Method for detecting analytes

Abstract

Disclosed is a method for detecting analytes in a biological sample, comprising following steps: a. contacting the biological sample with at least a first marker (100a) and at least a second marker (100b), wherein the at least first marker comprises a first affinity reagent (108a) configured to bind to a first analyte (102a) and a first nucleic acid label (104a) and wherein the at least second marker comprises a second affinity reagent (108b) configured to bind to a second analyte (102b) and a second nucleic acid label (104b), b. incubating the biological sample with said first marker and said second marker, in particular for a sufficient amount of time, to allow the at least first affinity reagent (108a) to bind to the first analyte (102a) and to allow the at least second affinity reagent (108b) to bind to the second analyte (102b), and c. adding at least one bridging strand (200) configured to bind to the first (104a) and second nucleic acid label (104b).

Description

[0001] Method for detecting analytes

[0002] Technical field

[0003] The invention relates to a method for detecting analytes with pairs or multiples of affinity reagents and can be used to improve the specificity of affinity reagents and immunoassays, to detect protein-protein, protein-RNA, protein-DNA, or molecular interactions and to assess posttranslational modifications.

[0004] Background

[0005] Affinity reagents are used in life science to bind targets or analytes with high affinity and specificity. A variety of assays for liquid or solid samples rely on detection reagents that comprise an affinity reagent and a detectable label, which may comprise a nucleic acid, e.g. an oligonucleotide, a (fluorescent) dye, an enzyme, a metal tag, a radioactive tag, or an affinity tag.

[0006] This allows the identification and / or localisation of the target analyte in the biological sample. An affinity reagent in the sense of this document may be for example an antibody, an antibody fragment, a nanobody, an aptamer, an aptabody, a polymeric binder, an affimer, an oligonucleotide probe (e.g. ISH or FISH probe), a drug / drug-like molecule or a toxin. An affinity reagent (AR) may also be a derivative of the above. An affinity reagent is usually configured to bind a target analyte or target with high specificity and affinity. However, many antibodies, which form the largest class of commercially available affinity reagents that bind proteins, are known to exhibit substantial cross- reactivity. In other words, many if not all antibodies do not only bind to their cognate target analyte, but also to a number of OFF-targets, i.e. targets that are not subject to the intended investigation. Both the cognate target against which an affinity reagent was produced or raised and the OFF-target may be collectively referred to as targets. A further particularly relevant class of affinity reagents are aptamers and their derivatives. Aptamers are typically short nucleic acid sequences that fold into 3- dimensional structures capable of binding a target or analyte with high affinity and specificity. In the sense of this document, an aptamer refers to a small affinity reagent that is typically based on nucleic acids - either naturally occurring RNA, DNA or artificial ones XNAs, but may in some cases also be based on a peptide backbone. In the sense of this document, aptamers also refers to derivatives of aptamers such as SOMAmers, aptabodies, or other aptamer-derivatives, for example modified with modifications that are typically found on proteins such as glycosylation. Aptamers are generated by SELEX, can be produced at low cost with very high batch-to-batch consistency, practically unlimited shelf-life at -20°C, can be easily modified and are well suited to o a cyclic staining and imaging process described the European patent application with the application number 23178065.1, the complete content thereof being incorporated herein by reference.

[0007] Both aptamers, antibodies, and nanobodies have dissociation constants (KD) in the pM to pM range and are used in a variety of affinity reagent-based assays or immunoassays, which are a pillar of life science research.

[0008] Markers in the sense of this document comprise at least one affinity reagent and at least one nucleic acid label, wherein the nucleic acid label may be unipartite or multipartite and may comprise nucleic acids of natural origin and / or non-natural nucleic acids also referred to as xeno nucleic acids (XNA).

[0009] A nucleic acid label may be a short oligonucleotide (5-200nt in length), a polynucleotide (>200nt), or a multipartite nucleic acid structure, such as a dendrimeric structure, a DNA-brick based structure, a DNA-origami-based structure like a rod, sheet, or arbitrarily shaped structure. Affinity reagents may be used for purification, isolation, and / or detection of target analytes from a variety of biological samples. In the sense of this document, a sample refers to a biological sample or specimen including for example blood, serum, plasma, tissue, bodily fluids (e.g. lymph, saliva, semen, interstitial fluid, cerebrospinal fluid), feces, solid biopsy, liquid biopsy, explants, cells (e.g. prokaryotes, eukaryotes, archea), suspension cell cultures, monolayer cell cultures, 3D cell cultures (e.g. spheroids, tumoroids, organoids derived from various organs such as intestine, brain, heart, liver, etc.), a lysate of any of the aforementioned, a virus. In the sense of this document, sample further refers to a volume surrounding a biological sample. For example in assays, where secreted proteins like growth factors, extracellular matrix constituents are being studied, the extracellular environment surrounding a cell up to a certain assay-dependent distance is also referred to as a sample. Specifically, affinity reagents brought into this surrounding volume are referred to in the sense of this document as being introduced into the sample.

[0010] Commonly used affinity reagent-based assays are for example western blots; immunoprecipitation, which may be used to purify analytes or to study proteinprotein interaction; enzyme-linked immunosorbent assays (ELISA) and various ELISA- style assays (e.g. sandwich immunoassays); multiplex immunoassays; lateral flow assays; flow cytometry; immunofluorescence assays; immunohistochemistry (IHC) and immunocytochemistry (ICC) assays; cell sorting assays; multiplex bead- or particle-based immunoaffinity assays like protein microarrays as well as single cell analysis and spatial biology techniques that rely on oligonucleotide-barcoded affinity reagents.

[0011] Detection in affinity reagent-based assays is usually performed by marking analytes with markers that generally comprise at least one affinity reagent and a (detectable) label, which may for example be an enzyme, a metal-tag, a Raman label, a fluorescent label or a hybrid label. A fluorescent label may comprise a fluorescent dye, a fluorophore, a fluorochrome, or a dye. A detectable label may be, in particular, an optically detectable label.

[0012] In the sense of this document, the terms "fluorescent dye", "fluorophore", "fluorochrome", "dye" are used interchangeably to denote a fluorescent chemical compound or structure and can be in particular one of the following: a fluorescent organic dye, a fluorescent quantum dot, a fluorescent dyad, a fluorescent carbon dot, graphene quantum dot or other carbon-based fluorescent nanostructure, a fluorescent protein, a fluorescent DNA origami-based nanostructure. From the organic fluorescent dyes in particular derivatives of the following are meant by the term "fluorescent dye": xanthene (e.g. fluorescein, rhodamine, Oregon green, Texas), cyanine (e.g. cyanine, indocarbocyanine, oxacarbocyanine, thiacarbocyanine, merocyanine), derivatives, squaraine rotaxane derivatives, naphthalene, coumarin, oxadiazole, anthracene (anthraquinones, DRAQ5, DRAQ7, CyTRAK Orange), pyrene (cascade blue), oxazine (Nile red, Nile blue, cresyl violet, oxazine 170), acridine (proflavine, acridine orange, acridine yellow), arylmethine (auramine, crystal violet, malachite green), tetrapyrrole (porphin, phthalocyanine, bilirubin), dipyrromethene (BODIPY, aza-BODIPY), a phosphorescent dye, or a luminescent dye. The following trademark groups denote commercially available fluorescent dyes, which may include dyes belonging to different chemical families such as CF dye (Biotium), DRAQ and CyTRAK probes (BioStatus), BODIPY (Invitrogen), EverFluor (Setareh Biotech), Alexa Fluor (Invitrogen), Bella Fluore (Setareh Biotech), DyLight Fluor (Thermo Scientific), Atto and Tracy (Sigma-Aldrich), FluoProbes (Interchim), Abberior Dyes (Abberior Dyes), Dy and MegaStokes Dyes (Dyomics), Sulfo Cy dyes (Cyandye), HiLyte Fluor (AnaSpec), Seta, SeTau and Square Dyes (SETA BioMedicals), Quasar and Cal Fluor dyes (Biosearch Technologies), SureLight Dyes (Columbia Biosciences), Vio Dyes (Milteny Biotec). From the group of fluorescent proteins in particular the members of the green fluorescent protein (GFP) family including GFP and GFP-like proteins (e.g DsRed, TagRFP) and their (monomerized) derivatives (e.g., EBFP, ECFP, EYFP, Cerulaen, mTurquoise2, YFP, EYFP, mCitrine, Venus, YPet, Superfolder GFP, mCherry, mPlum) are meant by the term "fluorescent dye" in the sense of this document. Further from the group of fluorescent proteins the term "fluorescent dye" in the sense of this document may include fluorescent proteins, whose absorbance or emission characteristics change upon binding of ligand such as BFPmsl or in response to changes in the environment such as redox-sensitive roGFP or pH- sensitive variants.

