Markers, markers and methods for analyzing biological samples

Abstract

In a first aspect, a marker for analyzing a biological sample is provided. The marker comprises a first marker moiety comprising a first nucleic acid backbone (100, 206) and a second marker moiety comprising a second nucleic acid backbone (102, 210). The first nucleic acid backbone (100, 206) and the second nucleic acid backbone (102, 210) are each configured to hybridize to the respective other. The marker further comprises at least one first tagged portion (208) and at least one second tagged portion (212). The marker further comprises at least one guest molecule (104) configured to form a complex with the host molecule (106). In other aspects, corresponding markers (200, 500, 600) and methods for analyzing a biological sample are provided.

Description

TECHNICAL FIELD

[0001] The present disclosure relates to a label and a corresponding labeler. In another aspect, a method for analyzing a biological sample is provided. BACKGROUND

[0002] Labels and labelers are often used when analyzing biological samples, for example in fluorescence microscopy. Labelers for fluorescence microscopy typically comprise an affinity reagent (AR), such as an antibody, a nanobody, an aptamer, an affimer, a polymeric binder, a toxin, and an oligonucleotide probe (e.g. a FISH probe), and an (detectable) label attached to the affinity reagent label, in order to enable the label (in particular by the affinity reagent) to specifically attach to a target analyte in the biological sample.

[0003] Detection in affinity reagent-based assays is typically performed by labeling the analyte with a labeler, which typically comprises at least one affinity reagent and a (detectable) label, which can be for example an enzyme, a metal tag, a Raman label, a fluorescent label, or a hybridization label. A fluorescent label can include for example a fluorescent dye, a fluorophore, a fluorochrome, a dye, a QDot, a PDot, or a polymeric dye. The detectable label can in particular be an optically detectable label.

[0004] Affinity reagents are used in life sciences to bind to targets or analytes with high affinity and specificity. Various assays on liquid or solid samples rely on detection reagents, which include affinity reagents and detectable labels, which can include nucleic acids (e.g. oligonucleotides), (fluorescent) dyes, enzymes, metal tags, radioactive tags, or affinity tags (e.g. HA-tag, Myc-tag, FLAG-tag). Aptamers, antibodies, and nanobodies all have dissociation constants (KD) in the range of pM to mM and are used in various affinity reagent-based assays or immunoassays, which are the backbone of life science research.

[0005] Various applications of labelers include multiplexing methods, which require a large number of distinguishable labelers, while at the same time, detection of only a small number of present analytes often requires labelers with bright labeling. In particular, when handling a large number of labelers, which can be assembled from individual components such as dyes and affinity reagents, it is of interest to reduce the complexity of the handling while maintaining flexibility and reliability of use. SUMMARY

[0006] It is an object to provide a label that enables efficient handling when analyzing a biological sample.

[0007] The above objects are achieved by the subject matter of the independent claims. Advantageous embodiments are set out in the dependent claims and the following description.

[0008] In the present context, a sample refers to a biological sample or specimen, including, for example, blood, serum, plasma, tissue, body fluids (e.g. lymph, saliva, semen, interstitial fluid, cerebrospinal fluid), fecal matter, solid biopsies, liquid biopsies, explants, cells (e.g. prokaryotic cells, eukaryotic cells, archaeal cells), suspended cell cultures, monolayer cell cultures, 3D cell cultures (e.g. spheroids, tumoroids, organoids derived from various organs such as intestine, brain, heart, liver, etc.), lysates of any of the above, viruses. In the present context, a sample further refers to the volume surrounding a biological sample. For example, in assays investigating secreted proteins such as growth factors, extracellular matrix components, the extracellular environment surrounding the cells is also referred to as sample, which is the volume surrounding the cells up to a certain assay-dependent distance. In particular, in the present context, affinity reagents introduced into said surrounding volume are referred to as introduced into the sample. For the present disclosure, particularly relevant sample types are formalin-fixed paraffin-embedded tissue sections or tissue microarrays as well as tissue cryosections.

[0009] This allows for the identification and / or localization of target analytes in a biological sample. In the present context, an affinity reagent can be, for example, an antibody, an antibody fragment, a nanobody, an aptamer, an aptabody, a polymer binder, an affimer, an oligonucleotide probe (e.g. ISH or FISH probe), a drug / drug-like molecule or a toxin. An affinity reagent (AR) can also be a derivative of the above-mentioned affinity reagents. Affinity reagents are typically configured to bind to a target analyte or target with high specificity and affinity. However, it is known that many antibodies, forming the largest class of commercially available affinity reagents binding proteins, exhibit considerable cross-reactivity. In other words, many antibodies bind not only to their cognate target analyte, but also to many OFF-targets, targets unrelated to the intended investigation. Both the cognate target and the OFF-targets against which the affinity reagent is produced or cultivated can be collectively referred to as targets.

[0010] Another particularly relevant class of affinity reagents are aptamers and their derivatives. Aptamers are typically short nucleic acid sequences folded into three-dimensional structures capable of binding targets or analytes with high affinity and specificity. In this document, aptamer refers to a low-affinity reagent, usually based on nucleic acids (naturally occurring RNA, DNA, or artificial nucleic acids XNA), but in some cases may also be based on a peptide backbone. In this document, aptamer also refers to aptamer derivatives, such as SOMAmer, aptabody, or other aptamer derivatives, for example, derivatives modified with modifications commonly found on proteins, such as glycosylation. Aptamers are generated via SELEX, can be produced at low cost with very high batch-to-batch consistency, have virtually unlimited shelf life at -20°C, are easily modified, and are well-suited for the cyclic staining and imaging methods described in European Patent Application No. 23178065.1, the entire contents of which are incorporated herein by reference. Aptamers, antibodies, and nanobodies all have dissociation constants (KD) in the pM to μM range and are used in a variety of affinity-based assays or immunoassays that are the backbone of life science research.

[0011] Typically, the marker can be linked to a primary affinity reagent or a secondary affinity reagent, which binds to the primary affinity reagent, thereby also introducing a specific degree of amplification. Affinity reagents can also be labeled with oligonucleotide barcodes, which can be addressed by using complementary oligonucleotide sequences that can be linked to the marker. Using oligonucleotide barcodes and adapters provides a simple and efficient method not only to link elements of the marker but also to incorporate additional functional elements, such as initiation sites, that can be amplified via enzymatic amplification (e.g., polymerase chain reaction PCR; loop-mediated isothermal amplification (LAMP); rolling circle amplification (RCA)) or via hybridization chain reaction (HCR). Additionally or alternatively, landing sites for the adapter can be incorporated, thereby allowing the formation of a dendritic structure. This allows for signal amplification, which is particularly desirable when analyzing low-abundance targets, such as PD1 or PD-L1, in tissue sections with high background autofluorescence. In the sense of this disclosure, a novel method for paired detection is provided, wherein the marker comprises at least a first label part and a second label part, the first label part and the second label part being directly or indirectly connected to a first affinity reagent and a second affinity reagent.

[0012] Another challenge in using such biomarkers lies in the cross-reactivity exhibited by affinity reagents, meaning they often bind not only to their target analyte (hereinafter also referred to as the target or associated target) but may also recognize, but usually with lower affinity, other so-called off-target targets. This is the case for antibodies and other protein-binding affinity reagents, as well as oligonucleotide probes (which are configured to bind to nucleic acid targets).

[0013] The markers, biomarkers, and methods described herein allow for the detection of proximity of multiple analytes in biological samples. The analytes may be parts of the same protein, in which case the method can be used to study post-translational modifications such as phosphorylation, ubiquitination, and acetylation; or the analytes may be different proteins, in which case the method can be used to determine their proximity in the sample as an indicator of protein-protein interactions. Furthermore, the method can also be used to probe multiple epitopes on the same target protein to enhance detection specificity, or to detect the presence of multiple epitopes on the same pathogen, such as *E. coli* or a virus, thereby identifying the corresponding strain with very high specificity. In other words, this disclosure provides a proximity determination method, which may be called the Host-Guest controlled Proximity Hybridization Assay (HGPHA).

