yields a nucleic acid duplex comprising at least one free 3’ end (i.e. at least one of the nucleic acid domains within the duplex has a 3’ end which can be extended) in case of a Proximity Extension Assay, or a free 5’ end and a free 3’-end that can be ligated together in case of a Proximity Ligation Assay. Addition or activation of a nucleic acid polymerase or ligase enzyme within the assay mix leads to extension or ligation, as the case may be. The extension / ligation product obtained is a reporter nucleic acid molecule as used herein, comprising a barcode sequence which indicates the presence of the analyte bound by the proximity probe pair from which the extension / ligation product was produced.In a proximity probe pair of the present invention, at least the first proximity probe is partially double-stranded and comprises a first hybridization oligonucleotide and a first conjugation oligonucleotide, wherein the conjugation oligonucleotide is conjugated to the analyte binding domain and the hybridization oligonucleotide is hybridized to the conjugation oligonucleotide as explained elsewhere herein.

[0169] The first hybridization oligonucleotide comprises a first end and a second end, wherein the first end (5’ end or 3’ end) comprises an anchor moiety or group for capture on a solid phase. The second end (5’ end or 3’ end, not being the end with the anchor moiety) is configured to directly or indirectly interact with a single or partly double-stranded second nucleic acid domain of the second proximity probe to generate a reporter nucleic acid molecule. This applies to the probe configurations below and is therefore not repeated for each version. In version 1 of Figure 1 , the second proximity probe is single stranded and comprises a second conjugation oligonucleotide (1104) conjugated to the second analyte binding domain. The first conjugation oligonucleotide of the first proximity probe is conjugated to the analytebinding domain by its 3' end and the second conjugation oligonucleotide of the second proximity probe is conjugated to the analyte-binding domain by its 5' end. The first hybridization oligonucleotide (1102) has a free 3’ end and can be extended using the second conjugation oligonucleotide as a template. Alternatively or additionally, as the second conjugation oligonucleotide also has a free 3' end it can therefore be extended using the first hybridization oligonucleotide as a template. In this version, the anchor moiety is positioned at the 5’ end of the first hybridization oligonucleotide (1102).

[0170] Version 2 of Figure 1 corresponds to version 1 , the difference being that the first conjugation oligonucleotide is conjugated to the first proximity probe by its 5’ end resulting in a different positioning of the first conjugation oligonucleotide and the first hybridization oligonucleotide (1202) within the first proximity probe. In this configuration, the free 3’ end of the first hybridization oligonucleotide (1202) may be extended using the second conjugation oligonucleotide (1204) as a template. Additionally or alternatively, as the conjugation oligonucleotide (1204) of the second proximity probe also has a free 3' end it can therefore be extended using the first hybridization oligonucleotide (1202) of the first proximity probe as a template.

[0171] In version 3 of Figure 1 , the second nucleic acid domain of the second proximity probe is also partially double-stranded comprising a second hybridization oligonucleotide (1304) and a second conjugation oligonucleotide. As in version 1 and 2, the anchor moiety is present in the 5’ end of the first hybridization oligonucleotide (1302). The first hybridization oligonucleotide (1302) may, through its free 3’ end, be extended using the secondhybridization oligonucleotide (1304) as a template. Additionally or alternatively, as the second hybridization oligonucleotide (1304) also has a free 3’ end, this can be extended using the first hybridization oligonucleotide (1302) as a template.

[0172] Version 4 of Figure 1 mirrors version 3 and extension will occur in the same manner as for version 3. In version 4, the first (1402) and second (1404) hybridization oligonucleotides have a region at their respective 5’-ends that may accommodate further functional sequence elements, such as sample specific identification sequences. This may be desired in embodiments where sample specific probe compositions are produced to incorporate sample identification sequence early in the protocol.

[0173] During the extension the conjugation oligonucleotide that is not being extended or used as a template may be displaced, or the generated reporter nucleic acid may be dissociated from the conjugation oligonucleotide, so that extension of the other strand may occur. This may be done e.g. by using a polymerase with strand displacement activity, denaturating conditions, such as increasing the temperature, decreasing the sale concentration etc., but is not limited thereto.

[0174] In versions 1-4 (PEA), the first hybridization oligonucleotide (1102, 1202, 1302, 1402) and the second nucleic acid domain (1104, 1204, 1304, 1404) are directly hybridized through a common 3’ overhang comprising common hybridization sites present in the 3’ ends of each of the nucleic acid domains. In these embodiments, the overhangs of the two nucleic acid domains form a duplex. If the 3’ ends of the two nucleic acid domains hybridise fully to one another, as shown, the duplex comprises two extendable 3’ ends., he 3’ ends may also be designed such that the extreme 3’ end of the nucleic acid domain that does not comprise an anchor moiety is blocked from being extended. This can be achieved by introducing a blocking group at the 3’-end or making the sequence of the 3’-end not complementary to the other, forming a flap (not shown in Fig 1), meaning that the duplex contains only one extendable 3’ end.