[0013] Further from the group of fluorescent proteins the term "fluorescent dye" in the sense of this document may include derivatives of cyanobacterial phycobiliprotein small ultra red fluorescent protein smURFP as well as fluorescent protein nanoparticles that can be derived from smURFP. An overview of fluorescent proteins can be found in the article of Rodriguez et al. in Trends Biochem Sci. 2017 Feb; 42(2): 111-129. A fluorescent dye in the sense of this document may further refer to a fluorescent quantum dot. A fluorescent dye in the sense of this document may further refer to fluorescent carbon dot, a fluorescent graphene quantum dot, a fluorescent carbon-based nanostructure as described in the article of Yan et al. in Microchimica Acta (2019) 186: 583 and Iravani and Varma 2020 in Environ Chem Lett. 2020 Mar 10 : 1-25. A fluorescent dye in the sense of this document may further refer to a fluorescent polymer dot (Pdot) or nanodiamond. A fluorescent dye in the sense of this document may further refer to a fluorescent dyad, such as a dyad of a perylene antenna and a triangelium emitter as described in the article of Kacenauskaite et al. in J. Am. Chem. Soc. 2021, 143, 1377-1385.

[0014] A fluorescent dye in the sense of this document may further refer to an organic dye, a dyad, a quantum dot, a polymer dot, a graphene dot, a carbon-based nanostructure, a DNA origami-based nanostructure, a nanoruler, a polymer bead with incorporated dyes, a fluorescent protein, an inorganic fluorescent dye, a SMILE, or a microcapsule filled with any of the aforementioned. A fluorescent dye in the sense of this document may further refer to a FRET-pair having at least one fluorescent dye as FRET donor and at least one fluorescent dye as a FRET acceptor, or a F RET-triple, which is used to generate a three component Forster resonance energy transfer. In particular, the FRET-pair or FRET-triplet is connected by a complementary linker or by a linking element.

[0015] A fluorescent dye in the sense of this document may further refer to a FRET n-tupel of physically connected dyes.

[0016] A fluorescent dye may also be a polymer dye.

[0017] The detectable label may be connected to the primary affinity reagent or a secondary affinity, which binds to the primary affinity reagent and thereby also introduces a certain degree of amplification. An affinity reagent may also be labeled with an oligonucleotide barcode, which can be addressed using a complementary oligonucleotide sequence that may be connected to a detectable label.

[0018] Fluorescent labels such as fluorescent proteins, fluorescent dyes, quantum dots, or DNA-oligonucleotide- or DNA-nanostructure-based fluorescent labels are commonly used for immunofluorescence assays that may be readout using imaging-based readout, i.e. microscopy or plate reading, or non-imaging based readouts such as for example cytometry. A label may also be a combinatorial label comprising a combination of dyes, a combination of Raman labels (e.g. polyynes), or a hybrid combinatorial label, for example a label comprising dyes and Raman labels as described in the European patent application with the application number 23168397.0, the complete content thereof being incorporated herein by reference. Such combinatorial labels may be used to enable multi- (20-200), high- (200-10,000), ultra-high-plex (10,000-100,000 or more analytes) analysis either in a single round or in a cyclic staining, imaging, inactivation process over multiple rounds. Years of scientific effort have resulted in an enormous amount of data obtained using a immunoassays. On July 13th 2023, PubMed contained 238,028 publications containing the word "immunofluorescence", with almost 10,000 publications appearing in 2022. Likewise, multiplex assays have gained significant traction over the past 20 years, as evidenced by the fact that 3,615 entries are found in PubMed for 2022 citing "multiplex assay".

[0019] The basic assumption underlying these efforts is that affinity reagents can be used to these ends, as they bind their targets with sufficient affinity and specificity.

[0020] Accumulating evidence shows, however, that this basic assumption does not stand up to scrutiny and that most affinity reagents are cross-reactive, i.e. they do not only bind to their target, but also to OFF-targets.

[0021] As fields like cytometry, plasma proteomics, and microscopy are evolving towards higher levels of plexity, the problem of cross reactivity has come more into focus and has been recognized by the scientific community as a key component of the so-called reproducibility crisis. In a position paper called "Reproducibility: Standardize antibodies used in research" and published by Bradbury and Pluckthin in Nature 518, Tl- 29 (2015) with 110 cosignatories, it was stated that fewer than half of around 6,000 routinely used commercial antibodies recognized only their specified targets.

[0022] More recent work by Schwenk et al. analysed 11,000 affinity-purified, monoclonal antibodies and found only 531 to produce a single band on a western blot. While a systematic and comprehensive analysis of the phenomenon has not been performed, the advent of antibody microarrays has generated new possibilities to probe crossreactivity of affinity reagents in a practical assay format. From a conceptual point of view it is therefore very well possible that we will never be able to make a genuinely specific affinity reagent, i.e. an affinity reagent that truly binds to only one target. Bradbury and Pluckthin further state that cosignatories were able to replicate the scientific results of only 6 of 53 landmark preclinical studies and went on to predict that this phenomenon is costing an estimated $350 million per year in the United States alone.

[0023] The advent of more multi-plex analysis technologies such as bead-based assays or protein microarrays brought the increasing realization that the cross-reactivity of affinity reagents, i.e. their binding to non-targets or OFF-targets is a fundamental limitation of this approach, with significant impact on life science research and society. Scientific community has been aware of the phenomenon of cross- reactivity for a long time, but the difficulty of mitigating its impact on measurements may have been underestimated.

[0024] Furthermore, this is not only an issue in terms of reproducibility of research findings, but also an issue for diagnostic testing, and the therapeutic application of antibodies and other affinity reagents. The use of affinity reagents in for example plasma proteomics assays or the analysis of the tumor microenvironment will become a key aspect of patient stratification.