[0014] Previously, a solution to this problem has been provided based on collecting cross-reactivity profiles and calculating cross-reactivity unmixing, as described in European Patent Application No. 23190568.8, the entire contents of which are incorporated herein by reference. In this document, a method is proposed that is particularly suitable for detecting the binding of paired affinity reagents to target analytes. This method can be used to measure a large number of protein-protein interactions in, for example, tissue samples, sometimes referred to as “spatial interoperomics.”

[0015] As fields such as cytometry, plasma proteomics, and microscopy advance to higher levels of complexity, cross-reactivity has become a concern and is considered by the scientific community to be a key component of the so-called reproducibility crisis. Bradbury and Pluckthin, along with 110 co-signers, in a position paper titled "Reproducibility: Standardize antibodies used in research" published in Nature 518, 27-29 (2015), stated that less than half of the approximately 6,000 commonly used commercial antibodies recognize only their designated targets. A recent study by Schwenk et al. (Toward Next Generation Plasma Profiling via Heat-induced Epitope Retrieval and Array-based Assays, Molecular & Cellular Proteomics, Vol. 9, No. 11, 2497–2507) analyzed 11,000 affinity-purified monoclonal antibodies and found that only 531 produced a single band on Western blotting. Although a systematic and comprehensive analysis of this phenomenon has not been conducted, the emergence of antibody microarrays in practical assays has opened up new possibilities for probing the cross-reactivity of affinity reagents. Therefore, conceptually, we may never be able to prepare truly specific affinity reagents—that is, those that truly bind to only one target. Bradbury and Pluckthin (Bradbury, A., Pluckthun, A. Reproducibility: Standardize antibodies used in research, Nature 518, 27–29 (2015). https: / / doi.org / 10.1038 / 518027a) further demonstrate that the signatories were able to replicate the scientific results of only 6 of 53 landmark preclinical studies, and subsequently predict that this phenomenon is costing an estimated $350 million annually in the United States alone.

[0016] Pair detection

[0017] Paired assays, which can detect the same analyte or multiple analytes, can be used in biochemical assays targeting a variety of methods. Typically, paired antibodies are combined with assays such as the Proximity Extension Assay (PEA) from OLINK (Uppsala, Sweden) for highly specific detection of the presence of analytes in liquids such as serum, or with the Proximity Linkage Assay (PLA) from Navinci (Uppsala, Sweden) for detecting the proximity of two different proteins. Matching paired antibodies for paired assays are readily available from suppliers such as Abcam, which currently offers more than 1800 paired antibodies.

[0018] The method disclosed in this paper, which can be called subject / object controlled proximity hybridization assay (hgPHA), is designed to allow for the faithful detection of analyte proximity in biological samples. (A) Increase the specificity of the assay by reducing false positive events caused by cross-reactivity or cross hybridization. (B) Query • Post-translation editing • Variable splicing, or • Protein hydrolysis processing.

[0019] Therefore, subject / guest controlled proximity hybridization assay (hgPHA) is valuable not only in detecting the proximity of two different molecules (e.g., two different proteins), but also in its potential to detect the same protein using paired assays, for example, by using two antibodies that recognize two different epitopes on the same protein or two sequence segments on the same RNA or DNA locus.

[0020] Subject-object controlled hybridization serves as a strategy to suppress duplex formation during target binding and removal of unbound affinity reagents and labeled moieties.

[0021] An important aspect of this disclosure can be considered as the ability to controllably form hybridization, or duplexes, between complementary first and second labeled portions using subject-object controlled hybridization. This is necessary to avoid aggregated aggregation mediated by the labeled portion of the affinity reagent, such as an antibody, in the absence of the target, which could otherwise interfere with the assay, and to enable duplex formation only after the unbound marker and labeled portion have been washed away. In this way, duplexes can only be formed when a pair of affinity reagents, such as antibodies, bind to two adjacent targets, where proximity is defined as being in a radius range of approximately 1–10, 5–20, 20–100, or 100–1000 nm, depending on the configuration of the assay. For example, for the co-detection of two surface markers on bacteria, spatial strictness can be selected to be low, such as 100–1000 nm. For post-translational modification assays, nanobodies, short linkers, and shorter first and second labeled portions can be used to achieve a spatial strictness (i.e., the analyte proximity required to produce a positive test result) in the range of 5–20 nm. For the highest steric rigor, one or both affinity reagents can be drugs, toxins, or other small molecules (e.g., hormones, neurotransmitters) known to bind to a target with high affinity and specificity. In this paper, high affinity refers to a KD in the range of μM to fM. In this paper, an affinity reagent is considered specific when regarded as such by a skilled biologist, biochemist, or molecular biologist. Since systematic whole-proteome binding studies of antibodies or other affinity reagents are not typically performed, it is clear in this paper that specific affinity reagents are likely to exhibit off-target binding, with binding strength generally weaker than that with their associated target.

[0022] Detection of double strand formation and thereby detection of marker formation

[0023] The markers disclosed herein, also referred to as hgPHA markers, comprise a first labeling portion and a second labeling portion. These labeling portions further comprise a first nucleic acid backbone and a second nucleic acid backbone, for example, oligonucleotide backbones configured to hybridize with each other. To inhibit the formation of a double strand between the first and second labeling portions, either or both are modified and comprise at least one guest molecule configured to complex with a host molecule, wherein both the guest molecule and the host molecule can be configured such that the complexation leads to a decrease in the strand invasion and the melting temperature of the double strand. The hgPHA assay according to this disclosure can be specifically configured such that… • Number of guest molecules per nt nucleic acid backbone • The melting temperature of the duplex under recombination (Tmc) and decocombination (Tmdc) conditions, and • The ionic strength of the composite and decomposition composite buffers, and • The concentration of potential additives such as formamide, dextran, competing guest molecules, and cap molecules, and • The temperature at which the labeled portion binds to the analyte in the biological sample, and • The temperature at which the double strands of the first and second marked portions form or hybridize. They are selected or adapted to be optimally suited to the sample type (e.g., lysates versus (vs.) cell samples, bacteria, cell cultures; versus tissue sections) and intended application. In other words, some samples tolerate high temperatures well in the 65–95°C range (e.g., diagnostic tests for bacterial or viral pathogens), while others, such as most tissue sections (e.g., FFPE tissue sections), should not be heated above 40–60°C due to the enhancement of autofluorescence. This is further elaborated below.

[0024] Compared with existing technologies, hgPHA has many advantages: (1) The change from recombination conditions (inhibiting double-strand formation) to decoombination conditions (allowing double-strand formation) is rapid and easy, for example, by simple rinsing, preferably by competition. (2) The detection mechanisms FRET and dequenching (labeling moiety, e.g., fluorescently labeled) are both robust and can be easily read on fluorometers, fluorescent gel recording systems, spectrometers, fluorometers, cell counters, FACS devices, and microscopes. (3) The first and second labeling moieties can be configured to allow single-molecule detection. (4) Only a small amount of actual operation time is required. (5) No expensive enzymes are required. (6) Moreover, hgPHA is compatible with a variety of enzymatic and non-enzymatic amplification methods. (7) In addition to changes in fluorescence intensity, hgPHA can also be used for FRET-based detection with instruments such as the STELLARIS confocal microscope from Leica Microsystems (Mannheim, Germany), which is equipped with a white light laser (WLL) pulsed light source and a FALCON fluorescence lifetime imaging module to measure changes in fluorescence lifetime (FLIM-FRET; further details below).

