Proximity probe library and usage

Abstract

The present invention relates to a library comprising a plurality of detection probes suitable for detecting a set of analytes of interest, each detection probe comprising an analyte-specific binding domain and a nucleic acid portion, the nucleic acid portion of the detection probe capable of generating a reporter nucleic acid molecule comprising a discrimination sequence that identifies each analyte of interest, the library comprising, for one or more analytes of interest, two or more sets of detection probes, each detection probe in a set capable of generating a reporter nucleic acid molecule comprising a discrimination sequence that differs from the discrimination sequences generated by other members of the same set. The present invention further relates to a method of constructing a panel using the library, as well as a computer program for implementing the method.

Description

[Technical Field]

[0001] The present invention relates to tools for biotechnology research, and in particular to products and methods for the rapid and efficient development of custom multiplexed panels of assays for analytes of interest. The present invention finds particular application in the development of flexible panels for use in the field of proteomics research. [Background technology]

[0002] Modern proteomics methods require the ability to detect a large number of different proteins (or protein complexes) in a small sample volume. To achieve this, multiplexed analyses must be performed. Common methods that can achieve multiplexed detection of proteins in a sample include proximity extension assay (PEA) and proximity ligation assay (PLA). PEA and PLA are described in WO 01 / 61037. For PEA, see WO 03 / 044231, WO 2004 / 094456, WO 2005 / 123963, WO 2006 / 137932, WO 2013 / 113699, WO 2021 / 191442, WO 2021 / 191448, WO 2021 / 191449, WO 2022 / 191450, and WO 2022 / 112300; Assarsson et al., PLoS 1, 2014, 9, 4, e95192; Lundberg et al., Molecular & Cellular Proteomics 10:10.1074 / mcp.M110.004978, 1-10, 2011; and Wik et al. al., 2021, Mol Cell Proteomics 20, 100168, all of which are incorporated herein by reference in their entireties.

[0003] If the protein of interest is present over a wide range of concentrations, as is common, this presents a challenge as the signal from the higher-concentration protein may overwhelm the signal from the lower-concentration protein, resulting in an inability to detect the protein present at low concentrations.

[0004] PEA and PLA are proximity assays that rely on the principle of "proximity probing." In these methods, an analyte is detected by binding multiple (i.e., two or more, generally two or three) probes that bind to the analyte and thereby generate a signal when they come into proximity (hence, "proximity probes"). Typically, at least one of the proximity probes comprises a nucleic acid domain (or moiety) linked to the analyte-binding domain (or moiety) of the probe, and signal generation involves interactions between the nucleic acid moiety and / or additional functional moieties carried by the other probe(s). Thus, signal generation depends on interactions between the probes (more specifically, between their nucleic acid or other functional moieties / domains) and therefore occurs only when the required probe binds to the analyte, thereby providing improved specificity to the detection system.

[0005] In PEA, nucleic acid moieties linked to the analyte-binding domains of a probe pair hybridize to each other when the probes are in close proximity (i.e., when they are bound to the same target molecule, or when they are bound to target molecules that are in close proximity, for example, in a complex, interaction, or aggregate, or when the two molecules coexist in close proximity) and are then extended using a nucleic acid polymerase. The extension product forms a reporter nucleic acid, the detection of which confirms the presence of a specific analyte (the analyte to which the associated probe pair binds) in the sample of interest. In PLA, nucleic acid moieties linked to the analyte-binding domains of a probe pair are in close proximity when the probes of the probe pair bind to their targets and can be ligated together, or they can together template the ligation of separately added oligonucleotides that can hybridize to the nucleic acid domains when they are in close proximity. The ligation products are then amplified and act as reporter nucleic acids. Multiplexed analyte detection using PEA or PLA can be achieved by including a unique barcode sequence in the nucleic acid moiety of each probe. Reporter nucleic acid molecules corresponding to specific analytes can be identified by the barcode sequence they contain. The methods of the present invention are particularly useful in multiplex PEA and PLA processes.