[0025] The International Working Group for Antibody Validation (IWGAV) has proposed five enhanced validation principles and provides a repository for antibody validation data at antibodypedia.org, which includes thousands of commercially available antibodies. These validation principles are (1) genetic validation using CRISPR or siRNA-mediated knock-out or knock-down of the target protein; (2) recombinant expression validation by overexpressing the target; (3) independent antibody validation; (4) orthogonal validation by comparing the Ab to either protein or corresponding RNA expression levels obtained by a method that does not use antibodies; and (5) capture mass spectrometry validation. To mitigate cross-reactivity and improve specificity in multi- and high plex proteomics the proximity extension assay (PEA) using pairwise detection with oligonucleotide- labeled antibodies has been developed. In this case simultaneous binding of two oligonucleotide-labeled antibodies allows the attached oligonucleotides to hybridize forming the duplex necessary for an extension reaction. The extended dsDNA product can then be detected by for example next generation sequencing (NGS).

[0026] A related technology the NaveniTM Proximity Ligation Technology developed by Navinci Diagnostics (Uppsala, Sweden) allows detection of oligonucleotide-labeled antibody pairs binding to targets that are colocalized in about a 40nm radius in tissue sections, i.e. the readout is imaging-based in this case and performed with a microscope. This technology is based on a combination of proximity ligation with a rolling circle amplification. In theory in this case an amplicon is formed only when both oligonucleotide labels of the antibody pair are in about a 40nm proximity. The amplicon is detected by hybridizing detector probes that are conjugated to an enzyme through a chromogenic reaction or through hybridization of fluorescent reporter probes. This technology can be used to improve specificity in multiplexed biomarker imaging by using antibody pairs to detect one analyte, i.e. 2:1 detection, or to analyse protein-protein interactions, or to analyse posttranslational modifications.

[0027] Earlier, the inventor of the approach presented herein proposed a solution of this problem that is based on collecting cross-reactivity profiles and computational crossreactivity unmixing as described in the European patent application with the application number 23190568.8, the complete content thereof being incorporated herein by reference. In the present document, the inventor proposes an assay format that is particularly suitable to detect the binding of pairs of affinity reagents to a target analyte. This method may be used to measure for example a high number of protein-protein-interactions in tissue samples, which is sometimes referred to as "spatial interactomics".

[0028] Forster Resonance Energy Transfer (FRET) has been widely used to measure proteinprotein interactions using both fluorescent proteins and fluorescent dyes. The spatial stringency of FRET is in the range of lOnm. In other words, the FRET efficiency E

[0029] E= l / (l+(r / / ?0)6) drops exponentially with an increase in donor and acceptor dye distance, such that effective FRET typically occurs when the donor and acceptor dye are within 1 to lOnm distance r to each other, wherein Rois the Forster Distance of the donoracceptor dye pair. Typically, the donor and acceptor dye or fluorophore are different fluorophores. For example the following dye pairs are commonly used FRET pairs: ATTO 425 - ATTO 520, ATTO 488 - ATTO 550, ATTO 488 - ATTO 565, ATTO 488 - ATTO 647N, ATTO 488 - ATTO 655, ATTO 520 - ATTO 647N, ATTO 532 - ATTO 647N, ATTO 532 - ATTO 655, ATTO 550 - ATTO 590, ATTO 550 - ATTO 647N, ATTO 565 - ATTO 590, ATTO 565 - ATTO 647N, ATTO 590 - ATTO 620, ATTO 590 - ATTO 647N, ATTO 590 - ATTO 680, ATTO 620 - ATTO 680, Alexa Fluor 488 - Alexa Fluor 555, Alexa Fluor 594 - Alexa Fluor 647, Fluorescein - Tetramethylrhodamine.

[0030] Likewise, commonly used fluorescent protein FRET pairs exists including but not limited to: ECFP-EYFP, mTurquoise2-mVenus, EGFP-mCherry, ml\leonGreen-mRuby3. Quantum dots may also be used as donor or acceptors. The use of quantum dots as FRET donors is particularly advantageous because of the high extinction coefficients, which leads to brighter FRET signals.

[0031] Various methods exist to measure the FRET efficiency including but not limited to measuring the intensity of the acceptor emission (sensitized emission), photobleaching FRET wherein the donor is bleached and bleaching rates differ depending on the presence or absence of the acceptor, fluorescence lifetime measurements that register the change in the lifetime of the donor (i.e. when FRET occurs the donor lifetime shortens; FRET-FLIM). Likewise, FRET may be assessed by measuring donor dequenching following the removal or bleaching of the acceptor (e.g. acceptor photobleaching). In addition to these methods FRET may also be measured by anisotropy imaging like for example in the case of Homo-FRET, i.e. in assays wherein the donor and acceptor are of the same fluorophore. While these aforementioned methods generally readout pluralities of donor and acceptor dye molecules, methods to measure single molecule FRET have likewise been developed. A simple method to measure FRET is ratiometric imaging of donor and acceptor intensity (sensitized emission), which can be performed on simple channel-based readouts like widefield microscopes, cytometers or plate readers. While measuring FRET efficiency or FRET signal in this way by measuring the donor and acceptor emission is simple, it is connected to the challenge of donor cross-talk into the acceptor emission channel, which may also be named the FRET channel. This is why more sophisticated methods like measurement of the donor dequenching were developed. Alternatively, spectral imaging offers a good solution to the cross-talk problem in FRET measurements, but suitable instrumentation may not always be available.

[0032] In tissue sections, however, it is difficult to perform FRET measurements, because of the high background autofluorescence. While FRET has been performed in tissue sections before, it is known in the field and also evident from the published data that FRET-based assays in tissue sections are limited by poor signal-to-noise or signal-to- background, because of the high background autofluorescence that is commonly found in tissue samples. Reference is made to Konig P, Krasteva G, Tag C, Konig IR, Arens C, Kummer W., FRET-CLSM and double-labeling indirect immunofluorescence to detect close association of proteins in tissue sections, Lab Invest. 2006 Aug;86(8):853-64. doi: 10.1038 / labinvest.3700443. Epub 2006 Jun 19. PMID: 16783395. The present invention, therefore, proposes an assay format that combines bright labelling for easy detection with high specificity in a way that is compatible with a cyclic staining process for high plex analysis of with improved specificity, for high plex analysis of molecular interactions and / or posttranslational modifications. The present invention is suited for any kind of analyte and AR.

[0033] Summary

[0034] It is an object to provide an assay format that combines bright labelling for easy detection with high specificity in a way that is in particular compatible with a cyclic staining process for high plex analysis with improved specificity, for high plex analysis of molecular interactions and / or posttranslational modifications. The present invention is suited for any kind of analyte and AR.

[0035] The aforementioned object is achieved by the method according to claim 1. Advantageous embodiments are defined in the dependent claims and the following description.

[0036] A method for detecting analytes in a biological sample comprises following steps: a. contacting the biological sample with at least a first marker and at least a second marker, wherein the at least first marker comprises a first affinity reagent configured to bind to a first analyte and a first nucleic acid label and wherein the at least second marker comprises a second affinity reagent configured to bind to a second analyte and a second nucleic acid label; b. incubating the biological sample with said first marker and said second marker, in particular for a sufficient amount of time, to allow the at least first affinity reagent to bind to the first analyte and to allow the at least second affinity reagent to bind to the second analyte; and c. adding at least one bridging strand configured to bind to the first nucleic acid label and the second nucleic acid label. Contacting the nucleic acid labels with the bridging strands enables determining whether or not the markers, in particular the nucleic acid labels, are in proximity to each other. Since the markers are bound to their respective analytes, the method enables determining whether or not these analytes are in proximity to each other.