[0175] In versions 5 and 6 (PLA) of Figure 1, the hybridization between the first (1502, 1602) and the second (1504, 1604) hybridization oligonucleotide is through a splint oligonucleotide (1506, 1606). This "splint oligonucleotide” bridges the gap between the nucleic acid domains, allowing them to interact with each other without direct hybridization, wherein each nucleic acid domain forms a duplex with the splint oligonucleotide. In this embodiment, the first and the second hybridization oligonucleotide form the duplex with the splint oligonucleotide. As mentioned elsewhere herein, the first and second hybridization oligonucleotide, even if not directly shown in the figures, can be ligated so that the 3’ and 5’ end of the respective hybridization oligonucleotides are positioned end to end alternatively that a gap is presentbetween the hybridization oligonucleotides when the splint oligonucleotide is hybridized to the first and second hybridization oligonucleotide respectively.

[0176] The splint oligonucleotide may be provided as a separate component of the assay. In other words it may be added separately to the reaction mix (i.e. added separately to the proximity probes to the sample containing the analytes). Alternatively, the splint oligonucleotide may be pre-hybridised to one of the nucleic acid domains of the proximity probes, i.e. hybridised prior to contacting the proximity probe with the sample.

[0177] Herein, when it is referred to herein a partially double-stranded nucleic acid domain, this refers to a nucleic acid domain comprising a hybridization oligonucleotide and a conjugation oligonucleotide, wherein the first and optionally the second nucleic acid domain of a proximity probe pair comprises said partially double-stranded nucleic acid domain.

[0178] As explained herein, when ligation is used for generating a reporter nucleic acid molecule a splint oligonucleotide is used together with a partially double-stranded nucleic acid domain. In this embodiment, the splint oligonucleotide can be seen as comprised in said partially double-stranded nucleic acid domain, wherein such an partially double-stranded nucleic acid domain comprises a hybridization oligonucleotide, a conjugation oligonucleotide and a splint oligonucleotide (wherein the splint oligonucleotide may be referred to as forming part of both the first and the second nucleic acid domain as it bridges both domains).

[0179] In version 5 of figure 1, a first and a second partially double-stranded nucleic acid domain is used within the proximity probe pair. A reporter nucleic acid molecule will be generated by ligation of the 5’ and the 3’ end of the first (1502) and the second (1504) hybridization oligonucleotide, respectively, using a splint oligonucleotide (1506) to template the ligation. The placement of the desired sequence elements, e.g. identification sequences and sequencing adapters, may be adjusted accordingly.

[0180] In version 6 of Figure 1 , a first and a second partially double-stranded nucleic acid domain is used within the proximity probe pair. The anchor moiety is positioned in the 3’ end of the first hybridization oligonucleotide (1602). This will not alter ligation which can occur in the same manner as for version 5 of Figure 5, i.e. the 3’-end of the second hybridization oligonucleotide (1604) is ligated to the 5’-end of the first hybridization oligonucleotide (1602), templated by the splint oligonucleotide (1606).

[0181] Probe designs 7 and 8 illustrate internal control reagents useful in the context of the present invention. Internal control reagents in the form of extension control reagents for use with prior art methods are described in US2023159983A1 and use a single binding moiety, in Figure 1 indicated as a coupling moiety. The control reagent is used to determine if the interactionbetween the nucleic acid domains of the proximity probe is functional, independently of the presence of an analyte of interest in the sample. Version 7 illustrates an extension control reagent useful to assess whether the extension step of a proximity extension protocol works as intended, It comprises a first nucleic acid domain comprising a first conjugation oligonucleotide (1701), a first hybridization oligonucleotide (1702) comprising an anchor moiety at the 5’-end, a second conjugation oligonucleotide (1703) and a second hybridization oligonucleotide (1704). Version 8 illustrates a ligation control reagent useful to assess whether the ligation step of a proximity ligation protocol works as intended, It comprises a first nucleic acid domain comprising a first conjugation oligonucleotide (1801), a first hybridization oligonucleotide (1802) comprising an anchor moiety at the 5’-end, a second conjugation oligonucleotide (1803), a second hybridization oligonucleotide (1804). When used in a proximity ligation protocol, a splint oligonucleotide (1806) will be present to template ligation.

[0182] With regards to the probe configurations discussed herein, and as otherwise suitable in the context of the present invention, reference is also made to co-pending application PCT / SE2026 / 010040 with the same applicant and claiming priority from European patent application 25155799.7.

[0183] Figure 2 discloses some of the probe configurations shown in Figure 1 in greater detail. Figure 2A is a probe design of the prior art, without an anchor moiety, and is also shown in Wiketal., 2021, Mol Cell Proteomics 20, 100168. Following the nomenclature adopted in Wik et al., the left hand probe is termed the “forward probe” and the right hand probe is termed the “reverse probe”. The nucleic acid moiety of the forward probe comprises the following sequence elements in 5’-3’ direction: a=lllumina P5 sequencing adapter; b=lllumina read 1 sequencing primer site (Rd1SP); fa=forward analyte identification sequence (termed “forward barcode” in Wik et al.); and h=hybridization sequence. The nucleic acid moiety of the reverse probe comprises the following sequence elements in 3’-5’ direction: h’=hybridization site able to hybridize to sequence element h of the forward probe; c= spacer; ra=reverse analyte identification sequence (termed “reverse barcode” in Wik et al.); and d=primer binding site for incorporating a sample index sequence and the Illumina P7 sequencing adapter in a separate amplification reaction.