[0025] Terms and Definitions

[0026] In this document, the terms “fluorescent dye,” “fluorophore,” “fluorescent pigment,” and “dye” are used interchangeably to refer to fluorescent chemical compounds or structures, and may specifically be one of the following: fluorescent organic dyes, fluorescent quantum dots, fluorescent dyads, fluorescent carbon dots, graphene quantum dots or other carbon-based fluorescent nanostructures, fluorescent proteins, or fluorescent DNA-based origami nanostructures. The term "fluorescent dye" refers to organic fluorescent dyes, specifically derivatives of the following: xanthones (e.g., fluorescein, rhodamine, Oregon green, Texas), anthocyanins (e.g., indocarbocyanine, oxacarbocyanine, thiacarbocyanine, terpinene), derivatives, rotaxane derivatives, naphthalene, coumarin, oxadiazole, anthracene (anthraquinones, DRAQ5, DRAQ7, CyTRAKOrange), pyrene (Cascade Blue), oxazine (Nile Red, Nile Blue, cresol violet, oxazine 170), acridine (proflavin, acridine orange, acridine yellow), arylmethine (auramine, crystal violet, malachite green), tetrapyrrole (porphyrin, phthalocyanine, bilirubin), dipyrrole methylene (BODIPY, aza-BODIPY), phosphorescent dyes, or luminescent dyes. The following trademark groups represent commercially available fluorescent dyes, which may include dyes belonging to different chemical families, such as CF dyes (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), Aberior 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), and Vio dyes (Milteny Biotec).In this document, the term "fluorescent dye" refers to the group of fluorescent proteins, specifically 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, -5-mPlum). Furthermore, from the perspective of the group of fluorescent proteins, the term "fluorescent dye" in this document can include fluorescent proteins whose absorption or emission characteristics change upon binding to a ligand such as BFPms1 or in response to environmental changes, such as redox-sensitive roGFP or pH-sensitive variants. Additionally, from the perspective of the group of fluorescent proteins, the term "fluorescent dye" in this document can include derivatives of the cyanobacterial phycobiliprotein small infrared fluorescent protein smURFP and fluorescent protein nanoparticles that can be derived from smURFP. An overview of fluorescent proteins can be found in the paper by Rodriguez et al., Trends Biochem Sci. 2017 Feb; 42(2):111–129. In this paper, fluorescent dyes can further refer to fluorescent quantum dots. In this paper, fluorescent dyes can further refer to fluorescent carbon dots, fluorescent graphene quantum dots, and fluorescent carbon-based nanostructures, as described in the papers by Yan et al., Microchimica Acta (2019) 186:583 and Iravani and Varma 2020, Environ Chem Lett. 2020 Mar 10:1-25. In this paper, fluorescent dyes can further refer to fluorescent polymer dots (Pdots) or nanodiamonds. In this paper, fluorescent dyes can further refer to fluorescent duals, such as duals of perylene antennas and triangelium emitters, as described in the paper by Kacenauskaite et al., J. Am. Chem. Soc. 2021, 143, 1377-1385. In this document, fluorescent dyes may further refer to organic dyes, duals, quantum dots, polymer dots, graphene dots, carbon-based nanostructures, DNA origami-based nanostructures, nanoscales, polymer beads containing dye-incorporated components, fluorescent proteins, inorganic fluorescent dyes, SMILEs, or microcapsules filled with any of the above. In this document, fluorescent dyes may further refer to FRET pairs having at least one fluorescent dye as a FRET donor and at least one fluorescent dye as a FRET acceptor, or to FRET-trinets used to generate three-component Foster resonance energy transfer. Specifically, the FRET pairs or FRET-trinets are connected by complementary joints or by connecting elements.In this paper, fluorescent dyes can further refer to FRET n-tuples of physically linked dyes. Fluorescent dyes can also be polymer dyes.

[0027] Foster resonance energy transfer (FRET) has been widely used to measure protein-protein interactions using both fluorescent proteins and fluorescent dyes. FRET is spatially strict within the 10 nm range. In other words, the FRET efficiency EE = 1 / (1 + (r / R0)^6) decreases exponentially with increasing donor-acceptor dye distance r, making efficient FRET typically occur when the donor and acceptor dyes are within a distance r of 1 to 10 nm, where R0 is the Foster distance of the donor-acceptor dye pair. Typically, the donor and acceptor dyes, or fluorophores, are distinct fluorophores. For example, the following dye pairs are commonly used FRET dye pairs: ATTO 425–ATTO 520, ATTO 488–ATTO 550, ATTO 488–ATTO 565, ATTO 488–ATTO 647N, ATTO488–ATTO 655, ATTO 520–ATTO 647N, ATTO 532–ATTO 647N, ATTO 532–ATTO 655, ATTO550–ATTO 590, ATTO 550–ATTO 647N, ATTO 565–ATTO 590, ATTO 565–ATTO 647N, ATTO590–ATTO 620, ATTO 590–ATTO 647N, ATTO 590–ATTO 680, ATTO 620–ATTO 680, AlexaFluor 488–Alexa Fluor 555, Alexa Fluor 594–Alexa Fluor 647, and fluorescein-tetramethylrhodamine. Similarly, commonly used fluorescent protein FRET pairs exist, including but not limited to: ECFP-EYFP, mTurquoise2-mVenus, EGFP-mCherry, and mNeonGreen-mRuby3. Quantum dots can also be used as donors or acceptors. Quantum dots are particularly advantageous as FRET donors due to their high extinction coefficient, resulting in a brighter FRET signal. Various methods exist for measuring FRET efficiency, including but not limited to measuring the intensity of acceptor emission (sensitized emission), photobleaching FRET (where the donor is bleached, and the bleaching rate varies depending on the presence or absence of the acceptor), and fluorescence lifetime measurements that record changes in donor lifetime (i.e., a shortening of donor lifetime when FRET occurs; FRET-FLIM). Similarly, FRET can be evaluated by measuring donor dequenching after acceptor removal or bleaching (e.g., acceptor photobleaching). In addition to these methods, FRET can also be measured by anisotropic imaging, such as in the case of Homo-FRET, i.e., in assays where the donor and acceptor have the same fluorophore. These methods typically read out multiple donor and acceptor dye molecules, and methods for measuring single-molecule FRET have also been developed.A simple method for measuring FRET is ratio imaging of donor and acceptor intensities (sensitized emission), which can be performed on simple channel-based readers such as wide-field microscopes, cell counters, or plate readers. While measuring FRET efficiency, or the FRET signal, in this manner by measuring donor and acceptor emission is straightforward, it is associated with the challenge of donor crosstalk into the acceptor emission channel (also known as the FRET channel). Therefore, more sophisticated methods, such as donor dequenching measurements, have been developed. Alternatively, spectral imaging offers a good solution to the crosstalk problem in FRET measurements, but suitable instruments may not always be available. However, FRET measurements are difficult to perform in tissue sections due to high background autofluorescence. Although FRET has been performed in tissue sections before, it is known in the art, and clearly evident from published data, that FRET-based measurements in tissue sections are limited by poor signal-to-noise ratios or signal-to-background ratios due to the high background autofluorescence often found in tissue samples. References. P, Krasteva G, Tag C IR, Arens C, Kummer W., FRET-CLSM and double-labeled indirect immunofluorescence assays were used to detect tight binding of proteins in tissue sections. Lab Invest. 2006 Aug; 86(8):853-64. doi:10.1038 / labinvest.3700443. Epub 2006 Jun 19. PMID:16783395.