[0006] Panels of such proximity assays are commercially available from Olink Proteomics AB (Uppsala, Sweden) under the trademarks Olink® Target, Olink® Focus, and Olink® Explore. These are fixed panels of up to 92 assays (Olink® Target and Focus) or up to approximately 3000 assays (Olink® Explore) divided into eight different panels. Each panel generally contains assays for proteins with known functions within a particular biological or physiological area, pathway, or organ in the body, such as inflammation, organ-specific proteins, cardiovascular, neurological, etc.

[0007] Enroth et al., Communications Biology 2(1), 2019, pages 1-12 (DOI: 10.1038 / s42003-019-0464-9) describe the use of the Olink panel and a custom-designed panel in the PEA assay to identify a novel, highly accurate plasma protein biomarker signature for ovarian cancer.

[0008] Currently available immunoassays that allow users to combine individual assays into multiplex assays have a combinability of 80–90%. This means that technical limitations prevent users from utilizing 10–20% of the theoretically possible complex multiplex panels. Furthermore, even when assays are combinable, they may need to be performed in separate reaction vessels. Summary of the Invention

[0009] The invention is set out in the accompanying claims. [Brief explanation of the drawings]

[0010] [Figure 1] FIG. 12 visualizes the distribution of discriminator sequences across a set of detection probes. DETAILED DESCRIPTION OF THE INVENTION

[0011] Definitions of Terms and Abbreviations All terms and abbreviations used herein shall be construed to have the meaning commonly given to them in the relevant art unless a different meaning is clearly intended. For clarity, some terms and abbreviations are defined below.

[0012] A "library" is a collection of items and / or sets of items. A library is generally ordered and structured so that all items and sets of items are separately searchable.

[0013] "Readout" is intended to refer to the process of quantifying the amount of reporter molecules with their respective unique identification sequences and correlating these amounts with the amount of each analyte of interest in the analytical sample. Thus, readout can be viewed as the step of quantitatively detecting the signal, or more specifically, the reporter molecules, in an assay.

[0014] The present invention provides means and methods for the flexible construction of multiplex biological assays based on the generation of reporter nucleic acids, where the amount of a particular reporter nucleic acid molecule corresponds to the amount of a particular analyte of interest in a sample. Such assays are known in the art and include, inter alia, PLA and PEA, as described above. However, there is a need for means and methods for the rapid, easy, and inexpensive construction of new panels with novel combinations of assays.

[0015] Multiplexing biological assays, such as proximity assays, means performing multiple assays in parallel, preferably in the same reaction vessel, such as a test tube or a well of a microtiter plate. Multiplexing therefore has the potential to greatly increase sample throughput and reduce the required instrument footprint.

[0016] It is not easy to combine multiple biological assays in the same container for the same sample, because many aspects may cause the interference between assays.For the multiplexed assay that is based on correlating the amount of reporter nucleic acid molecules that contain identification sequence with the analyte of interest, one specific interference that needs to be solved is to avoid the overlap of identification sequence, so that no identification sequence is connected to more than one analyte of interest.That is, each identification sequence is unique to the analyte of interest in any given panel of assays.

[0017] The discriminating sequence may be, for example, a unique sequence (commonly referred to as a "barcode sequence" or simply "barcode") that is sequence-specifically detected, e.g., sequenced for identification in a readout step, or that provides a specific binding (hybridization) site for a probe or primer used in detection, e.g., a unique primer binding site that can be used for readout using quantitative PCR (qPCR).

[0018] A common solution to the above redundancy problem is to provide a fixed panel of assays, where any redundancy issues are resolved during the development of specific fixed panels prior to market release. Fixed multiplex assay panels of various sizes and assay contents are ideal for broad screening-to-targeted discovery solutions for many research applications and research questions. provides a discovery solution.

[0019] However, there may be situations where there is no existing fixed panel suitable for the research question at hand, and the user wants to design a panel of assays for a particular set of analytes of interest. In each such case, to avoid duplication within the newly designed panel, it is possible to assign a unique identification sequence to each detection probe, but this is time-consuming both in the design process and in physical manufacturing. It would be advantageous to have a library of ready-made detection probes with pre-assigned unique identification sequences available.

[0020] It may also be desirable to limit the available pool of discriminator sequences, for example, if the discriminator sequences contain unique primer binding sites, it may be desirable to limit the number of unique primer binding sites so that the same set of readout primers can be used for any panel, regardless of the assay selected.