[0184] The configuration in Figure 2B corresponds to version 1 in Figure 1 , wherein the first proximity probe (forward probe) is partially double-stranded. The sequence element references in Fig 2B refer to equivalent sequence elements as in Fig 2A. in this configuration, the universal sequence element is represented by b and b’. The forward probe (first proximity probe) has a first conjugation oligonucleotide comprising the sequence b’ coupled (covalentlyor non-covalently) to it at its 5’-end. b' is the reverse complement of universal sequence element b, so that b and b’ can hybridize to form a duplex. The first hybridization oligonucleotide of the forward probe comprises the same sequence elements b, fa, and h as the nucleic acid moiety of the forward probe in Fig 2A. The conjugation oligonucleotide of the reverse probe (second proximity probe) comprises the same sequence elements d, ra, c and h’ as the nucleic acid moiety of the reverse probe in Fig 2A.

[0185] The configuration in Figure 2C corresponds to version 3 in Figure 1 , wherein also the second proximity probe (reverse probe) is partially double-stranded. The sequence element references in Fig 2C refer to equivalent sequence elements as in Fig 2Aand 2B. In this configuration, the second conjugation oligonucleotide also comprises a universal sequence element, d’, which is the reverse complement to d in the second hybridization oligonucleotide in the in the same manner as for the first conjugation oligonucleotide.

[0186] The configuration in Figure 2D corresponds to version 4 in Figure 1, wherein the hybridization oligonucleotides have a 5’-overhang to accommodate further sequence elements. The sequence element references in Fig 2D refer to equivalent sequence elements as in Fig 2A-2C. In this configuration, the first hybridization oligonucleotide further comprises a first (or forward) sample specific identification sequence, fs, and the second hybridization oligonucleotide comprises a second (or reverse) sample identification sequence, rs. The second hybridization oligonucleotide further comprises the P7 sequencing adapter, e, at the 5’-end.

[0187] The configuration in Figure 2E corresponds to version 5 of Figure 1 , adapted for ligation and wherein also the second proximity probe is partially double-stranded. The sequence element references in Fig 2E refer to equivalent sequence elements as in Fig 2A-2C. In addition, a splint oligonucleotide is illustrated comprising sequence elements hf’ and hr’ which are hybridization sequences that are the reverse complement of hybridization sequences hf and hf present in the forward and reverse probe (first and second proximity probe), respectively, are included. As seen in Figure 2E, a gap may be present between the first and the second hybridization oligonucleotide as the reverse complements hf’ and hr’ are not presented side by side in the splint oligonucleotide. The splint oligonucleotide can be double-stranded in this part, shown in Fig 2E as the short strand s’. The sequence portion s in the splint oligonucleotide may comprise a sample identification sequence, as described in Hammond et al. 2012, PLoS ONE 7(7): e40405, or it may be a single stranded stretch (i.e. s’ is not present), or it may solely be a phosphodiester bond, bringing the 3’ end of the first hybridization oligonucleotides and the 5’-end of the second hybridization oligonucleotide into a position for direct ligation. While not shown in Fig 2E, the first hybridization oligonucleotidemay be designed with a larger 5’ overhang to accommodate a forward sample identification sequence, fs, and the second hybridization oligonucleotide may be designed with a larger 5’ overhang to accommodate a reverse sample identification sequence, rs, analogous to the design shown in Fig 2D.

[0188] Figure 3 illustrates some steps of a method according to the second aspect of the present invention. The figure illustrates reporter molecule generation through extension, but the skilled person will understand that the same general workflow may be used with ligation for reporter molecule generation, as explained elsewhere herein. Initially, in step i) a probeanalyte complex is allowed to be formed comprising a matched first and a second proximity probe of a proximity probe pair bound to their analyte (302) of interest present in a sample. In this illustration, both the first and the second proximity probe is partially double-stranded comprising a first and a second hybridization oligonucleotide and a first and a second conjugation oligonucleotide, respectively. An anchor moiety, for capture on a solid phase, is comprised in one end of the first hybridization oligonucleotide. Once the first and the second proximity probe have bound to their analyte, a ligation or an extension reaction is performed generating a reporter nucleic acid molecule (ii) and displacing the immune complexes comprising analyte and analyte-binding domains with conjugation oligonucleotides from the reporter nucleic acid molecule generated by extension or ligation.