[0028] Therefore, this disclosure proposes an assay method that combines easily detectable bright labeling with high specificity, compatible with cyclic staining methods for high-plexity analysis that improve specificity, for high-magnification analysis of molecular interactions and / or post-translational modifications. This disclosure is applicable to any type of analyte and AR.

[0029] In a first aspect, a marker for analyzing biological samples is provided. The marker comprises a first labeling portion comprising a first nucleic acid backbone and a second labeling portion comprising a second nucleic acid backbone. Each of the first and second nucleic acid backbones is configured to hybridize with its respective other nucleic acid backbone. Specifically, the first and second nucleic acid backbones may be at least partially complementary to each other. Hybridization of the first and second nucleic acid backbones can result in the formation of a double-stranded nucleic acid. The marker further comprises at least one first labeling portion and at least one second labeling portion. The marker further comprises at least one guest molecule configured to form a complex with a host molecule.

[0030] The marker can be used, for example, to detect the proximity between two target molecules.

[0031] Specifically, the label can be configured to be optically detectable at least when the first labeled portion and the second labeled portion are in close proximity. For example, the at least one first labeled portion and / or the at least one second labeled portion can be optically detectable, such as a fluorophore, and the optical properties of at least one of the first labeled portion and the second labeled portion can be varied depending on the distance between the first labeled portion and the second labeled portion. Specifically, the at least one first labeled portion and the at least one second labeled portion can have the same optical properties, such as excitation wavelength, emission wavelength, and fluorescence lifetime, or they can have different optical properties. The first labeled portion and the second labeled portion can be attached to the same nucleic acid backbone of the same labeled portion, or to the nucleic acid backbone of different labeled portions of the label.

[0032] The guest molecule is preferably covalently attached to one of the nucleic acid backbones. Specifically, the label may comprise multiple guest molecules attached to one or two nucleic acid backbones. In this case, one guest molecule may bind to one host molecule. Specifically, the at least one guest molecule is not a nucleotide and / or is not a labeled portion. In one specific embodiment, there may be one guest molecule for every ten nucleotides of one of the nucleic acid backbones.

[0033] The guest molecule is preferably configured to selectively form a complex with the host molecule. Therefore, the guest molecule can be configured to form a complex with the host molecule only under specific recombination conditions. Similarly, the guest molecule can be configured to disintegrate from the host molecule only under disintegration conditions different from the recombination conditions. In the simplest case, the recombination conditions are characterized by the presence or high concentration (e.g., in the μM-mM range) of the host molecule, while the disintegration conditions are characterized by a low concentration or absence of the host molecule. Complex formation can include the binding of the guest molecule to the host molecule. For example, complex formation can be based on intermolecular forces, such as hydrogen bonds, van der Waals forces, or dipole interactions. Preferably, the complex formed between the host molecule and the guest molecule is configured to disrupt or prevent hybridization between the first and second nucleic acid backbones. Specifically, the complex can lower the melting temperature of the double strands of the hybridized first and second nucleic acid backbones.

[0034] Preferably, the at least one guest molecule is configured to form a complex with the host molecule under recombination conditions. Similarly, the guest molecule and / or the host molecule is configured to decomplex under decomplexation conditions. Such decomplexation and / or recombination conditions may include changing the concentration of the host molecule or adding a competing guest molecule, changing the pH, salt, and / or temperature. This enables the selective formation of a complex between the host molecule and the guest molecule. Specifically, recombination of the at least one guest molecule with the host molecule can lead to strand invasion and dissociation of the double strands between the first and second nucleic acid backbones.

[0035] Preferably, the optical properties of at least one of the first and second marked portions change according to the distance between the first and second marked portions, specifically according to the distance between the first and second marked portions. Specifically, the change in optical properties can be detected to determine the distance between the marked portions. The change in optical properties can be proportional to the distance between the marked portions.

[0036] Preferably, the complex is configured to invade the duplex and lower its melting temperature.

[0037] Preferably, each of the first and second nucleic acid backbones comprises at least one of a natural nucleic acid and a nucleic acid analogue. Specifically, the first and / or second nucleic acid backbones may be substantially composed of nucleic acid analogues. The first and / or second nucleic acid backbones may comprise a single nucleic acid molecule.

[0038] Specifically, the first and second nucleic acid backbones are at least partially complementary to each other to hybridize and form a double strand. Guest-host complex formation can hinder this hybridization. The guest molecule is preferably attached (covalently) to the phosphate backbone or ribose or nucleobase of the respective nucleic acid backbone. In some embodiments, the first or second nucleic acid backbone may comprise an XNA or a heteronucleic acid, such as a peptide nucleic acid, in which case the guest molecule may be coupled to the peptide backbone of the PNA.

[0039] Preferably, one of the first and second nucleic acid backbones is substantially composed of a nucleic acid sensitive to degradation agents, such as a natural nucleic acid, while the other of the first and second nucleic acid backbones is substantially composed of a nucleic acid analog resistant to degradation agents. This enables, for example, the selective degradation of one or both of the first and second nucleic acid backbones during iterative staining.

[0040] This degrading agent can be DNase I or a restriction enzyme. In some embodiments, the nucleic acid backbone may include cleavage sites configured to be chemically cleaved (e.g., reductive cleavage with TCEP, hydrogen peroxide) or physically cleaved (e.g., by ultraviolet light, temperature). It is clear that these examples are provided herein merely for illustrative purposes. Those skilled in the art will readily find further chemicals and / or enzymes that can be used to degrade the marker or portions thereof.

[0041] Typically, nucleic acid analogs are compounds that are structurally similar to naturally occurring RNA and DNA. Nucleic acids are chains of nucleotides, consisting of three parts: a phosphate backbone, a pentose sugar (ribose or deoxyribose), and one of the four nucleobases. Analogs can alter any of these parts. Nucleic acid analogs can be artificial or xenobiotic nucleic acids. While natural nucleic acids (DNA and RNA) are generally sensitive to degrading agents such as nucleases, nucleic acid analogs are typically resistant to degrading agents such as nucleases.

[0042] Furthermore, if the affinity reagent contains nucleic acids or is composed primarily of nucleic acids, these nucleic acids can similarly be nucleic acid analogs that are resistant to degradation agents.

[0043] Preferably, the at least one first labeled portion and / or the at least one second labeled portion are optically detectable. This enables efficient detection of the labeled material, for example, by microscopy, specifically by fluorescence microscopy. For example, the first labeled portion and / or the second labeled portion may contain a fluorophore, such as a fluorescent protein, an organic or inorganic fluorescent molecule, or fluorescent nanoparticles.

[0044] Preferably, the at least one first labeled portion and / or the at least one second labeled portion preferably comprise the same fluorescent dye. Specifically, the fluorescence characteristics of the at least one first labeled portion and / or the at least one second labeled portion are substantially the same. Alternatively, the at least one first labeled portion comprises at least one first fluorescent dye, and the at least one second labeled portion comprises at least one second fluorescent dye, wherein the at least one first fluorescent dye and the at least one second fluorescent dye have different characteristics. These characteristics may differ regarding the excitation, emission, and / or fluorescence lifetime of the at least one first fluorescent dye and the second fluorescent dye.

[0045] Preferably, the at least one first labeled portion and the at least one second labeled portion are configured to perform nonradiative energy transfer between them, specifically when they are in close proximity to each other. For example, the first labeled portion and the second labeled portion can form a fluorescence resonance energy transfer (FRET) pair, one of which is an FRET donor and the other is an FRET acceptor. This enables determining whether the first labeled portion and the second labeled portion are in close proximity to each other based on the FRET efficiency between the FRET pair. Typically, FRET efficiency is highly dependent on the distance between the FRET donor and the FRET acceptor. In this context, proximity can be characterized as being in the range of 1 to 10 nm. In a specific embodiment, when FRET does not occur, the FRET donor and the FRET acceptor may have different optical properties, such as emission wavelength or excitation wavelength. This enables accurately determining whether energy transfer occurs between the FRET acceptor and donor pair. Preferably, the FRET donor, as the first labeled portion, can be aligned on the first labeled portion or the first nucleic acid backbone, while the FRET acceptor, as the second labeled portion, can be aligned on the second labeled portion or the second nucleic acid backbone. Therefore, when the first nucleic acid backbone and the second nucleic acid backbone hybridize at least partially to form a double strand, a FRET pair can be formed, as further described below.