[0021] The present invention aims to provide a library of detection probes that facilitates the flexible design of panels of assays that can be rapidly generated using pre-made detection probes, optionally with a limited pool of discriminatory sequences.

[0022] The present invention further aims to facilitate the rapid and easy compilation and production of custom multiplexed panels of biological assays based on linking nucleic acid molecules capable of generating reporter nucleic acid molecules comprising discriminating sequences to analytes of interest, where each of the analytes of interest is assayed based on the abundance of reporter nucleic acid molecules comprising discriminating sequences unique to the particular analyte of interest.

[0023] To this end, the present invention provides, in one aspect, a library comprising a plurality of detection probes for detecting a plurality of analytes of interest, each detection probe comprising an analyte-specific binding domain and a nucleic acid portion, the nucleic acid portion of the detection probe capable of generating a reporter nucleic acid molecule comprising a discrimination sequence that identifies the respective analyte of interest, the library comprising, for one or more analytes of interest, a set of two or more detection probes, each detection probe in a set capable of generating a reporter nucleic acid molecule comprising a discrimination sequence that differs from the discrimination sequences generated by other members of the same set.

[0024] By appropriately adjusting the number of members for each set of probes, libraries provide a convenient means to facilitate the combination of detection probes for any or most subsets of analytes of interest, where all detection probes in the combination have a unique identification sequence.

[0025] In other words, the provision of a library according to the present invention allows for the rapid selection of a panel of assays for detecting multiple analytes of interest by selecting detection probes from the library for inclusion in the panel such that each selected probe can generate a reporter nucleic acid molecule having an identification sequence that is unique within the selected panel. Methods for such selection are included within the scope of the present invention.

[0026] Thus, for a given analyte, the library can contain sets of detection probes (i.e., different detection probes for a particular analyte) that can generate reporter molecules with different identification (ID) sequences, from which a user can select detection probes for inclusion in an assay panel. It is not necessary to include a different set of detection probes for each and every analyte; rather, such different sets of detection probes need to be included for at least some analytes, or for many or multiple analytes. Thus, a library can be viewed as containing multiple or multiple sets of detection probes, where a set of detection probes includes two or more detection probes for detecting a given analyte (i.e., the same analyte). In other words, a library contains separate sets of detection probes for one or more analytes. A set of detection probes is specific for a particular analyte (i.e., the same analyte is detected by the set), but within a set, the reporter nucleic acid molecules generated by the detection probes are different (or, more specifically, include different ID sequences).

[0027] A detection probe, as discussed herein, comprises at least one analyte-specific binding domain and at least one nucleic acid moiety. The analyte-specific binding domain and nucleic acid moiety may be covalently or non-covalently bound to each other. A detection probe may further comprise one or more components or moieties. Thus, it may be a single probe comprising one analyte-specific binding domain and one nucleic acid moiety, or a probe provided with two or more moieties, or a group or set of probes for use together (i.e., in combination) to detect an analyte. Thus, a detection probe may comprise or be provided by a proximity probe. Proximity probes are typically used in pairs, although in certain embodiments, more than two proximity probes, e.g., three proximity probes, may be used together to detect a given analyte. One or more pairs of proximity probes are said to be matched or cognate to each other.

[0028] The analyte of interest can be any analyte that one desires to detect. In embodiments, the analyte is a protein. However, the analyte may be any biological or chemical entity that one desires to detect. As indicated above, proximity probes are used in the art to detect a wide variety of analytes, which may include interactions, complexes, and the like. Thus, each of a pair (or more) proximity probes can bind to the same target molecule (but at different sites, such that each individual proximity probe can bind to its respective target binding site contemporaneously (i.e., simultaneously)), or to different molecules (e.g., when the target analyte is an interaction and each proximity probe binds to a different member of the interaction, or when a post-translational modification of a given protein is detected). In fact, the target analyte may be the co-localization of two molecules in close proximity.

[0029] The reporter nucleic acid molecule can be generated from the nucleic acid portion(s) of the detection probe in a variety of ways. This may include amplification, extension, ligation, and / or cleavage. In relation to proximity probes, possibilities are discussed in the documents cited above. The discriminator sequence or a portion thereof may be included in the detection probe (or one or more portions or components thereof) or may be generated by generation of the reporter nucleic acid molecule.