[0189] Subsequently, in step iii) the reporter nucleic acid molecule, comprising the anchor moiety, is captured on the solid phase. This is followed by one or more washing steps (step iv) of the solid phase to remove impurities such as any unbound probes, immune complexes and free analytes from the reaction. During this step, the reporter nucleic acid molecule remains bound to the solid phase. In step (v), an amplification of the reporter nucleic acid molecules is performed, using the reporter nucleic acid molecules captured on the solid phase as templates and generating a mixture comprising amplified reporter nucleic acid molecules. As shown in step v), the amplified reporter nucleic acid molecules remain in solution while the amplification occurs, and the solid phase may subsequently be removed. The amplified reporter nucleic acid molecules may thereafter form the basis for further handling and analysis as described elsewhere herein.

[0190] Figure 4 illustrates some steps of one embodiment of the method according to the third aspect as disclosed herein, wherein the anchor moiety on the first hybridization oligonucleotide is used to capture the probe analyte complex on a solid phase. Figure 4 illustrates the method using probe pairs without sample identification sequences. It may however be adapted so that one or both of the proximity probes of a proximity probe paircomprise a sample identification sequence and wherein multiple samples are pooled before detection and analysis, as will be described below.

[0191] In step I, a proximity probe pairs specific to the analytes of interest (3102, 3104) are incubated with the sample. The samples contain analytes (3102, 3104, 3202) and matched pairs of forward (306) and reverse (308) probes, wherein both probes in a matched pair bind the same analyte. The forward probe (first proximity probe, 306) comprises a partially doublestranded nucleic acid domain comprising a first hybridization oligonucleotide and a first conjugation oligonucleotide. The reverse probe (second proximity probe) may also, but need not be, partially double-stranded, in that situation comprising a second hybridization oligonucleotide and a second conjugation oligonucleotide. In this embodiment, it is illustrated a second proximity probe comprising a second conjugation oligonucleotide (not doublestranded).

[0192] In step II, the probes are allowed to bind to their respective analyte molecules.

[0193] Steps I and II can be performed separately on separate samples using sample-specific probe compositions. In this embodiment, at least one of the forward and reverse probes, preferably both carry a sample identification sequence identifying the sample-specific probe composition. If the method is intended to measure only one analyte), then no analyte identification sequences are necessary as the detected reporter molecules comprising the sample identification sequence(s) to 100% originate from detection of that same analyte. If the method is intended to measure more than one analyte, at least one of the forward and reverse probes, preferably both, comprise analyte identification sequences as well. The forward probes (306, first proximity probes) have an anchor moiety present in one end of the first hybridization oligonucleotide. The anchor moiety is attached already at the start of the protocol, or even at manufacture.

[0194] When the probe-analyte complexes have formed in the separate samples, all samples are pooled prior to step III.

[0195] In step III, the (optionally pooled) sample-probe combinations are brought into contact with a solid phase (312) comprising a capture group (314) to which the anchor moiety of the first proximity probe can bind. Unbound probes and other reagents are washed away in step IV. In step IV, the matched proximity probe pairs and the immune complexes (probe-analyte complexes) are still captured on the solid phase via the first proximity probe In step V, a reporter nucleic acid molecule is generated through ligation or extension while the probeanalyte complex is still bound to the solid phase. During extension or ligation, the conjugation oligonucleotides bound to the analytes will be displaced from, or allowed to dissociate from, the generated reporter nucleic acid molecules. Optionally one or more washing steps (stepVI) are then performed to remove impurities in the form of unbound analyte, immune complexes bound to the conjugation oligonucleotides, etc. This is subsequently followed by amplification (step VII) of the reporter nucleic acid molecules, in the same way as the method according to the second aspect of the invention. It is also conceivable that the matched proximity probe pairs and the immune complexes (probe-analyte complexes) of step IV are released from the solid phase and eluted from the reaction. Extension or ligation to produce a reporter nucleic acid molecule is performed away from the solid phase (analogous to step V,). This is subsequently followed by amplification of the reporter nucleic acid molecules (analogous to step VII) and further handling and analysis as discussed elsewhere herein. Furthermore, as discussed elsewhere herein, it is known in the art of proximity extension assays to include a control reagent known as “extension control” for quality control of the extension step of the proximity extension reaction, and also for normalization of expression levels of the analytes in the sample, cf. Assarsson et al. 2014 and Wik et al. 2021. The extension control is composed of an antibody coupled to a pair of oligonucleotides comprising unique identification sequences. These oligonucleotides are always in proximity, so that this control is expected to give a constant signal independently of the immunoreaction. This control monitors variation in the extension and amplification / detection step and is used to adjust the signal from each sample with respect to extension and amplification. Analogous ligation control reagents may be used in proximity assays based on ligation, rather than extension, for generation of reporter molecules.