[0037] Preferably, an unbound first marker and an unbound second marker are removed before the bridging strand is added. Thus, specificity can be improved, in particular because unbound markers are e.g. washed away and therefore do not contribute to a detection step.

[0038] Preferably, at least one first measurement, readout, or image of the biological sample is acquired following step b and prior to step c and at least one second measurement, readout, or image is acquired. In this way the FRET efficiency or FRET signal or FRET intensity can be assessed before and after the application of the bridging strands.

[0039] According to a preferred embodiment, each of the first nucleic acid label and the second nucleic acid label comprises an oligonucleotide backbone.

[0040] Preferably, each of the first nucleic acid label and the second nucleic acid label comprises at least one dye which is covalently conjugated to the oligonucleotide backbone. As an alternative to such a direct binding, predetermined nucleotide sequences in form of barcodes / attachment sites may be provided which enable the dyes to be indirectly connected to the oligonucleotide backbone.

[0041] Preferably, each of the first nucleic acid label and the second nucleic acid label comprises at least one dye which is bound to the oligonucleotide backbone by a reporter probe.

[0042] Preferably, the one of the first nucleic acid label and the second nucleic acid label comprises at least one FRET donor dye and the other of the first nucleic acid label and the second the nucleic acid label comprises at least one FRET acceptor dye. This enables determining whether or not the labels are in proximity with a spatial accuracy based on Forster distance of the FRET pair.

[0043] Preferably, one of the first nucleic acid label and the second nucleic acid label comprises a fluorescence quencher and the other of the first nucleic acid label and the second nucleic acid label comprises a fluorescent dye. This enables whether or not the labels are in proximity based on the quenching of the dye by the quencher.

[0044] Preferably, one of the first nucleic acid label and the second nucleic acid label comprises a fluorescent protein or fluorescent protein fragment.

[0045] Preferably, one of the first nucleic acid label and the second nucleic acid label comprises a first reactant group and the other of the first nucleic acid label and the second nucleic acid label comprises a second reactant group, wherein the first reactant group and the second reactant group are configured to allow the formation of a fluorescent compound in a controllable manner. For example, the first and second reactant groups may be respective click chemistry groups such as azide, alkyne, DBCO, or other reactive moieties such as amino groups, or NHS groups.

[0046] Preferably, one of the first nucleic acid label and the second nucleic acid label comprises a first reactant group and a second reactant group configured to form a fluorescent dye following to an externally applied stimulus.

[0047] Preferably, one of the first nucleic acid label and the second nucleic acid label comprises a first reactant group and a second reactant group configured to form a fluorescent dye by a Cu(l) catalyzed azide- alkyne cycloaddition (CuAAC) following to the application of Cu(l). Preferably, one of the first nucleic acid label and the second nucleic acid label comprises a first reactant group and a second reactant group configured to form a fluorescent dye upon illumination with UV light.

[0048] Preferably, the first nucleic acid label and the second nucleic acid label are a multipartite nucleic acid-based structure.

[0049] Preferably, a plurality of bridging strands is used to spatially align the first nucleic acid label and the second nucleic acid label.

[0050] Preferably, the first nucleic acid label and the second nucleic acid label and a plurality of bridging strands are configured such that the addition of the bridging strands leads to the formation of a folded DNA structure, wherein FRET donor dye molecules and FRET acceptor dye molecules are aligned to each other in substantially a pairwise or otherwise regular arrangement configured to maximize FRET efficiency and / or the FRET signal.

[0051] Preferably, at least a part of the first nucleic acid label and the second nucleic acid label are amplified before reporter probes are bound to the amplika of said first nucleic acid label and second nucleic acid labels.

[0052] Preferably, the biological sample is analysed for diagnostic purposes.

[0053] Preferably, the first nucleic acid label and the second nucleic acid label are connected to the first affinity reagent and the second affinity reagent via hybridization.

[0054] Preferably, the first marker and the second marker are configured to bind to distinct, in particular different, epitopes on the same target protein. Preferably, the first marker and the second marker are configured to bind to distinct, in particular different, protein targets.

[0055] Preferably, the first marker and the second marker are configured to bind to distinct, in particular different, sequence targets on the same mRNA target.

[0056] Preferably, one of the first marker and the second marker is configured to bind to a nucleic acid target sequence and the other of the first marker and the second marker is configured to bind to a protein.

[0057] Preferably, the at least one FRET donor dye of one of the first nucleic acid and the second nucleic acid label is excited and the emission of the at least one FRET acceptor dye of the other one of the first nucleic acid and the second nucleic acid label is detected.

[0058] Preferably, a FRET efficiency is assessed by donor or acceptor photobleaching.

[0059] Preferably, the FRET between donor and acceptor emission intensity and / or lifetime is measured prior to and following to addition of the at least one bridging strand.

[0060] Preferably, the FRET between donor and acceptor emission intensity and / or lifetime is measured following to the addition of a removal agent that is configured to at least one of to remove, to unbind, to cleave, or to substantially degrade the at least one bridging strand.

[0061] Preferably, the bridging strands are configured such that they dynamically associate with the first nucleic acid label and the second nucleic acid label. In particular, dynamic association may be achieved by bridging strands that have only short complementarities, such as 9 nucleotide complementarity. This may lead to a blinking FRET. Preferably, the bridging strands comprises a guest-modified nucleotide or nucleobase, wherein the guest molecule is configured to allow complexation with a cucurbituril host molecule. This enables melting off the bridging strands by putting in cucurbituril, since the host-guest complex will invade the DNA duplex. Thus, this enables removing the bridging strands.

[0062] Preferably, the bridging strands comprise a backbone comprising L-DNA and D-DNA.

[0063] Preferably, one of the first nucleic acid label and the second nucleic acid label comprises L-DNA and the other one of the first nucleic acid label and the second nucleic acid label comprises D-DNA. This enables selective degradation of either the first nucleic acid label or the second nucleic acid label. This in turn enables measuring the dequenched donor emission in the absence of the acceptor dye.

[0064] Short Description of the Figures

[0065] Hereinafter, specific embodiments are described referring to the drawings, wherein:

[0066] Figure 1 Schematic of markers used in the method.

[0067] Figure 2 Schematic showing bridging strands connecting nucleic acid labels.

[0068] Figure 3 Schematic of the "molecular zipper" and FRET efficiency.

[0069] Figure 4 Schematic of the label removal.

[0070] Figure 5 Overview of applications of the method.

[0071] Figure 6 Schematic of the markers with nucleic acid labels.

[0072] Figure 7 Schematic of the markers with nucleic acid labels and bridging strands.

[0073] Figure s Schematic of the markers with nucleic acid labels and bridging strands and dye-conjugated oligonucleotides. Figure 9 Schematic of the markers with a dendrimeric structure.

[0074] Figure 10 Schematic of the markers used to detect nucleic acid-protein interactions.

[0075] Figure 11 Schematic of nucleic acid-protein interaction detection.

[0076] Figure 12 Schematic of the markers used to detect nucleic acid sequences.

[0077] Figure 13 Schematic of nucleic acid target detection.

[0078] Figure 14 Schematic of amplification.

[0079] Figure 15 Schematic showing bridging strands binding to different predetermined subsequences.

[0080] Detailed Description

[0081] Figure 1 shows schematically a first marker 100a and a second marker 100b. The first marker 100a comprises a first affinity reagent 108a and a first nucleic acid label 104a. The second marker 100b comprises a second affinity reagent 108b and a second nucleic acid label 104b.