[0030] As is known in the art, the analyte-specific binding domain of a detection probe can be any entity capable of specifically binding to a target analyte (or a portion thereof) and coupling to a nucleic acid moiety. As known to those skilled in the art, a binding domain being "specific" for a particular analyte means that it recognizes the analyte with low cross-reactivity (off-target binding) with other potentially present analytes in the relevant application and experimental context. A framework for determining binder specificity has been established by the International Working Group for Antibody Validation (Uhlen et al. Nat Methods, 2016 Oct;13(10),823-827, incorporated herein by reference).

[0031] Typically, the analyte-specific binding domain may be a protein, such as an antibody or antigen-binding portion thereof, including, but not limited to, monoclonal antibodies, recombinant monoclonal antibodies, and polyclonal antibodies, as well as antigen-binding antibody derivatives and fragments. However, the analyte-specific binding domain may also be of any nature, including lectins, soluble cell surface receptors, proteins combinatorially derived from phage display or ribosome display, peptides, carbohydrates, molecularly imprinted polymers (MIPs), nucleic acids, such as aptamers or nucleic acid molecules comprising the complementary sequence of a target nucleic acid, or combinations thereof.

[0032] Reagents useful as analyte-specific binding domains are commercially available from a number of manufacturers that provide ready-made reagents or develop new binding reagents for particular analytes and specific needs. Such manufacturers include Thermo Fisher Scientific (Boston, MA, USA), Abcam (Cambridge, UK), Bio-Techne (Minneapolis, MN, USA), Proteogenix (Schiltigheim, France), Sino Biological (Beijing, China), Agrisera (Vännäs, Sweden), Novaptech (Pessac, France), and Aptamer Group (York, UK), among others. Reagents useful as analyte-specific binding domains can also be developed independently of commercial sources according to protocols well known to those skilled in the art. Such protocols are described, for example, in "Monoclonal Antibody Production" (National Academy Press, Washington, DC, USA, 1999), Carey-Hanly et al. (ILAR Journal, Volume 37, Issue 3, 1995, Pages 93-118), and Ilgu and Nilsen-Hamilton (Analyst. 2016 March 7;141(5):1551-1568). Reagents may also include antibody derivatives or fragments such as Fab, Fab', F(ab'), Fv fragments; diabodies; single domain antibodies (sdAb, Desmyter et al. (1996) Nat. Structure Biol. 3:803-811), nanobodies, single chain Fvs (scFv, Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85, 5879-5883), bivalent scFvs (di-scFvs), tandem scFvs, triabodies, diabodies, single chain diabodies (sCDbs), bispecific T cell engagers (BiTEs, Kufer et al. (2004) Trends Biotechnol. 22:238-244), and dual affinity retargeting molecules (DARTs, diabodies further stabilized via a C-terminal disulfide bridge).The specificity of the analyte-specific reagent for the intended detection assay can be assessed using the framework proposed by the International Working Group for Validating Antibodies cited above.

[0033] Furthermore, the analyte-specific binding domain can bind to the analyte directly or indirectly: in other words, the detection probe can be a primary reagent that binds directly to the analyte, or a secondary reagent that binds indirectly by binding to an intermediate molecule (primary reagent) that is itself directly bound to the analyte.

[0034] In addition to the analyte-specific binding domain, the detection probes used in the present invention also contain a nucleic acid portion, also referred to herein as an oligonucleotide. The oligonucleotide must be long enough to contain the necessary functional elements for use in the detection assay for which the detection probe is intended to be used. That is, at least a sequence capable of generating an identification sequence in the reporter molecule. This is typically 5-20 nucleotides, e.g., 5-10, 5-15, 10-15, or 15-20 nucleotides. The oligonucleotide may also contain sequences related to primer sites and / or sequencing adapters for readout, as known in the art. Generally, the oligonucleotide has a length in the range of 20-100 nucleotides, but can be shorter or longer as required for the specific detection assay for which the detection probe is intended to be used.