[0046] In an alternative embodiment, the first and second labeled portions may be of the same type, meaning they have the same optical properties. In this case, self-quenching may occur between the first and second labeled portions, specifically when the proximity of the first and second labeled portions leads to nonradiative energy transfer between them. This can significantly reduce the emission efficiency and fluorescence intensity detectable from the first and second labeled portions. Therefore, in this case, the proximity of the first and second labeled portions can be detected by the reduced fluorescence intensity when they are in close proximity. In this embodiment, the first and second labeled portions may be aligned on the same nucleic acid backbone, i.e., on either the first or second nucleic acid backbone, as further described below.

[0047] Referring to the above, when the first labeled portion and the second labeled portion are configured to transfer nonradiative energy between them, the first labeled portion and the second labeled portion can attach to their respective first or second nucleic acid backbones. Therefore, the first labeled portion can attach to the first nucleic acid backbone, and the second labeled portion can attach to the second nucleic acid backbone.

[0048] Alternatively, the first labeled portion and the second labeled portion may attach to the same nucleic acid backbone. Therefore, the first labeled portion and the second labeled portion may attach to either the first nucleic acid backbone or the second nucleic acid backbone. In this case, the first labeled portion and the second labeled portion are preferably hydrophobic labeled portions.

[0049] Typically, hydrophobic labeled moieties tend to aggregate in aqueous solutions. Specifically, adjacent hydrophobic labeled moieties on a flexible single-stranded nucleic acid backbone can lead to aggregate formation. This aggregation can result in self-quenching or changes in the optical properties of these labeled moieties (such as their absorption wavelengths and / or specifically their fluorescence emission or fluorescence intensity) compared to unaggregated hydrophobic labeled moieties. The formation of a double strand between the first and second nucleic acid backbones when they are in close proximity leads to an increase in the rigidity or stiffness of the nucleic acid backbone. The measure of the rigidity or stiffness of the nucleic acid backbone, specifically the double-stranded structure, is the durability length (L). P Duration length is a mechanical parameter that quantifies the rigidity of a polymer: the higher the duration length, the higher the rigidity of the polymer. In the presence of monovalent or divalent salts, double-stranded nucleic acid structures often have duration lengths of 30 to 55 nm, while single-stranded nucleic acids are more flexible, with duration lengths of 1.5 to 3 nm. Therefore, double-stranded nucleic acids have higher duration lengths and correspondingly more linear forms.

[0050] Specifically, when the first labeled portion and the second labeled portion attach to opposite ends of the first or second nucleic acid backbone, the increased rigidity of the respective nucleic acid backbone can lead to an increase in the distance between the first labeled portion and the second labeled portion. Therefore, previously aggregated labeled portions separate through hybridization with the first and second nucleic acid backbones. This separation may result in detectable changes in their optical properties.

[0051] Examples of commonly used hydrophobic labeled portions for analyzing biological samples include ATTO 390, ATTO 425, ATTO 550, ATTO Rho12, ATTO 633, and ATTO 647N.

[0052] Preferably, the at least one first labeled portion and the at least one second labeled portion are attached to the first nucleic acid backbone or the second nucleic acid backbone. For example, the labeled portions may be attached to, and preferably covalently attached to, the phosphate backbone of the respective nucleic acid backbone. Specifically, the first labeled portion and the second labeled portion may be attached to opposite ends of one of the first nucleic acid backbone and the second nucleic acid backbone.

[0053] In an alternative implementation, the at least one first labeled portion is attached to the first nucleic acid backbone, while the at least one second labeled portion is attached to the second nucleic acid backbone.

[0054] Preferably, the first nucleic acid backbone extends along a first direction, and a plurality of the first labeled portions are arranged on the first nucleic acid backbone along the first direction, and / or the second nucleic acid backbone extends along a second direction, and a plurality of the second labeled portions are arranged on the second nucleic acid backbone along the second direction. This enables the generation of an optically detectable signal proportional to the distance between the first labeled portions and the second labeled portions. Specifically, the respective labeled portions are arranged sequentially along their respective directions.

[0055] Preferably, the spacing between each marked portion and any adjacent marked portion is substantially equal. This enables the generation of an optically detectable signal proportional to the distance between the first and second marked portions. When the marker comprises multiple marked portions, the spacing between each first marked portion and any adjacent first marked portion is substantially equal, and / or the spacing between each second marked portion and any adjacent second marked portion is substantially equal. Preferably, the substantially equal spacing can be in the range of 0.33 nm to 33 nm.

[0056] Preferably, the marker comprises a plurality of guest molecules, wherein the guest molecules are uniformly spaced along the first nucleic acid backbone and / or the second nucleic acid backbone. This allows for effective control of hybridization between the first and second nucleic acid backbones by means of the host molecule. Specifically, the guest molecules are arranged along the first or second direction. Preferably, one guest molecule is provided for every 5 to 10 nucleotides of each nucleic acid backbone. Preferably, the length of the nucleic acid backbone is in the range of 20-120 nt, specifically up to about 120 nt, allowing for high-yield and high-quality synthesis. In some cases, the nucleic acid backbone can be longer and contains, for example, 120 nt-600 nt, which can be achieved by concatenation or chemical linkage. Reference is made to European Patent Application No. 24151783.8, the entire contents of which are incorporated herein by reference.

[0057] Preferably, the at least one guest molecule is one of 1-adamantanemethylamine, ferrocenemethylamine, 1,4-phenylenediamine, and 4-tert-butylbenzylamine. Xiao et al., 2022 (Controllable DNA hybridization by host–guest complexation-mediated ligand invasion. Nature Communications 13:5936) provide further details on suitable host and guest molecules.

[0058] Preferably, the label comprises at least one host molecule. Providing the label with at least one guest molecule and a host molecule enables the avoidance of random or spontaneous hybridization between the first and second nucleic acid backbones. Specifically, this enables the control of hybridization between the first and second nucleic acid backbones. This makes it easier and more efficient to introduce the labeled portion into biological samples. Specifically, the label may comprise multiple host molecules to form a complex with at least each guest molecule. The host molecule may be configured to form a complex with the guest molecule.

[0059] Preferably, the host molecule is cucurbit[n]urea, specifically cucurbit[7]urea. Specifically, the host molecule does not include nucleotides or nucleic acids.

[0060] Regarding cucurbit[7]urea, the particularly preferred host molecule used in the methods disclosed in this document, the following publication is cited: A reference scale of cucurbit[7]uril binding affinities. Alnajjar et al., 2021 Org. Biomol. Chem., 2021, 19, 8521-8529. As detailed by Alnajjar et al. using competitive titration, a wide range of suitable molecules can be guests for CB[7], spanning the millimolecular (pM) (e.g., TBA) to femtomolar (fM) (e.g., AMADA) range (compare Scheme 1 and Table 1). Several fluorescent dye molecules that may be (mainly) labeled in this document can be guests for CB[7], but do not have very high affinity binding. In this document, nM binding constant can be considered as high affinity, pM as very high affinity, and fM and above as ultra-high affinity; unless otherwise explicitly stated, when referring to “high affinity”, this includes both very high and ultra-high affinity.