[0196] Thus, in a fourth aspect, the present invention relates to a control reagent that is adapted for use as an extension control or ligation in a method according to the present invention. In this aspect, the invention relates to a control reagent comprising a first nucleic acid domain and a second nucleic acid domain coupled together to be in constant proximity, the first nucleic acid domain is partially double-stranded, comprising:

[0197] i) a first conjugation oligonucleotide and,

[0198] ii) a first hybridization oligonucleotide

[0199] together providing a double-stranded part of the first nucleic acid domain,

[0200] wherein the first conjugation oligonucleotide is coupled to the second nucleic acid domain to keep the first and second nucleic acid domains in constant proximity, wherein the first conjugation oligonucleotide and the first hybridization oligonucleotide are configured to hybridize to each other through paired universal sequence elements present in the conjugation oligonucleotide and the hybridization oligonucleotide, wherein the first and / or second nucleic acid domain of the control reagent comprise a control reagent identification sequence identifying the control reagent,wherein the first hybridization oligonucleotide and the second nucleic acid domain together are capable of directly or indirectly interacting to generate a reporter nucleic acid molecule, and

[0201] wherein the first hybridization oligonucleotide comprises, at its 5’ or 3’ end, an anchor moiety for capture onto a solid phase.

[0202] In an embodiment, the first conjugation oligonucleotide is configured to be displaced from the first hybridization oligonucleotide upon the generation of a reporter nucleic acid molecule. In an embodiment, the first hybridization oligonucleotide of the first nucleic acid domain is configured to be extended using the second nucleic acid domain as a template and optionally the second nucleic acid domain is configured to be extended using the first hybridization oligonucleotide of the first nucleic acid domain as a template.

[0203] In an embodiment, the second nucleic acid domain of the control reagent is partially doublestranded, comprising: i) a second conjugation oligonucleotide and, ii) a second hybridization oligonucleotide, together providing a double-stranded part of the second nucleic acid domain, wherein the second conjugation oligonucleotide and the second hybridization oligonucleotide are configured to hybridise to each other through paired universal sequence elements present in the conjugation oligonucleotide and in the hybridization oligonucleotide.

[0204] In an embodiment, the second conjugation oligonucleotide is coupled to the first nucleic acid domain to keep the first and second nucleic acid domains in constant proximity, and configured to be displaced from the second hybridization oligonucleotide upon the generation of a reporter nucleic acid molecule.

[0205] In an embodiment, the first and the second hybridization oligonucleotides are configured to be extended using each other as templates.

[0206] In an embodiment, the second hybridization oligonucleotide is blocked in its 3'end to prevent extension of said second hybridization oligonucleotide.

[0207] In an embodiment, the first and the second hybridization oligonucleotide are configured to hybridize to a splint oligonucleotide, wherein said splint oligonucleotide comprises paired hybridization sites for the first and second hybridization oligonucleotide, optionally wherein the splint oligonucleotide is partially double-stranded.

[0208] The first and second nucleic acid domain may be coupled together in any manner that keeps the domains in constant proximity under normal storage and reaction conditions. In an embodiment, the first and second nucleic acid domains are coupled together through a phosphodiester bond (i.e., the first and second nucleic acid domain are two parts of the samenucleic acid sequence). In a further embodiment, the first and second nucleic acid domains are coupled together through mutual coupling to a protein. Preferably, any protein comprised in the control reagent does not significantly interfere in the detection methods according to the present invention. Preferred proteins include antibodies that are not specific to any analyte, or external control, expected to be present in a sample to be analyzed. Polyclonal antibodies from goat or rabbit may be used. The first and second nucleic acid domain of the control reagent may also be coupled together through a polymer linker or spacer, such as a flexible carbon chain (e.g. C3, C6, or C12 spacer), polyethylene glycol (PEG) spacers, biotinstreptavidin linker, etc.

[0209] Production of proximity probes

[0210] Proximity probes, as used in the present invention are generally composed of an analytebinding domain and a nucleic acid domain.

[0211] 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 has been established by an International Working Group for Antibody Validation (Uhlen et al., Nat Methods, 2016 Oct; 13(10), 823-827, incorporated herein by reference).

[0212] Typically, the analyte-specific binding domain may 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.

[0213] 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, UnitedKingdom). 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.

[0214] 22: 238-244), and Dual Affinity Retargeting molecules (DARTs, diabodies additionally stabilized through a C-terminal disulfide bridge). The specificity of analyte-specific 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.

[0215] 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.

[0216] As described further above, in addition to the analyte-specific binding domain, a detection probe (proximity probe) as used in the present invention also comprises a nucleic acid domain. The nucleic acid domain must be long enough to comprise the necessary functional elements used in the detection assay for which the detection probe (proximity 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 nucleic acid domain may also contain sequences related to primer sites for amplification and / or sequencing adaptors for readout, as known in the art. Generally, the nucleic acid domain 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 (proximity) probe is intended to be used. Herein, at least one of the nucleic acid domains is partially double-stranded, as explained elsewhere herein.

[0217] Conjugation of a nucleic acid domain to an antibody can be performed in several ways known to the skilled person, e.g. as reviewed by Dugal-Tessier et al.

[0218] (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 orconvenient 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).

[0219] The nucleic acid domain 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 domain and analyte binding domain through the linking group. The linking group, when present, is in many embodiments biologically inert.

[0220] 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.

[0221] 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.

[0222] 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.

[0223] The nucleic acid domain of the detection (proximity) 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. PNAor other derivatives containing non-nucleotide backbones.

[0224] All embodiments described herein are applicable in all aspects of the invention as well as in any combination. All prior publications cited in the present specification are incorporated by reference in their entirety.