[0035] Conjugation of nucleic acid moieties to antibodies can be carried out by several methods known to those skilled in the art, for example, as reviewed by Dugal-Tessier et al. (J. Clin. Med. 2021, 10, 838). Commercial kits for preparing antibody-oligonucleotide conjugates are also readily available from many sources. The oligonucleotide may be coupled to the analyte-binding domain by any means known in the art, which may be desired or convenient, and may be direct or indirect (e.g., via a linking group). For example, the domains may be associated with each other by covalent bonds (e.g., chemical crosslinking) or non-covalent bonds, such as streptavidin-biotin-based coupling (biotin is provided on one domain, particularly the oligonucleotide domain, and streptavidin is provided on the other domain).

[0036] The oligonucleotide and the analyte-binding domain are joined together directly via a bond or indirectly via a linking group. When a linking group is used, such group can be selected to provide covalent attachment of the nucleic acid moiety and the analyte-binding domain via the linking group. When present, the linking group, in many embodiments, is biologically inert. In representative embodiments, the linking group is generally at least about 50 daltons, usually at least about 100 daltons; if the linking group contains a spacer, it may be 1,000 daltons or larger, e.g., up to 1,000,000 daltons, but generally not more than about 500 daltons, usually not more than about 300 daltons. Typically, such linkers include a spacer group terminating at either end with a reactive functional group capable of covalently binding to the nucleic acid domain or the analyte-binding domain. Spacer groups of interest can include aliphatic and unsaturated hydrocarbon chains, spacers containing heteroatoms such as oxygen (ethers such as polyethylene glycol) or nitrogen (polyamines), peptides, carbohydrates, and cyclic or acyclic systems that may contain heteroatoms. The spacer group may also consist of a ligand that binds to the metal 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-dioxaoctane diacid, ethylenediamine-N,N-diacetic acid, 1,1'-ethylenebis(5-oxo-3-pyrrolidinecarboxylic acid), and 4,4'-ethylenedipiperidine.

[0037] Potentially reactive functional groups include nucleophilic functional groups (amines, alcohols, thiols, hydrazides), electrophilic functional groups (aldehydes, esters, vinyl ketones, epoxides, isocyanates, maleimides), and functional groups capable of cycloaddition reactions, disulfide bond formation, or metal bonding. Specific examples include primary and secondary amines, hydroxamic acids, N-hydroxysuccinimidyl esters, N-hydroxysuccinimidyl carbonates, oxycarbonylimidazoles, nitrophenyl esters, trifluoroethyl esters, glycidyl ethers, vinyl sulfones, and maleimides.

[0038] Specific linker groups that can be utilized in the present proximity probes include heterofunctional compounds such as azidobenzoyl hydrazide, N-[4-(p-azidosalicylamino)butyl]-3'-[2'-pyridyldithio]propionamide, bis-sulfosuccinimidyl suberate, dimethyl adipimidate, disuccinimidyl tartrate, N-maleimidobutyryloxysuccinimide ester, N-hydroxysulfosuccinimidyl-4-azidobenzoate, N-succinimidyl ... Examples of suitable succinimidyl esters include 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), and 4-(N-maleimidomethyl)-cyclohexane-1-carboxylic acid N-hydroxysuccinimide ester (SMCC).

[0039] The nucleic acid domain of the detection probe can be composed of ribonucleotides and / or deoxyribonucleotides, as well as synthetic nucleotide residues capable of participating in Watson-Crick or similar base pairing interactions. Thus, the nucleic acid domain can be DNA or RNA, or a combination or any modification thereof, such as PNA or other derivatives containing a non-nucleotide backbone.

[0040] In one embodiment, the detection probe is produced by coupling a universal oligonucleotide to an analyte-specific binding domain, followed by hybridization of a tag oligonucleotide to the universal oligonucleotide, the tag oligonucleotide comprising a sequence capable of generating a distinguishing sequence in the reporter molecule, a sequence complementary to the universal oligonucleotide to facilitate hybridization, and any other functional sequences necessary to perform the detection assay for which the detection probe is intended. Methods for producing such detection probes are described, inter alia, in WO 2017 / 068116.