[0082] The first affinity reagent 108a is configured to specifically bind to a first target analyte 102a of a biological sample (not shown in Figure 1). The second affinity reagent 108b is configured to specifically bind to a second target analyte 102b of the biological sample. The affinity reagents 108a, 108b are antibodies, for example. Alternatively, the affinity reagents 108a, 108b may be an antibody fragment or an aptamer. The target analytes 102a, 102b may be different proteins of the biological sample, for example.

[0083] In particular, the target analytes 102a, 102b may interact with each other, such that they are clustered together in close proximity to each other, as shown in Fig. 1.

[0084] The first nucleic acid label 104a comprises a plurality of first dyes 106a. The second nucleic acid label 104b comprises a plurality of second dyes 106b. The first and second dyes 106a, 106b are directly or indirectly connected to the respective nucleic acid label 104a, 104b. Each of the first and second dyes 106a, 106b is preferably configured to form a Forster / fluorescence resonance energy transfer (FRET) pair with the respective other one of the first and second dyes 106a, 106b. The first and second nucleic acid labels 104a, 104b may each comprise an oligonucleotide backbone, to which the respective dyes 106a, 106d are connected. The oligonucleotide backbones may comprise 100 to 10000 nucleotides, preferably 100 to 500 nucleotides or 1000 to 10000 nucleotides.

[0085] The oligonucleotide backbone of the first nucleic acid label 104a and the oligonucleotide backbone of the second nucleic acid label 104b essentially do not have sequences complementary to each other. Thus, the first nucleic acid label 104a and the second nucleic acid label 104b do not directly hybridise with each other.

[0086] The dyes 106a, 106b may either be directly (covalently) connected to the respective oligonucleotide backbone (the first nucleic acid label 104a and the second nucleic acid label 104b) or the dyes 106a, 106b may each comprise an oligonucleotide barcode, which may hybridise to a complementary sequence of the respective oligonucleotide barcode (the first nucleic acid label 104a and the second nucleic acid label 104b).

[0087] In Figure 1, the markers 100a, 100b have been introduced into the biological sample comprising the target analytes 102a, 102b and allowed to bind to their respective target 102a, 102b. This may be an initial step of a method for detecting analytes in a biological sample. Since the target analytes 102a, 102b are in close proximity to each other, the affinity reagents 108a, 108b, and the respective markers 100a, 100b, bound to the target analytes 102a, 102b are similarly in close proximity to each other.

[0088] The first and second nucleic acid labels 104a, 104b, in particular their respective dyes 106a, 106b and their respective oligonucleotide backbones, do not bind to each other. The distance between the labels 104a, 104b is therefore generally dictated by the distance between the target analytes 102a, 102b the markers 100a, 100b are bound to. In particular, on average, the distance between the labels 104a, 104b can be estimated to correlate to the distance (15 nm in Fig. 1) between the affinity reagents 108a, 108b bound to the target analytes 102a, 102b. In addition, the distance between the labels 104a, 104b varies over time due to random motion of each individual label 104a, 104b, despite the respective marker 100a, 100b being bound to the target analytes 102a, 102b. Thus, on average over time, the labels 104a, 104b, in particular their respective dyes 106a, 106b, are at a distance at which FRET efficiency between all first dyes 106a and second dyes 106b collectively is low (in particular when no bridging strands are present). Thus, on average, the dyes 106a, 106b do not form FRET pairs and the optical properties of both dyes 106a, 106b, such as excitation and emission characteristics, are not influenced by each other and may be detected individually, for example, by means of a fluorescence microscope. Thus, the markers 100a, 100b may be readout in a readout step, in particular, to determine emission characteristics of the dyes 106a, 106b. Based on this, a FRET efficiency for the dyes 106, 106b may be determined. For example, the distance between the labels 104a, 104b may be composed of the distance between the affinity reagents 108a, 108b (15 nm in Fig. 1) and the distance of the binding sites of the affinity reagents 108a, 108b on the target analytes 102a, 102b (X nm in Fig. 1). The exemplary 15 nm distance may be reduced by using affinity reagents 108a, 108b that are smaller than antibodies, for example, antibody fragments.

[0089] In other words, as the labels 104a, 106a are "freely moving", the FRET efficiency, FRET signal, i.e. the intensity of the acceptor emission, of all first dyes 106a and second dyes 106b collectively is low, as only some of them are within FRET distance at any given time, even if the target analytes 102a, 102b are in close proximity. In this context "freely moving" shall in particular be understood in the sense that the nucleic acid backbones of the labels 104a, 106a is preferably a single stranded DNA or XNA oligonucleotide. Such oligonucleotides are known to be very flexible. For this reason it can be expected that at any point in time only some of the first dyes 106a and the second dyes 106b are in close proximity. The FRET signal or FRET efficiency may be measured in various ways known to someone skilled in the art including but not limited to sensitized emission, acceptor photobleaching (also referred to as donor dequenching), ratiometric FRET, fluorescence lifetime (e.g. by measuring the shortening of the lifetime of the FRET donor). The method described in this document can be used with microscopes, plate readers and cytometers for example. Ideally the FRET signal or efficiency is measured using a spectral imaging device in a ratiometric fashion this is sometimes referred to as lambda FRET.

[0090] Figure 2 schematically shows a further step of the method for detecting analytes in a biological sample, preferably following the initial step shown in Fig. 1. In this further step bridging strands 200 are contacted with the first marker 100a and the second marker 100b. The bridging strands 200 each comprise at least a first sequence that is complementary to a part of the oligonucleotide backbone of the first nucleic acid label 104a and a second sequence that is complementary to a part of the oligonucleotide backbone of the second nucleic acid label 104b. Preferably, a plurality of bridging strands is contacted with the markers 100a, 100b. Each bridging strand of the plurality of the bridging strands may comprise first and second sequences complementary to either the first or second nucleic acid labels 104a, 104b and that differ from other bridging strands. In particular, each subsequent bridging strand of the plurality of the bridging strands may have a first sequence that is complementary to a complementary sequence of the first nucleic acid label 104a that is adjacent in a particular direction to a complementary sequence that a previous bridging strand's first sequence is complementary to. Similarly, that subsequent bridging strand may have a second sequence that is complementary to a complementary sequence of the second nucleic acid label 104b that is adjacent in a particular direction to a complementary sequence that the previous bridging strand's first sequence is complementary to. The bridging strands 200 may be configured similar to a zipper, in that they enable a continuous bringing into proximity of the first nucleic acid label 104a with the second nucleic acid label 104b along their lengths as subsequent bridging strands 200 hybridise to their respective complementary sequences on the labels 104a, 104b.

[0091] The degree to which the labels 104a, 104b are brought into proximity along their lengths is proportional to the degree of interaction between the first dyes 106a and the second dyes 106b. In particular, when the first and the second labels 104a, 104b are brought into proximity by means of the bridging strands 200, the first dyes 106a and the second dyes 106b may form FRET pairs. Thus, the bringing into proximity of the first and second labels 104a, 104b may be determined by an increase in the FRET efficiency or FRET signal.

[0092] The target analytes 102a, 102b, as shown in Figs. 1 and 2, are in close proximity to each other. Thus, the labels 104a, 104b of the markers 100a, 100b bound to the target analytes 102, 102b are in similarly close proximity to each other and due to the binding of the labels 104a, 104b to each other by means of the bridging strands 200, the FRET efficiency of the dyes 106a, 106b is high, in particular higher than in the case where the bridging strands 200 are not present as described for Fig. 1.