[0225] The figures of the present disclosure are not to scale and provided for illustrative purposes, and shall not be considered as limiting the invention.

[0226] 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.

[0227] EXPERIMENTAL SECTION

[0228] Example 1

[0229] This example relates to an implementation of the invention describing the advantages of proximity probes wherein one hybridization oligonucleotide comprises an anchor group at the 5’-end and wherein this is used to capture the reporter nucleic acid molecules onto a solidphase. The examples show that the invention allows for larger sample volumes as compared to homogenous proximity assays.

[0230] Material and methods

[0231] A general reference is made to Assarsson et al., 2014, for details regarding common reagents and process steps referred to in the below.

[0232] Proximity probe pairs were prepared for three analytes, AGRP, IL-6 and aB42, essentially as described in US10781473, except that the hybridization oligonucleotide on one of the probes in each pair was biotinylated in the 5’-end, corresponding to the design in Figure 1, version 3 of the present specification. An extension control comprising a goat IgG antibody not specificto any of the assessed analytes and with conjugation and hybridization oligonucleotides as shown in Figure 1, version 7 was also prepared, to provide immediate proximity independent of antigen binding, as described in Assarsson et al. The hybridization oligonucleotides were adapted for qPCR readout.

[0233] A 96 well incubation plate with dried probes and extension control was prepared as described in WO2025012398. Briefly, forward and reverse probes for the three analytes, extension control, Olink Target 48 incubation solution (Olink Proteomics AB, Uppsala, Sweden), trehalose as a stabilizer and MilliQ water were mixed and dispensed into a standard 96-well microtiter plate and vacuum dried (0 mBar, 35 °C for 2h). The wells were covered by plastic film and stored at 4 °C until use.

[0234] Standard plasma samples from healthy controls were used and spiked with low levels of aB42 to increase detectability, as normal levels of aB42 are low in healthy individuals.

[0235] Different sample volumes were tested and varied within fixed-volume immunoreactions to remove confounders from reaction volumes increasing, and probe concentrations decreasing, when more sample is added. Three fixed volume immunoreactions of 4, 8, and 16 pL were used wherein each immunoreaction volume was made up of plasma sample and Phosphate Buffered Saline (PBS) with 0.1 % BSAto make up the volume difference. Dilution series shown in tables below.

[0236]

[0237] Table 1 : 4 pL volume

[0238]

[0239] Table 2: 8 pL volume

[0240]

[0241] Table 3: 16 pL volume

[0242] Samples were added to the incubation plate as described above in duplicates, and in quadruplicates for negative control, “1 in 4”, “1 in 8” and “1 in 16” and incubated overnight at room temperature to allow for proximity probes to bind to their respective analytes.

[0243] An extension mix comprising MilliQ water, PEA Enhancer, PEA Solution and PEA Enzyme (all Olink Proteomics AB, Uppsala Sweden) was prepared and 96 pL of the stock solution was added to each well of the incubation plate. The extension reaction was done at 50 °C for 20 minutes and then cooled to 10 °C. Extension was stopped by addition of 10 pL 100 mM EDTAto each reaction.

[0244] 4 pL of washed streptavidin-coated magnetic beads were added to each well and incubated for 45 minutes to capture extension products. The incubation reaction was gently vortexed every 15 minutes.

[0245] The incubation plate was briefly spun at 1 ,000 rpm and then placed on a magnetic plate to immobilize magnetic beads. All liquid was removed, while ensuring that beads did not dry out, and beads were washed with 150 pL wash buffer. Wash buffer was removed, 96 pL PCR mix was added and plate vortexed to mix all beads. Extension products were amplified (PCR protocol: (95 °C 30 s, 54 °C 60 s, 60 °C 60 s)*17 cycles, 10 °C hold). Plate was spun at 4,000 rpm for 5 minutes and placed on a magnetic plate for 2 minutes. 50 pL supernatant comprising amplified reporter molecules (i.e. amplified extension products) was transferred to new plate to remove magnetic beads. Extension products were quantified using qPCR. Results

[0246] Results are shown in Figure 5. In panel A, the Ct values for the extension control and the respective analytes are shown. Panel B shows the Ct signal / noise (Ct values for the negative control).

[0247] Discussion

[0248] The amount of extension control is the same in all reactions, meaning that the signal for extension control ideally should be stable over all incubation volumes. It is noted that signal appears to drop for sample volumes of 12 pL and 16 pL. This indicates that the extension reaction is slightly less efficient at these sample volumes. Analyte signal values may be adjusted or normalized taking this drop in extension efficacy into account, although this has not been done in the current preliminary assessment of the utility of the invention.It is clear from the results as shown in Fig 5A that the signal for all analytes increase with increased sample volume. Also the signal to noise increases with increased sample volume (Fig 5B).

[0249] The analyte aB42 is present in plasma in very low abundance. Even though the samples were spiked with a low amount of aB42 protein to increase the concentration, the amount of aB42 in 1 , 2 or 4 pL sample appears to be below apparent limit of detection (LOD). However, at sample volumes of 6 pL or more, the signal to noise does increase as expected.