[0061] Regarding the selection of guest molecules, it is important to note that the method can be carried out in a manner in which the guest molecules have an affinity in the range of μM to nM. For example, in this case, the complexing buffer contains CB at a concentration of μM or mM [7]. This is in contrast to the work from Xiao et al. 2022 (Controllable DNA hybridization by host–guest complexation-mediated ligand invasion, Nature Communications 13, 5936). Figure 5 The conditions used in b (10 μM CB[7], 30 μM FC guest competitor, 15 nt duplex of cytosine with 3 FC-guest modified) were similar, which confirmed the formation of the cyclic duplex.

[0062] In other words, during recombination conditions, the concentration of the host molecule is high, causing most guest molecules to recombine with the host molecule and eliminating double-strand formation, which could otherwise mediate the binding of the first and second nucleic acid backbones in solution, leading to false positives. After the marker binds, the decomposition conditions are applied. In the simplest case, this can be a simple rinse of the host molecule with a standard washing buffer such as PBS, PDT, or a blocking buffer (which may contain, for example, BSA, dextran, salmon sperm, or contaminated DNA). Rinsing or buffer exchange effectively reduces the concentration of the host molecule. Alternatively or additionally, a decomposition buffer can be used to achieve decomposition conditions containing competing guest molecules, which may be the same or different guest molecules. For example, if FC (ferrocene or a ferrocene derivative) is used as the guest molecule, AD (adamantane or an adamantane derivative), which has a higher affinity for CB[7], can be used in the decomposition buffer. Further on this point, cited by Xiao et al., 2022, it is known that “1-adamantanemethylamine (AD), ferrocenyl methylamine (FC), 1,4-benzenedimethanamine (BA) and 4-tert-butylbenzylamine (TB)” have a wide range of binding affinities across “CB[7]” (Ka values ​​of the cationic derivatives of these guest molecules previously reported: ~10 for AD). 14 M -1 For FC = ~10 12 M -1 For BA = ~10 9 M-1 For TB of ~10 6 M -1 )(a wide range of binding affinities on CB[7](Ka values ​​ofpreviously reported cation derivatives of these guest molecules were~10 14 M - 1 for AD, ~10 12 M -1 for FC, ~10 9 M -1 for BA and ~10 6 M -1 "for TB)," and "as shown by analysis by isothermal titration calorimetry (ITC) (supplement)" Figure 1 The binding constants between these four guest molecules and CB[7] are: 1.64 × 10⁻⁶ for AD. 9 M -1 For FC = 4.70 × 10 8 M -1 For BA, it is 8.88 × 10 5 M -1 For TB, which is 3.98 × 10⁴ M⁻¹, it is significantly reduced mainly due to the pH effect and the interaction between the sodium cation and the carbonyl end of CB[7] (as shown by theisothermal titration calorimetry (ITC) analysis (Supplementary Fig. 1), the binding constants between CB[7] and these four guest molecules were 1.64 × 10⁴ M⁻¹. 9 M -1 for AD, 4.70×10 8 M -1 for FC, 8.88×10 5 M -1 for BA and 3.98×10 4M-1 for TB, which were significantly reduced mostly due to the effects of pH and the interactions between sodium cations and the carbonylportals of CB[7]”. Therefore, it will be clear to those skilled in the art that the combinations of guest and host molecules and their concentrations, the concentrations of guest molecule competitors and salt and other buffer components provided in this document are provided for illustrative purposes only, and many other configurations may be chosen to implement the method without departing from the invention.

[0063] The difference in melting temperature (Tm) between the recombination and decomposition conditions under which the first and second labeled portions attach to their respective targets can be adjusted to the desired point by: (A) the selection of the guest molecules, (B) the number of guest molecules coupled to the first and / or second labeled portions, and (C) changing the salt concentration, or adding competitive guest molecules or other additives, such as formamide, dextran sulfate, or other agents, to the recombination, decomposition, washing, or blocking buffer.

[0064] Regarding cucurbituril as the host molecule: In the context of this application, it should be understood primarily as an endo guest, i.e., a molecule that is wholly or partially contained within the host molecule, in addition to the CB guest molecule. There are also molecules that can cap CB molecules, which can be used as other additives because they can modify the binding and release kinetics of the endo guest molecule. An example of such a molecule is hexaiodobenzene bound to the 7-terminus of CB by computational analysis, as described by Lambert et al. in npjComputational Materials (2022) 8:21 Supramolecular gating of guest release from cucurbituril using de novo design.[7]

[0065] The following compositions are provided for illustrative purposes. Those skilled in the art will readily find new additives and modify salt concentrations, inhibitor concentrations, dispersants, etc., to suit the needs of specific applications or biological samples without departing from the invention.

[0066] Exemplary blocking buffer: Basic components: 5% standard goat serum / PBS, or 5% standard donkey serum / PBS, or 1% BSA / PBS 137mM–500mM NaCl 0.2-10 mg / ml salmon sperm DNA (e.g., from THERMO #AM9680) 1-10 nmol / ml mixed oligonucleotides ssDNA (e.g., from IDT, Iowa) Other optional additives: DMSO Cleaning agents (Triton-X100, NP-40, saponins, Tween-20)

[0067] Exemplary decomposition buffer (allows double strand formation):

[0068] In the simplest case, the decomposition buffer can be simply PBS or a blocking buffer to wash away the host molecules.

[0069] Alternatively, the decomplexing buffer may further comprise a guest molecule competitor. This may be the same as or different from the competitor used to modify the nucleic acid backbone of the labeled moiety. For example, if ferrocene (FC) is used to modify the label, then FC or a different guest molecule, such as adamantane (AD), may be included in the decomplexing buffer. 1% BSA / PBS 137mM–500mM NaCl 0.2-10 mg / ml salmon sperm DNA (e.g., from THERMO #AM9680) 1-10 nmol / ml mixed oligonucleotides ssDNA (e.g., from IDT, Iowa) 10μM-1mM adamantane

[0070] Exemplary composite buffer (inhibits double-strand formation): 1% BSA / PBS 137mM–500mM NaCl 0.2-10 mg / ml salmon sperm DNA (e.g., from THERMO #AM9680) 1-10 nmol / ml mixed oligonucleotides ssDNA (e.g., from IDT, Iowa) 10μM-10mM CB[7]

[0071] Example 1: Subject-guest controlled proximity hybridization assay (HGPHA) for direct detection of analyte proximity in cells:

[0072] preparation: (A) Preparation of oligonucleotide-barcoded primary antibody pairs. These can be obtained directly from various suppliers or generated using an available antibody-oligonucleotide conjugation kit (e.g., Oligonucleotide Conjugation Kit ab218260 from abcam (Cambridge, UK)). Oligonucleotides with suitable functional groups can be purchased from IDT (Coralville, USA). Similarly, oligonucleotide-barcoded primary antibodies can be obtained using site-specific conjugation. (B) Prepare a first and second labeled portion comprising complementary barcodes to allow subsequent conjugation to the antibody pair mentioned in (A) via hybridization. This is a particularly simple and efficient method to work with the method described above. However, the first and second labeled portions can also be directly covalently conjugated using the kits described above or other chemicals such as click chemistry and suitable bifunctional adapters (e.g., NHS-PEG(n)-N3 adapters). (C) Depending on the expression level of the target, signal amplification may be required. This can be easily achieved in at least two different ways: (1) by using dendritic oligonucleotide-based structures or other DNA carrier structures, such as nanorulers, DNA origami-based structures, DNA brick-based structures; (2) By using enzymatic methods, such as rolling circle amplification (RCA) or loop-mediated isothermal amplification (LAMP). Alternatively, the barcode sequence can be amplified using polymerase chain reaction (PCR).