[0093] In an alternative, one of the first dye 106a and the second dye 106b may be a fluorescent dye and the other one of the first dye 106a and the second dye 106b may be a quencher molecule. Thus in this case, when any one of the first dyes 106a is brought into proximity of one of the second dyes 106b, a fluorescent emission of the fluorescent dye may be quenched by the quencher molecule. This quenching enables determining, if the first label 104a and the second label 104b are in close proximity to each other, as well as the degree of proximity.

[0094] Preferably, the readout is performed by acquiring two measurements or two images one before application of the bridging strands and one after. This may be especially important, when the assay is based on quenching as described in the previous paragraph. But also, when the assay is based on an increase in FRET efficiency, it is generally advisable to measure FRET efficiency by one of the methods cited above before and after application of the bridging strands.

[0095] Similar assays, such as Proximity Ligation and Proximity Extensions assays are known. The present solution is distinct from these known methods, in particular, in that the nucleic acid labels 104a, 104b are neither ligated nor extended. Rather, the nucleic acid labels 104a, 104b are zipped like a zipper along the length of their respective oligonucleotide sequences by application of bridging strands 200 configured to bind to predetermined attachment sites on both of the oligonucleotides the labels 104a, 104b. The degree of zipping of this molecular zipper can be readout by using excitonic dyes such as a dye-quencher pair or a FRET-pair. The benefit of the present invention compared to such assays as the Proximity Ligation and Proximity Extensions Assays in particular is that it requires no enzymes, which improves cost efficiency in production and shipping, time-to-result is shorter and a signal can be measured that is dependent on the distance of target analytes. In the other cases it is more of binary ligation or not, amplicon or no amplicon result.

[0096] As schematically shown in Fig. 2 following the addition of the bridging strands 200 the first nucleic acid label 104a and the second nucleic acid label 104b are indirectly connected to each other via the bridging strands 200 and the first dye 106a and the second dye 106b are brought into a proximity which is close enough to achieve a high FRET efficiency. The proximity or distance is typically below lOnm, in particular, in a range between 1 to 8 nm.

[0097] Fig. 3 shows schematically an alternative spatial arrangement of the target analytes 102a, 102b relative to each other. In this case, the target analytes 102, 102b are separate from each other and at a distance from each other, in particular, at a distance from each other that is greater than the distance according to Figs. 1 and 2. In particular, this is in comparison to Figs. 1 and 2, in which the target analytes 102a, 102b are in close proximity to each other, for example due to binding to each other or interacting with each other.

[0098] Due to the distance between the target analytes 102a, 102b as shown in Fig. 3, the markers 100a, 100b are at a similar distance to each other, in particular, at an increased distance compared to Figs. 1 and 2. This causes the labels 104a, 104b to be only partially bound to each other, despite the presence of the bridging strands 200. Thus, the FRET efficiency is reduced in comparison to the case where the target analytes 102a, 102b are in close proximity, as shown in Fig. 2.

[0099] In particular, the spatial stringency of the method described with reference to Figs. 1 to 3 depends largely on the size of the affinity reagents 108a, 108b. For an antibody pair spatial stringency would be in the range of a 40nm radius, i.e. if the targets proteins are in that radius, the antibodies may bring the conjugation sites of the first and second nucleic acid labels 104a, 104b in close proximity. Conjugation sites are the sites on the affinity reagents to which the nucleic acid labels are conjugated. The FRET efficiency resulting from the proximity between the labels 104a, 104b is not a binary ON / OFF, but depends on the degree of proximity between the labels 104a, 104b or zipping. This is a key aspect, as this means that the proximity of the target analytes 102a, 102b determines the strength of FRET, in particular the strength of the measured FRET signal. In this sense, it is particularly preferred to use small affinity reagents 108a, 108b that are, for example, aptamers, antibody fragments or nanobodies with the methods described in this document. An alternative to FRET between the dyes 106a, 106b would be quenching, this may be named an inverted readout. In this case the first and second dye 106a, 106b may be the same dye. Alternatively, the second dye may be a quencher. In the case of such an inverted readout, the strength of quenching following to the addition of the bridging strands, would be taken as a measure for the interaction between the dyes of the first and second label. In this case, preferably, the readout is performed by acquiring two measurements or two images, one before application of the bridging strands and one after.

[0100] Figure 4 shows schematically a further step of the method for detecting analytes, in particular after a preceding measuring step (of at least one of the situations as shown in Fig. 1 to 3) has been performed. In this step, the first and second labels 104a, 104b are inactivated, in particular removed from the affinity reagents 108a, 108b of the markers 100a, 100b. For example, the nucleic acid labels 104a, 104b may be digested by means of an enzyme 400 such as DNase I. Specifically, this may digest the oligonucleotide backbones of the labels 104a, 104b. Thus, the labels 104a, 104b are removed from the affinity reagents 108a, 108b, as shown in the top view of Fig. 4. The removal of the labels 104a, 104b causes removal of the dyes 106a, 106b and therefore removal of a corresponding detectable optical signal. A washing step might need to be applied to remove the labels 104a, 104b and / or the dyes 106a, 106b which are no longer connected to the affinity reagents 108a, 108b.

[0101] Alternatively, the bridging strands 200 may be digested by means of the enzyme 400. This causes the unbinding of the labels 104a, 104b, as shown in the bottom view of Fig. 4.

[0102] Alternative approaches to label inactivation include UV-cleavage and PT-DNA / iodine cleavage.

[0103] The labels 104a, 104b and / or the bridging strands 200 may be selectively degraded, for example, by using selectively degradable nucleic acid compositions comprising D- DNA and L-DNA.

[0104] Examples of suitable inactivation methods are disclosed e.g. in the European patent applications with the application numbers 23205852.9, 23178065.1, and 23169125.4, the complete contents thereof are incorporated herein by reference.

[0105] The inactivation of the markers 100a, 100b, for example as described above, are particularly suitable when used in combination with cyclical staining methods. In this case, the markers 100a, 100b comprising the affinity reagents 108a, 108b may initially be contacted with the target analytes 102a, 102b and an optical readout of the target analytes 102a, 102b with the markers 100a, 100b may be generated. Subsequently, the markers 100a, 100b, in particular the labels 104a, 104b, may be inactivated or removed and a further set of markers or labels may be used to stain or mark the target analytes 102a, 102b in a further round of imaging.

[0106] In a further example, a plurality of first and second markers may be used, wherein multiple subsets of first and second markers comprise first and second nucleic acid labels, which may be inactivated by a first, second, and third restriction enzyme to allow interrogation subsets in a cyclic process. This allows to analyse a large number of first and second marker pairs, to, for example, probe a large number of proteinprotein interactions or to probe a large number of proteins and posttranslational modifications to analyse entire signalling cascades for example.

[0107] In this case the nucleic acid label of the acceptor may be cleaved off and another readout may be performed, analogous to acceptor photobleaching, which enables better analysis of FRET eff. This may be named acceptor removal FRET.

[0108] Figure 5 shows schematically further examples of the method for detecting analytes in a biological sample. In the top panel of Fig. 5, the first and second affinity reagents 108a, 108b of the markers 100a, 100b are bound to the respective first and second target analytes 102a, 102b, as described above. This enables assaying the proximity or interaction between the target analytes 102a, 102b.

[0109] For improved clarity, in Fig. 5 no labels are shown, only the affinity reagents of respective markers.