[0041] When proximity probes are used as detection probes, each nucleic acid portion of a matched pair or more proximity probes may contain a discrimination sequence or partial discrimination sequence. The reporter molecule produced may contain the discrimination sequence from each of the matched proximity probes. In other words, the discrimination sequence of the reporter molecule may be a combination or composite of the discrimination sequences of the individual nucleic acid portions of the matched proximity probes. The discrimination sequences of the individual matched proximity probes may be the same or different. In embodiments, each discrimination sequence in a matched proximity probe is indicative of or corresponds to an analyte of interest. However, it is not necessary that the particular discrimination sequence of an individual proximity probe be indicative of the analyte of interest; it is the discrimination sequence of the reporter nucleic acid molecule that is indicative of the analyte of interest. As noted above, the reporter discrimination sequence may be a combination or composite. Alternatively, the discrimination sequence of the reporter nucleic acid molecule may be derived from the nucleic acid portion of a single proximity probe (although it will be understood that interaction of the nucleic acid portions of matched proximity probes is required for the reporter nucleic acid molecule to be formed).

[0042] In an embodiment, the analyte-specific binding domain is not a nucleic acid that binds by hybridization. In another embodiment, the analyte-specific binding domain is not a nucleic acid. In another embodiment, the detection probe is not composed entirely of nucleic acid. In another embodiment, the detection probe is not a gene-specific probe. In another embodiment, the detection probe is not a padlock probe. In another embodiment, the detection probe is not for use in microscope-based optical detection methods.

[0043] As described above, the detection probes in a set include detection probes specific to (or directed toward) the same analyte of interest. Use does not require that each detection probe in the set be used to detect that analyte. Thus, in embodiments, the detection probes in a set are not designed or intended for use together in a method for detecting the analyte.

[0044] In one embodiment of a library according to the invention, each detection probe comprises a matched pair of proximity probes, each proximity probe comprising an analyte-specific binding domain and a nucleic acid moiety, wherein both analyte-specific binding domains of each matched pair of proximity probes are capable of specifically binding to the same analyte of interest, and the nucleic acid moieties of the matched pair of proximity probes are capable of together forming a reporter nucleic acid molecule comprising a discrimination sequence that identifies each analyte of interest.

[0045] This embodiment is particularly relevant for practicing the invention in proximity assays such as proximity extension assays and proximity ligation assays.

[0046] Similar to the proximity assays developed by Olink Proteomics AB and others mentioned and referenced above, the libraries of the present invention may be utilized in a variety of commercially available assays available in the art or in development.

[0047] In one embodiment, each set of detector probes contains five or fewer detector probes. Each set of detector probes may independently contain one, two, three, four, or five detector probes. In some embodiments, all sets of detector probes have the same number of members. In some embodiments, all sets of detector probes have two members. In some embodiments, all sets of detector probes have three members. In some embodiments, all sets of detector probes have four members. In some embodiments, all sets of detector probes have five members. In a preferred embodiment herein, all sets of detector probes have three members.

[0048] In some embodiments, the library comprises two or more sets of detection probes for at least 25% of the analytes of interest, hi some embodiments, the library comprises two or more sets of detection probes for at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the analytes of interest.

[0049] In some embodiments, the number of unique discriminating sequences in the library is less than the number of members of the set of analytes of interest.

[0050] In some embodiments, the library is adapted for the selection of an n-plex panel of detection probes, where the adaptation includes including 1.5n to 10n unique identifying sequences in the library. That is, the library is configured so that a user can select n analytes of interest (n being any integer) and construct a panel of n detection probes, each specific to one analyte. The library accommodates this by including enough unique identifying sequences in the library to ensure that as high a percentage of possible panels as possible can actually be compiled, while not including too many unique identifying sequences to keep production costs low. While including 1.5n to 3n unique identifying sequences in the library has been found to be a reasonable trade-off between these two requirements, in certain circumstances it may be preferable to include more unique identifying sequences, for example, up to 5n, 7n, or 10n.

[0051] It follows that a library adapted to select panels of a particular plex grade, n, will also be suitable for selecting panels of slightly lower and higher plex grades. For example, a library adapted to select 21-plex panels will also be well suited to selecting 15-plex and 24-plex panels. In some embodiments, n is an integer between 1 and 100.