[0073] Main operating procedures: 1. Wash the cells twice. 2. Fix with 4% formaldehyde for 10 minutes and wash 3 times. 3. Permeate with 0.1% TX-100 / PBS for 15-20 min and wash 3×. 4. Block in the blocking buffer for 45 minutes. 5. Dilute the oligonucleotide barcode-conjugated primary antibody in blocking buffer and apply it for 1-2 hours (or overnight at 4°C). Dilutions are typically in the range of 1:100-1:500. 6. With stirring, wash thoroughly 4× with PBS or blocking buffer to remove unbound primary antibodies. 7. Dilute the first and second labeled portions containing the complementary barcode sequence (i.e., the barcode complementary to that on the primary antibody) in a composite buffer. 8. The first and second labeled portions, diluted in a composite buffer, are applied to the sample and hybridized to their respective barcodes on the primary antibody that binds to the target. This step can be performed at room temperature for 5–60 min or at higher temperatures. 9. Wash 4x with a compound buffer while stirring to remove unbound first and second labeled portions. DAPI may also be added during this step for nuclear staining. 10. Optionally, the sample is readout and / or imaged for the first time before applying the dissociation conditions. This readout / image can serve as a baseline and can improve the quality of the assay, but is not necessary in many cases. 11. Equilibrate the sample to decomposition conditions by washing it 4x with a suitable volume (e.g., 4-5 flow cell volumes) of decomposition buffer or rinsing the sample. 12. Hybridization of the first and second labeled portions is allowed to occur under decomposition conditions, which typically may take 5-60 minutes. 13. Optionally, apply a drop of sealing agent to a microscope slide and invert a coverslip containing cells or a biological sample onto the drop. Gently press the sample with tweezers to distribute the sealing agent well, but do not compress the sample. Once cured, the sample is ready for microscopic observation. 14. For optical-grade substrates, dishes, or flow cells, samples can be imaged directly in decomposition buffer, PBS, or any other imaging buffer of choice, provided that the imaging buffer does not contain any substance that interferes with the formation of the double strand between the first and second labeled portions. 15. Read out and / or image the sample. 16. Optionally, before and after the formation of a double chain between the first and second marker portions, i.e. under recombination and decolombination conditions, the pixel intensity is compared to calculate the multiple change or ratio of the (FRET) signal at the pixel level, thereby assessing the proximity of the marker and, consequently, the proximity of the analyte.

[0074] Example 2: Subject-guest controlled proximity hybridization assay (HGPHA) for indirect detection of analyte proximity in cells: 1. Wash the cells twice and carefully place the coverslip with the upturned cells into the humidified chamber using tweezers. 2. Fix with 4% formaldehyde for 10 minutes and wash 3 times. 3. Permeate with 0.1% TX-100 / PBS for 15-20 min and wash 3×. 4. Block with 5% regular goat serum / PBS or 1% BSA / PBS for 45 min (no washing required). 5. Dilute the primary antibody in blocking buffer and apply it for 2 hours (or overnight at 4°C). Dilutions are typically in the range of 1:100–1:500. 6. Thoroughly wash 4× samples with stirring to remove unbound primary antibodies. 7. Wash 1x with a composite buffer while stirring to equilibrate the sample to composite conditions. (1). Option A: Incubate with the corresponding paired secondary antibodies containing the respective first and second labeled portions diluted in a complexing buffer for 1–3 h. Then aspirate the secondary antibodies and, if necessary, incubate with Hoechst or DAPI before proceeding to washing step 8. Note: Under complexing conditions, perform binding of the antibody containing the labeled portion to avoid hybridization of the labeled portion in solution, which may produce false positive results. (2). Option B: Incubate with the corresponding paired secondary antibodies containing oligonucleotide barcodes diluted in a blocking buffer for 1–3 h. Then aspirate the secondary antibodies and wash thoroughly with a compound buffer 4x. Dilute a first and second labeled portion containing suitable complementary barcode sequences in a compound buffer and apply to the sample, the aforementioned barcode sequences being configured to mediate attachment with the barcoded secondary antibodies. Allow the first and second labeled portions to hybridize with their respective secondary antibodies. Typically, incubate at room temperature for 5–60 h. min is sufficient. Proceed to step 8 to wash away any unbound markers. 8. Thoroughly wash 4x with a compound buffer while stirring to remove unbound secondary antibodies, without even performing nuclear staining. 9. Optionally, the sample may be read out and / or imaged before applying the dissociation conditions. This readout / image may serve as a baseline and may improve the quality of the assay, but is not necessary in many cases. 10. Equilibrate the sample to decomposition conditions by washing it 4x with a suitable volume (e.g., 4-5 flow cell volumes) of decomposition buffer or rinsing the sample. 11. Hybridization of the first and second labeled portions is allowed to occur under decomposition conditions, which typically may take 5-60 minutes. 12. Optionally, apply a drop of sealing agent to a microscope slide and invert a coverslip containing cells or a biological sample onto the drop. Gently press the sample with tweezers to distribute the sealing agent well, but do not compress the sample. Once cured, the sample is ready for microscopic observation. 13. For optical-grade substrates, dishes, or flow cells, samples can be imaged directly in decomposition buffer, PBS, or any other imaging buffer of choice, provided that the imaging buffer does not contain any substance that interferes with the formation of the double strand between the first and second labeled portions. 14. Optionally, before and after the formation of a double chain between the first and second marker portions, i.e., under recombination and decolombination conditions, pixel intensities are compared to calculate the multiple change or ratio of the (FRET) signal at the pixel level, thereby evaluating the proximity of the markers. Then, the proximity of the analyte is assessed.

[0075] Example 3: Subject-guest controlled neighbor-near hybridization assay (HGPHA) for tissue sections:

[0076] HGPHA assays for tissue sections are based on a multiplexed IF protocol from Gerdes et al. (2013), "Highly Multiplexed Single-Cell Analysis of Formalin-Fixed, Paraffin-Embedded Cancer Tissue, PNAS". While this is a robust protocol for circulating immunofluorescence of FFPE tissue sections and tissue microarrays, other protocols exist and may be equally suitable. For these protocols to be suitable for HGPHA assays, care must be taken to ensure the binding of the first and second labeled portions to the target under multiplexing conditions to avoid false positives. Specific protocol steps depend on whether the user intends to use the following assay. A. Direct detection using directly labeled primary antibodies B. Direct detection using barcoded primary antibodies C. Indirect detection using directly labeled secondary antibodies D. Indirect detection using barcoded secondary antibodies.

[0077] The following embodiments are provided for (A), and must be adapted accordingly to suit other options. Those skilled in the art, upon reading the description of the methods provided herein, will readily understand how to adjust the operating scheme for each of these options, and how to modify the operating scheme without departing from the invention.

[0078] Preparation: (1) Collect tissue biopsies according to standard procedures known to the technician and fix them with a fixative such as 4% PFA in PBS or formalin. The fixation buffer may further contain a cleaning agent such as Tween 20, Triton X-100, saponins, or NP-40, depending on the tissue, at a concentration ranging from 0.01% to 1%. Fixation time is 1 to 3 hours at room temperature or overnight at 4°C. (2) The tissue can then be embedded for cryosectioning or proceed to a paraffin embedding and sectioning workflow, which includes stepwise dehydration. (3) Although frozen sections can be used directly for downstream blocking, there are also dewaxing, rehydration and epitope repair procedures that can be applied to tissue microarrays (TMA) for sections that are formalin-fixed and paraffin-embedded (FFPE).

[0079] Note: Blocking buffers used for tissue work contain higher concentrations of blocking agents compared to those used for cells; similarly, higher concentrations of cleaning agents can be used. For example, 10% (wt / vol) donkey serum and 3% (wt / vol) BSA.