[0110] In the second from top panel of Fig. 5, both the first and second affinity reagents 108a, 108b are, in an alternative, specifically bound to the same target analyte 500. Specifically, the first affinity reagent 108a is bound to a first area 502a or epitope of the target analyte 500 and the second affinity reagent 108b is bound to a second area 502b or epitope of the target analyte 500. This enables a proteomic assay, a bulk multiplex immunoassay, a Fluorospot assay, or a posttranslational modifications assay with improved specificity due to the requirement of two affinity reagents 108a, 108b binding to the target analyte 500.

[0111] In the second from bottom panel of Fig. 5, the affinity reagent 108b is specific to the target analyte 102b, as described above. A nucleic acid affinity reagent 504 is specific to a nucleic acid target analyte 506. Thus, the nucleic acid affinity reagent 504 binds specifically to the nucleic acid target analyte 506, for example by means of a complementary nucleic acid sequence. In turn, the target analyte 102b may similarly bind (specifically) to the nucleic acid target analyte 506, for example by intermolecular forces such as hydrogen bonding, dipole-dipole interactions, and van der Waals forces. This enables assaying the protein-nucleic acid interaction between the target analyte 102b and the nucleic acid target analyte 506.

[0112] In the bottom panel of Fig. 5, the nucleic acid affinity reagent 504 is bound to the nucleic acid target analyte 506. A second nucleic acid affinity reagent 508 may similarly be specifically bind to the nucleic acid target analyte 506. This enables assaying the nucleic acid target analyte 506 with improved specificity.

[0113] The affinity reagents 504, 508 may comprise natural or non-natural nucleic acids (e.g. DNA, LNA, morpholino, PT-DNA, PNA).

[0114] Figures 6 to 8 schematically show steps of a method for detecting analytes in a biological sample. Fig. 6 shows schematically step 1: The affinity reagents 108a, 108b are introduced to the sample and allowed to bind to their targets 102a, 102b, each affinity reagent 108a, 108b is connected to a oligonucleotide backbone 600a, 600b of the labels 104a, 104b

[0115] Fig. 7 shows schematically step 2: The bridging strands 200 are contacted with the oligonucleotide backbones 600a, 600b in order to bind the backbones 600a, 600b together.

[0116] Fig. 8 shows schematically step 3: At least one of the dye 106a and one of the dye 106b are contacted with the oligonucleotide backbones 600a, 600b in order to attach the dyes 106a, 106b to the respective backbones 600a, 600b. The dyes 106a, 106b may be conjugated to respective oligonucleotides 800a, 800b that are complementary to sequences of the oligonucleotide backbones 600a, 600b of the labels 104a, 104b in order to attach the dyes 106a, 106b to either the backbone 600a or the backbone 600b. This generates the labels 104a, 104b. The dyes 106a, 106b are configured to excitonically interact (e.g. FRET, quench). This may alternatively be performed before, during, or after step 2. The oligonucleotides 800a, 800b are examples of reporter probes, by which the dyes 106a, 106b may be bound to the respective backbones 600a, 600b.

[0117] Figure 9 schematically shows an alternative to step 3 of Fig. 8. In this case, a plurality of dyes 106a, 106b, in particular eleven dyes 106a, 106b, are attached, in particular covalently, to each of the oligonucleotides 800a, 800b. The oligonucleotides 800a, 800b may be bound to each other in pairs by secondary bridging strands 900. To that end, the secondary bridging strands 900 may comprise a first sequence complementary to a part of the oligonucleotide 800a and a second sequence complementary to a part of the oligonucleotide 800b. This enables generating bright labels. In particular, the labels shown in Fig. 9 with the oligonucleotides 800a, 800b are examples of multipartite structures. Figures 10 and 11 schematically show how the method for detecting analytes in a sample may be used to target a RNA-protein, or DNA-protein interaction. To that end two markers 11007100b are contacted with the target analytes 506, 102b.

[0118] Figures 12 and 13 schematically show how the method may be used to improve the specificity of fluorescent in situ hybridization (ISH). In this case, two markers 1300a, 1300b are contacted with the target analyte 506. An optical signal may only be detected, when both markers 1300a, 1300b are bound to the target nucleic acid 506 (mRNA or target DNA). This embodiment of the method may also be used to study the 3-dimensional architecture of the genome in the nucleus. It is known that the accessibility and 3- dimensional organization of the genome is tightly regulated. The methods disclosed in this document enable studying the spatial arrangement of multiple loci in the nuclei of large numbers of cells in tissue sections, which will allow correlating differences in 3D genome architecture to different cell types and cell states. This may be particularly important in the context of stratifying patient populations as the genome architecture of, for example tumour cells, can be expected to be substantially different from (A) healthy cells and (B) to contain valuable information for patient monitoring and stratification. Likewise, the method lends itself well to studying gene integrations, which is of key importance for the development, quality control of cell and gene therapy products as well as for the monitoring of cell and gene therapy patients. In the context of cell therapy development and genetic modification by (A) integrating genetic modifications or (B) cutting out parts of the genome this method may be used to identify clones that have desired modifications in desired locations and / or to avoid selecting clones that have too few, too many, or ill-positioned integrations. In this case one of the markers may comprise a degenerate primer capable of binding to multiple locations in the genome. Figure 14 schematically shows an alternative to introducing markers with longer oligonucleotide labels into samples. It may be preferable, in some applications to use shorter oligonucleotide labels and expand them by rolling circle amplification 1400, or by loop-mediated amplification (LAMP) for example. This may lead to very strong amplification, which may be desired for low abundance targets.

[0119] Figure 15 schematically shows bridging strands that each may target a pair of corresponding positions (numbered from one to six in Fig. 15) on the first and second nucleic acid label 104a, 104b. A first set of bridging strands may lead to a first alignment in a first round, may be removed and then a second set of bridging strands may lead to a second alignment in a second round. The excitation / emission characteristics of dyes in the first spatial alignment and in the second spatial alignment may differ, as e.g. disclosed in the European patent application with the application number 23192184.2, the complete content thereof is incorporated herein by reference. Preferably, the sets of bridging strands may align inter-label or intralabel positions of dyes.

[0120] Identical or similarly acting elements are designated with the same reference signs in all Figures. As used herein the term "and / or" includes any and all combinations of one or more of the associated listed items and may be abbreviated as

[0121] Although some aspects have been described in the context of an apparatus, it is clear that these aspects also represent a description of the corresponding method, where a block or device corresponds to a method step or a feature of a method step. Analogously, aspects described in the context of a method step also represent a description of a corresponding block or item or feature of a corresponding apparatus. Reference signs

[0122] 100a7100b, 1100, 1300a, 1300b Marker

[0123] 102a, 102b Target analyte

[0124] 104a, 104b Nucleic acid label

[0125] 106a, 106b Dye

[0126] 108a, 108b, 504, 508 Affinity reagent

[0127] 200 Bridging strand

[0128] 300 Molecular zipper

[0129] 400 Enzyme

[0130] 500 Target analyte with epitopes

[0131] 502a, 502b Epitope

[0132] 506 Nucleic acid target analyte

[0133] 600a, 600b Oligonucleotide backbone of label

[0134] 800a, 800b Oligonucleotide of dye

[0135] 900 Secondary bridging strand

Claims

Claims1. A method for detecting analytes in a biological sample, comprising following steps: a. contacting the biological sample with at least a first marker (100a) and at least a second marker (100b), wherein the at least first marker comprises a first affinity reagent (108a) configured to bind to a first analyte (102a) and a first nucleic acid label (104a), and wherein the at least second marker comprises a second affinity reagent (108b) configured to bind to a second analyte (102b) and a second nucleic acid label (104b), b. incubating the biological sample with said first marker and said second marker, in particular for a sufficient amount of time, to allow the at least first affinity reagent (108a) to bind to the first analyte (102a) and to allow the at least second affinity reagent (108b) to bind to the second analyte (102b), and c. adding at least one bridging strand (200) configured to bind to the first nucleic acid label (104a) and the second nucleic acid label (104b).