[0052] One advantage of the present invention is that the readout of assay results can be standardized across multiple assay panels, and the identification sequence can be reused to correlate with different analytes of interest in panels of different configurations. In the readout step, the amount of reporter nucleic acid molecules with specific identification sequences is quantified and assigned to the corresponding analyte of interest for the particular panel of assays being readout.

[0053] As is known in the art, different detection modes for readout are possible. These include sequencing. Thus, for example, the identification sequence can be a barcode that is sequenced. Any sequencing method can be used, including sequencing-by-synthesis and sequencing-by-hybridization. Thus, depending on the nature of the ID sequence, any suitable method for identifying the ID sequence can be used, and this can include the use of hybridization probes and / or primers. For example, the detection (readout) step can include amplifying a reporter nucleic acid using one or more primers, at least one of which binds to the ID sequence. Alternatively, the detection method can include amplifying the reporter and detecting the amplicon with a specific hybridization probe that binds to the ID sequence (or its complement). In sequencing-by-hybridization, the barcode can be decoded using labeled hybridization probes, including in a combinatorial manner.

[0054] Sequencing advantageously allows high level multiplexing and is a convenient detection method.As mentioned above, any form of sequencing can be used, including any sequencing by synthesis method, for example, pyrosequencing, reversible dye terminator sequencing and ion torrent sequencing.In particular, high-throughput sequencing method is used, and in particular, massively parallel DNA sequencing is used.For example, Illumina® NovaSeq® system can be used to carry out massively parallel DNA sequencing using reversible dye terminator method.

[0055] In another embodiment, the ID sequence is a primer binding site, for example for a PCR primer, although other amplification methods may be used.

[0056] In yet a further embodiment, the ID sequence may be a restriction site (i.e. a nucleotide sequence recognised by a restriction enzyme). In this embodiment, the nucleic acid domains of the proximity probes may contain different restriction sites (so as to be recognised and cleaved by different restriction enzymes). Thus, different combinations of restriction enzymes can be applied to distinguish between different reporter nucleic acids.

[0057] PCR-based amplification methods are advantageous, and the readout can advantageously include quantitative PCR (qPCR) or real-time PCR. Amplicons can be detected using any convenient protocol, including the use of dyes and stains, or labels, such as intercalating dyes, or labeled probes that bind to amplicons. These include molecular beacons, such as probes with FRET labels.

[0058] For example, if readout is performed by qPCR, it is preferable to provide a limited set of qPCR primers that function for all panels of assays, regardless of the assay content of the various panels. It is also preferable to keep the number of qPCR primers low to reduce costs, the risk of mismatch binding, and other biological artifacts. In the present invention, it is possible to select a set of detection probes in which all detection probes generate reporter molecules with unique identification sequences, and all of these identification sequences also correspond to a limited set of qPCR primer binding sites, so that a corresponding limited set of qPCR primers can be used to readout the panel. In this way, the same set of qPCR primers can be used to readout any panel compiled from a library according to the present invention.

[0059] Thus, in one embodiment, unique identifying sequences in a library can be generated that correspond to a set of qPCR primers that can be used to read out any panel compiled from a library according to the present invention.

[0060] In one embodiment, the discriminator sequence is a binding site for a qPCR primer.

[0061] In one embodiment, the identification sequence is a barcode sequence.

[0062] In one embodiment, the set of analytes of interest comprises between 100 and 30,000 analytes of interest.

[0063] In a further aspect, the present invention relates to a method of constructing a panel of detection probes for a group of analytes of interest, wherein the group of analytes of interest is a subset of a larger set of analytes of interest, the method comprising the step of selecting, from a library according to the present invention, one detection probe per analyte in the group of analytes for inclusion in the panel, such that each selected detection probe can generate a reporter nucleic acid molecule having an identification sequence that is unique within the constructed panel.

[0064] In some embodiments, the detection probes comprise a matched pair of proximity probes.

[0065] The information of which unique discriminatory sequences represent which analytes of interest must be taken into account in the readout of a panel, and therefore this information is preferably documented and transferred to the user when selecting a panel, and implemented in the readout of a particular panel.

[0066] In a further aspect, the present invention relates to a computer program comprising instructions for causing a computer to carry out the method according to the present invention.