[0250] The results thus show that capture of the reporter molecules on a solid phase prior to amplification is a way to increase sensitivity for proximity assays by significant increase in sample volumes.

Claims

CLAIMS1. A system for detection of at least one analyte of interest in a sample, said system comprising at least one proximity probe pair, each proximity probe pair comprising:a. a first proximity probe comprising a first analyte binding domain coupled to a first nucleic acid domain, and;b. a second proximity probe comprising a second analyte binding domain coupled to a second nucleic acid domain,wherein the first and second analyte binding domains of said proximity probe pair are capable of simultaneously and specifically binding to an analyte of interest present, or suspected of being present, in the sample,in each proximity probe pair, the first nucleic acid domain is partially doublestranded comprising:i) a first conjugation oligonucleotide and,ii) a first hybridization oligonucleotidetogether providing a double-stranded part of the first nucleic acid domain, wherein the first conjugation oligonucleotide is conjugated to the analyte binding domain of the first proximity probe via its 5’ end or 3’ end,wherein the first conjugation oligonucleotide and the first hybridization oligonucleotide are configured to hybridise to each other through paired universal sequence elements present in the conjugation oligonucleotide and the hybridization oligonucleotide,wherein the first hybridization oligonucleotide and the second nucleic acid domain together are capable of directly or indirectly interacting to generate a reporter nucleic acid molecule when said first and second proximity probes are both bound to the same analyte, andwherein the first hybridization oligonucleotide comprises, at its 5’ or 3’ end, an anchor moiety for capture onto a solid phase.

2. The system of claim 1 , wherein the first conjugation oligonucleotide conjugated to the analyte-binding domain is configured to be displaced from the first hybridization oligonucleotide upon the generation of a reporter nucleic acid molecule.

3. The system of claim 1 or 2, wherein the first hybridization oligonucleotide of the first nucleic acid domain is configured to be extended using the second nucleic acid domain as a template and optionally wherein second nucleic acid domain is configured to be extended using the first hybridization oligonucleotide of the first nucleic acid domain as a template.

4. The system of any one of claims 1 to 3, wherein the second nucleic acid domain of the second proximity probe of the proximity probe pair is partially double-stranded comprising: i) a second conjugation oligonucleotide and, ii) a second hybridization oligonucleotide, together providing a double-stranded part of the second nucleic acid domain, wherein the second conjugation oligonucleotide and the second hybridization oligonucleotide are configured to hybridise to each other through paired universal sequence elements present in the conjugation oligonucleotide and in the hybridization oligonucleotide.

5. The system of claim 4, wherein the second conjugation oligonucleotide conjugated to the second analyte-binding domain is configured to be displaced from the second hybridization oligonucleotide upon the generation of a reporter nucleic acid molecule.

6. The system of claim 4 or 5, wherein the first and the second hybridization oligonucleotides are configured to be extended using each other as templates.

7. The system of claim 6, wherein the second hybridization oligonucleotide is blocked in its 3'end to prevent extension of said second hybridization oligonucleotide.

8. The system of claim 4 or 5, wherein the first and the second hybridization oligonucleotide are configured to hybridize to a splint oligonucleotide, wherein said splint oligonucleotide comprises paired hybridization sites for the first and second hybridization oligonucleotide.

9. The system of any one of the preceding claims, further comprising a polymerase or a ligase.

10. The system of any one of the preceding claims, wherein the first and / or second analyte-binding domain of each proximity probe is or comprises a binding moiety 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 the complementary sequence for a target nucleic acid, or combinations thereof.

11. A method for detecting at least one analyte of interest in a sample, said method comprising the steps of:i) Providing a system of any one of claims 1 to 10,ii) Contacting a sample with the system of step i),Hi) Allowing the system of step ii) to interact with the sample to generate matched pairs of proximity probes bound to their analyte of interest,iv) Generating a reporter nucleic acid molecule from interacting first and second nucleic acid domains of the system of step iii) to obtain a reporter nucleic acid molecule comprising the anchor moiety,v) Bringing the reporter nucleic acid molecule of step iv) into contact with a solid phase, wherein said solid phase comprises a capture group configured to capture the anchor moiety present in the reporter nucleic acid molecule, vi) Allowing the reporter nucleic acid molecule to be captured onto the solid phase via the anchor moiety,vii) Washing the solid phase one or more times,viii) Performing an amplification reaction of the reporter nucleic acid molecule captured on the solid phase wherein the amplification reaction results in the production of a reaction mixture comprising amplified reporter nucleic acid molecules free from the solid phase,ix) Detecting the at least one analyte of interest through detection of the amplified reporter nucleic acid molecules.