2. The method according to claim 1, wherein an unbound first marker and an unbound second marker is removed before the at least one bridging strand (200) is added.

3. The method according to one of the preceding claims, wherein each of the first nucleic acid label (104a) and the second nucleic acid label (104b) comprises an oligonucleotide backbone.

4. The method according to claim 3, wherein each of the first nucleic acid label (104a) and the second nucleic acid label (104b) comprises at least one dye (106a, 106b) which is covalently conjugated to the respective oligonucleotide backbone (600a, 600b).

5. The method according to claim 3, wherein each of the first nucleic acid label (104a) and the second nucleic acid label (106b) comprises at least one dye (106a, 106b) which is bound to the respective oligonucleotide backbone by a reporter probe .

6. The method according to one of the preceding claims, wherein one of the first nucleic acid label (104a) and the second nucleic acid label (104b) comprises at least one FRET donor dye (106a) and the other comprises at least one FRET acceptor dye (106b).

7. The method according to one of the preceding claims, wherein one of the first nucleic acid label (104a) and the second nucleic acid label (104b) comprises a fluorescence quencher and the other comprises a fluorescent dye.

8. The method according to one of the preceding claims, wherein one of the first nucleic acid label (104a) and the second nucleic acid label (106b) comprises a fluorescent protein or fluorescent protein fragment.

9. The method according to one of the preceding claims, wherein one of the first nucleic acid label (104a) and the second nucleic acid label (104b) comprises a first reactant group and the other of the first nucleic acid label (104a) and the second nucleic acid label (104b) comprises a second reactant group, wherein the first reactant group and the second reactant group are configured to allow the formation of a fluorescent compound in a controllable manner.

10. The method according to one of the preceding claims, wherein one of the first nucleic acid label (104a) and the second nucleic acid label (104b) comprises a first reactant group and the other of the first nucleic acid label (104a) and thesecond nucleic acid label (104b) comprises a second reactant group configured to form a fluorescent dye following to an externally applied stimulus.

11. The method according to one of the preceding claims, wherein one of the first nucleic acid label (104a) and the second nucleic acid label (104b) comprises a first reactant group and the other of the first nucleic acid label (104a) and the second nucleic acid label (104b) comprises a second reactant group configured to form a fluorescent dye by a Cu(l) catalyzed azide-alkyne cycloaddition (CuAAC) following to the application of Cu(l).

12. The method according to one of the preceding claims, wherein one of the first nucleic acid label (104a) and the second nucleic acid label (104b) comprises a first reactant group and the other of the first nucleic acid label (104a) and the second nucleic acid label (104b) comprises a second reactant group configured to form a fluorescent dye upon illumination with UV light.

13. The method according to one of the preceding claims, wherein the first nucleic acid label (104a) and the second nucleic acid label (106b) are a multipartite nucleic acid-based structure.

14. The method according to one of the preceding claims, wherein a plurality of bridging strands (200) is used to spatially align the first nucleic acid label (104a) and the second nucleic acid label (104b).

15. The method according to one of the preceding claims, wherein the first nucleic acid label (104a) and the second nucleic acid label (104b) and a plurality of bridging strands (200) are configured such that the addition of the bridging strands (200) leads to the formation of a folded DNA structure, wherein FRET donor dye molecules and FRET acceptor dye molecules are aligned to eachother in substantially a pairwise or otherwise regular arrangement configured to maximize FRET efficiency.

16. The method according to one of the preceding claims, wherein at least a part of the first nucleic acid label (104a) and the second nucleic acid label (104b) are amplified before reporter probes are bound to the amplika of said first nucleic acid label and second nucleic acid label.

17. The method according to one of the preceding claims, wherein the biological sample is analysed for diagnostic purposes.

18. The method according to one of the preceding claims, wherein the first nucleic acid label (104a) and the second nucleic acid label (104b) are connected to the respective first affinity reagent (108a) and second affinity reagent (108b) via hybridization.

19. The method according to one of the preceding claims, wherein the first affinity reagent (108a) and the second affinity reagent (108b) are configured to bind to distinct epitopes (502a, 502b) on the same target protein (500).

20. The method according to one of the preceding claims 1 to 18, wherein the first affinity reagent (108a) and the second affinity reagent (108b) are configured to bind to distinct protein targets (102a, 102b).

21. The method according to one of the preceding claims 1 to 18, wherein the first affinity reagent (108a) and the second affinity reagent (108b) are configured to bind to distinct sequence targets on the same mRNA target (506).

22. The method according to one of the preceding claims 1 to 18, wherein one of the first affinity reagent (108a) and the second affinity reagent (108b) isconfigured to bind to a nucleic acid target sequence and the other is configured to bind to a protein.

23. The method according to one of the preceding claims, wherein the at least one FRET donor dye of one of the first nucleic acid label (104a) and the second nucleic acid label (104b) is excited and the emission of the at least one FRET acceptor dye of the other one of the first nucleic acid label (104a) and the second nucleic acid label (104b) is detected.

24. The method according to one of the preceding claims, wherein at least one of a FRET efficiency, a FRET signal, a FRET intensity between donor and acceptor, a fluorescence intensity of the donor (lD), a fluorescence intensity of the acceptor (lA), a fluorescence lifetime of the donor and a fluorescence lifetime of the acceptor is assessed by donor or acceptor photobleaching.

25. The method according to one of the preceding claims, wherein at least one of a FRET signal, a FRET intensity between donor and acceptor, a fluorescence intensity of the donor (lD), a fluorescence intensity of the acceptor (lA), a the fluorescence lifetime of the donor and a fluorescence lifetime of the acceptor is measured prior to and following to addition of the at least one bridging strand (200).

26. The method according to one of the preceding claims, wherein at least one of a FRET signal, a FRET intensity between donor and acceptor, a fluorescence intensity of the donor (lD), a fluorescence intensity of the acceptor (lA), a fluorescence lifetime of the donor and the fluorescence lifetime of the acceptor is measured following to the addition of a removal agent that is configured to remove, to unbind, to cleave, or to substantially degrade the at least one bridging strand (200).

27. The method according to one of the preceding claims, wherein the bridging strands (200) are configured such that they dynamically associate or associate in a manner controllable by an external cue with the first nucleic acid label (104a) and the second nucleic acid label (104b).

28. The method according to one of the preceding claims, wherein the bridging strands (200) comprises a guest-modified nucleotide or nucleobase, wherein the guest molecule is configured to allow complexation with a cucurbituril host molecule.

29. The method according to one of the preceding claims, wherein the bridging strands (200) comprise a backbone comprising L-DNA and D-DNA.

30. The method according to one of the preceding claims, wherein one of the first nucleic acid label (104a) and the second nucleic acid label (104b) comprises L- DNA and the other one comprises D-DNA.

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