[0067] Assays using panels composed of libraries according to the invention can be performed as known in the art, as described, inter alia, in Assarsson et al., PLoS 1, 2014, 9, 4, e95192; Lundberg et al., Molecular & Cellular Proteomics 10:10.1074 / mcp.M110.004978, 1-10, 2011; and Wik et al., 2021, Mol Cell Proteomics 20, 100168.

[0068] The present invention is further described in the following illustrative examples, which are intended merely to facilitate understanding of the invention and are not to be construed as limiting the scope of the invention as defined in the appended claims.

[0069] All prior publications cited herein are incorporated by reference in their entirety. [Example]

[0070] A set of 200 human proteins was selected as analytes of interest based on their overall relevance to biological pathways related to inflammation. It was determined that the available plex grade must be 21, i.e., any selected panel must consist of a maximum of 21 detection probes.

[0071] A matched pair of proximity probes commonly used in PEA was used as detection probes, and the qPCR primer binding site was used as the discrimination sequence.For each analyte of interest, a set of three detection probes was prepared, each with a unique discrimination sequence.For the entire library, 45 unique discrimination sequences were used that correspond to the qPCR primers contained in the qPCR readout primer for Olink® Target 48 (Olink Proteomics AB).

[0072] Each set of detection probes was assigned a unique set of three distinct discriminator sequences; that is, no two sets of detection probes had the same combination of discriminator sequences, and no two members of the same set had the same discriminator sequence. The distribution of discriminator sequences across the detection probe sets is visualized in Figure 1.

[0073] Simulations were performed to determine how many of all possible 21-plex panels could be generated from the library, and an achievement rate of 99.9% was found, i.e., only 0.1% of possible panels could not be delivered using the example library.

Claims

1. 1. A library comprising a plurality of detection probes suitable for detecting a set of analytes of interest, each detection probe comprising an analyte-specific binding domain and a nucleic acid portion, the nucleic acid portion of the detection probe capable of generating a reporter nucleic acid molecule comprising a discrimination sequence that identifies a respective analyte of interest, the library comprising, for one or more analytes of interest, two or more sets of detection probes, each detection probe in the set capable of generating a reporter nucleic acid molecule comprising a discrimination sequence that differs from the discrimination sequences generated by other members of the same set.

2. 2. The library of claim 1, wherein each detection probe comprises a matched pair of proximity probes, each proximity probe comprising an analyte-specific binding domain and a nucleic acid moiety, wherein both analyte-specific binding domains of each matched pair of proximity probes are capable of specifically binding to the same analyte of interest, and wherein the nucleic acid moieties of the matched pair of proximity probes are capable of together forming a reporter nucleic acid molecule comprising an identification sequence that identifies each analyte of interest.

3. 3. The library of claim 1 or 2, wherein each set of detection probes comprises five or fewer detection probes.

4. The library of any one of claims 1 to 3, wherein the library comprises two or more sets of detection probes for at least 25% of the analytes of interest.

5. The library of any one of claims 1 to 4, wherein the number of unique discriminating sequences in the library is less than the number of members of the set of analytes of interest.

6. 6. The library of any one of claims 1 to 5, wherein the library is adapted for selection of an n-plex panel of detection probes, and the adaptation comprises including between 1.5n and 10n unique identification sequences in the library.

7. The library according to claim 6, wherein n is an integer from 1 to 100.

8. The library of any one of claims 1 to 7, wherein the identification sequence is a binding site for a qPCR primer.

9. The library according to any one of claims 1 to 8, wherein the identification sequence is a barcode sequence.

10. The library of any one of claims 1 to 9, wherein the set of analytes of interest comprises between 100 and 30,000 analytes of interest.

11. 11. A method of constructing a panel of detection probes for a group of analytes of interest, wherein the group of analytes of interest is a subset of a larger set of analytes of interest, the method comprising selecting one detection probe per analyte in the group of analytes for inclusion in the panel from a library according to any one of claims 1 to 10, such that each selected detection probe can generate a reporter nucleic acid molecule having an identification sequence that is unique within the constructed panel.

12. The method of claim 11 , wherein the detection probes comprise a matched pair of proximity probes.

13. A computer program comprising instructions for causing a computer to carry out the method according to claim 11 or 12.

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