12. A method for detecting at least one analyte of interest in a sample, said method comprising the steps of:i) Providing a system of any one of claims 1 to 10,ii) Contacting a sample with the system of step i),iii) Allowing the system of step ii) to interact with the sample to generate matched pairs of proximity probes bound to their analyte of interest,iv) Bringing the matched pairs of proximity probes of step iii) into contact with a solid phase, wherein said solid phase comprises a capture group configured to capture the anchor moiety present in the first nucleic acid domain, v) Allowing the matched pairs of proximity probes to be captured onto the solid phase through the anchor moiety present in the first nucleic acid domain, vi) Optionally washing the solid phase one or more times,vii) Generating a reporter nucleic acid molecule from interacting nucleic acid domains of the system of step iii) to obtain a reporter nucleic acid molecule captured on the solid phase through the anchor moiety, orreleasing the matched pairs of proximity probes from the solid phase followed by generation of a reporter nucleic acid molecule,viii) Optionally washing the solid phase one or more times,ix) Performing an amplification reaction of the reporter nucleic acid molecule released from or captured on the solid phase wherein the amplification reaction results in the production of a reaction mixture comprising amplified reporter nucleic acid molecules free from the solid phase,x) Detecting the at least one analyte of interest through detection of the amplified reporter nucleic acid molecules.

13. The method of claim 11 or 12, wherein the reporter nucleic acid molecules of step iv) of claim 11 and step vii) of claim 12, respectively, are generated through an extension reaction of the interacting nucleic acid domains.

14. The method of claim 11 or 12, wherein the reporter nucleic acid molecules of step iv) of claim 11 and step vii) of claim 12, respectively, are generated through a ligation reaction of the interacting nucleic acid domains.

15. The method of any one of claims 11 to 14, wherein a plurality of analytes is detected and wherein the reporter molecules obtained comprises analyte identification sequences.

16. The method of any one of claims 11 to 15, wherein a plurality of samples is analysed and wherein the reporter molecules comprise sample identification sequences.

17. The method of claim 16, wherein the plurality of samples are pooled after performing step iii) of the method of claim 11 or 12, and wherein the pooled samples are brought into contact with a solid phase.

18. The method of any one of claims 12 to 17, wherein said amplification reaction is performed by Polymerase Chain Reaction (PCR) or by an isothermal amplification reaction.

19. A control reagent for a proximity assay, said reagent comprising a first nucleic acid domain and a second nucleic acid domain coupled together to be in constant proximity, the first nucleic acid domain is partially double-stranded, comprising: i) a first conjugation oligonucleotide and,ii) a first hybridization oligonucleotidetogether providing a double-stranded part of the first nucleic acid domain, wherein the first conjugation oligonucleotide is coupled to the second nucleic acid domain to keep the first and second nucleic acid domains in constant proximity, wherein the first conjugation oligonucleotide and the first hybridization oligonucleotide are configured to hybridize to each other through paired universal sequence elements present in the conjugation oligonucleotide and the hybridization oligonucleotide, wherein the first and / or second nucleic acid domain of the control reagent comprise a control reagent identification sequence identifying the control reagent,wherein the first hybridization oligonucleotide and the second nucleic acid domain together are capable of directly or indirectly interacting to generate a reporter nucleic acid molecule, andwherein the first hybridization oligonucleotide comprises, at its 5’ or 3’ end, an anchor moiety for capture onto a solid phase.

20. The control reagent for a proximity assay according to claim 19, wherein the first conjugation oligonucleotide is configured to be displaced from the first hybridization oligonucleotide upon the generation of a reporter nucleic acid molecule.

21. The control reagent for a proximity assay according to claim 19 or 20, wherein the first hybridization oligonucleotide of the first nucleic acid domain is configured to be extended using the second nucleic acid domain as a template and optionally the second nucleic acid domain is configured to be extended using the first hybridization oligonucleotide of the first nucleic acid domain as a template.

22. The control reagent for a proximity assay according to any one of claims 19 to 21 wherein the second nucleic acid domain of the control reagent is partially doublestranded, comprising: i) a second conjugation oligonucleotide and, ii) a second hybridization oligonucleotide, together providing a double-stranded part of the second nucleic acid domain, wherein the second conjugation oligonucleotide and the second hybridization oligonucleotide are configured to hybridise to each other through paired universal sequence elements present in the conjugation oligonucleotide and in the hybridization oligonucleotide.

23. The control reagent for a proximity assay according to any one of claims 19 to 22 wherein the second conjugation oligonucleotide is coupled to the first nucleic acid domain to keep the first and second nucleic acid domains in constant proximity, and configured to be displaced from the second hybridization oligonucleotide upon the generation of a reporter nucleic acid molecule.

24. The control reagent for a proximity assay according to any one of claims 19 to 23 wherein the first and the second hybridization oligonucleotides are configured to be extended using each other as templates.

25. The control reagent for a proximity assay according to any one of claims 19 to 24 wherein the second hybridization oligonucleotide is blocked in its 3 'end to prevent extension of said second hybridization oligonucleotide.

26. The control reagent for a proximity assay according to any one of claims 19 to 23 wherein the first and the second hybridization oligonucleotide are configured to hybridize to a splint oligonucleotide, wherein said splint oligonucleotide comprises paired hybridization sites for the first and second hybridization oligonucleotide.

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