[0080] Main operating procedures: (1) Formalin-fixed paraffin-embedded (FFPE) tissue samples or tissue arrays were incubated at 65°C. Bake for 1 hour. (2) Dewaxing with Histochoice cleaner (Amresco) or similar products. (3) Rehydration is carried out by cleaning with a reduced ethanol concentration (ethanol series). (4) Perform antigen retrieval (Note: Several recognized methods exist and are known to those skilled in the art). (5) Incubate in PBS containing 0.3% Triton X-100 at room temperature for 10 min (Note: Depending on the organization type, other permeability conditions may be equally or better suited. (6) Block with a blocking buffer at room temperature for 45 minutes. (7) Dilute the oligonucleotide-barcoded primary antibody to the optimal concentration (typical range 0.1-10 μg / mL) in blocking buffer and apply at room temperature for 1-3 h or at 4 °C. Spend the night there. (8) Thoroughly wash 4x with stirring. (9) Wash thoroughly with PBS or blocking buffer 4× with stirring to remove unbound primary antibodies. (10) Dilute the first and second labeled portions containing the complementary barcode sequence (i.e., the barcode complementary to the primary antibody) in a composite buffer. (11) The first and second labeled portions, diluted in a composite buffer, are applied to the sample and allowed to hybridize with their respective barcodes on the primary antibody that is bound to the target. This step can be performed at room temperature for 5–60 min with stirring or at a higher temperature. (12) Wash 4x with a compound buffer while stirring to remove unbound first and second labeled portions. DAPI may also be included during this step for nuclear staining. (13) Optionally, the sample is read out and / or imaged before applying the dissociation conditions. This readout / image can be used as a baseline and can improve the quality of the assay, but is not necessary in many cases. (14) Equilibrate the sample to decomposition conditions by washing the sample 4x with a suitable volume (e.g., 4-5 flow cell volumes) of decomposition buffer while stirring or by rinsing the sample. (15) Hybridization of the first and second marker portions is allowed to occur under decomposition conditions, which may typically take 5-60 minutes. (16) Optionally, apply a drop of sealing agent to a microscope slide and invert a coverslip containing cells or biological samples onto the drop. Gently press the sample with tweezers to distribute the sealing agent well, but do not compress the sample. Once cured, the sample is ready for microscopic observation. (17) For optical grade substrates, dishes or flow cells, samples can be imaged directly in decomposition buffer, PBS or any other imaging buffer of choice, provided that the imaging buffer does not contain any substance that interferes with the formation of the double strand between the first and second labeled portions. (18) Optionally, before and after the formation of a double chain between the first and second marker portions, i.e. under recombination and decolombination conditions, the pixel intensity is compared to calculate the multiple change or ratio of the (FRET) signal at the pixel level, thereby assessing the proximity of the marker and, in turn, the proximity of the analyte.

[0081] While the provided embodiments are all microscopic or imaging embodiments in which samples are imaged, it is clear that the invention can also be used for non-imaging detection. For example, the method can be used to detect the presence of two analytes on cells such as immune cells, bacteria, or non-cellular particles such as viruses or exosomes. Therefore, the method can be adapted to analyze or sort cells or particles using flow cytometry and fluorescence-activated cell sorting (FACS). Similarly, readout can be performed via plate reading (FRET signal). Alternatively, the method can be configured for lateral flow assays and can be used to detect pathogens or hormones with very high specificity.

[0082] This method can be further performed in a cyclical manner, with one cycle consisting of staining, readout / imaging, and inactivation.

[0083] Further discussion on composite conditions and solution composite conditions

[0084] The nucleic acid backbone of the first and second labeled portions can preferably be 20-120 nt in length and contain one or more guest molecules, for example, 1-5 guest molecules per 10 nt (nt = nucleotide).

[0085] On the other hand, a biomarker is provided for analyzing biological samples containing multiple target analytes. The biomarker comprises a marker as described above, the marker having a first labeling portion and a second labeling portion. The biomarker further comprises a first labeling portion and a second labeling portion, the first labeling portion comprising a first affinity reagent and the first labeling portion, and the second labeling portion comprising a second affinity reagent and the second labeling portion. Each of the first affinity reagent and the second affinity reagent is configured to specifically bind to one of the target analytes of the biological sample.

[0086] The biomarker can efficiently determine the distance or proximity between two target analytes. It can also accurately determine the presence and / or location of two or a single target analyte in a biological sample. The biological sample can be a cellular sample, such as a tissue section. Further embodiments include biopsy samples, such as liquid biopsies or tissue biopsies.

[0087] The affinity reagent for the marker can be an antibody, an antibody fragment, an amino acid-based or nucleic acid-based aptamer, or a linear nucleic acid. This enables the detection of a wider variety of target analytes. Specifically, the nucleic acid backbone of the labeled portion is attached to, preferably covalently attached to, its respective affinity reagent.

[0088] The affinity reagents of the markers can bind to a single target analyte or two target analytes. In a first case, the first affinity reagent can be configured to specifically bind to a first region or epitope of the single target analyte, and the second affinity reagent can be configured to specifically bind to a second region or epitope of the single target analyte. By requiring both marker portions to bind to the single target analyte, the presence and / or location of the single target analyte can be precisely determined, specifically with increased specificity. In a second case, the first affinity reagent can be configured to specifically bind to a first target analyte, and the second affinity reagent can be configured to specifically bind to a second target analyte. This enables the determination of the distance or proximity between the first and second target analytes.

[0089] Preferably, the marker comprises an anchor oligonucleotide configured to anchor the marker to a solid support. This enables the marker to be used, for example, in flow assays. Specifically, the anchor oligonucleotide may be (covalently) attached to either the first or second affinity reagent. The anchor oligonucleotide may include an affinity moiety configured to specifically bind to a corresponding affinity moiety arranged on the solid support.

[0090] On the other hand, a method for analyzing a biological sample is provided. The method includes introducing at least one of the aforementioned biomarkers into the biological sample. The step of introducing the at least one biomarker may optionally include a waiting period to allow the at least one biomarker to bind to its respective target analyte in the biological sample. After the introduction of the at least one biomarker, any biomarkers not bound to the target analyte may optionally be removed from the biological sample, for example, by washing. Furthermore, optionally, recombination conditions are applied to the biological sample at least during the introduction of the at least one biomarker and the removal of the unbound biomarkers. The recombination conditions enable the formation of a complex between the guest molecule and the host molecule, thereby preventing hybridization of the first and second nucleic acid backbones of the biomarker. The method further includes applying decomplexing conditions to the biological sample, specifically after the introduction of the at least one biomarker into the biological sample. The decomplexing conditions may include, for example, adjusting the salt concentration, adjusting the temperature, and / or adding a competing guest molecule not attached to the biomarker. This dissociates or removes the guest-host complex and allows hybridization of the first and second nucleic acids of the biomarker portion. The method further includes generating optical readouts of the biological sample containing the marker, for example by means of a microscope, such as a fluorescence microscope.

[0091] Specifically, the at least one biomarker introduced into the biological sample comprises at least one host molecule. Therefore, it can form the complex with the guest molecule. The step of introducing the at least one biomarker may include adding individual portions of the at least one biomarker to the sample, specifically, adding them separately over time.

[0092] Typically, multiple biomarkers can be introduced into the biological sample. These biomarkers are specific to their respective target analytes or paired target analytes to simultaneously identify a large number of (different or identical) target analytes. Preferably, the target analytes are identified and / or located within the biological sample based on the marking, specifically the marked portion, of the biomarker associated with the target analyte in an optical reading. The biomarkers can be physically constructed prior to introduction into the biological sample. Alternatively, affinity reagents can be barcoded using oligonucleotide barcodes and initially introduced into the biological sample, and the biomarkers containing complementary barcoded oligonucleotides can be introduced into the biological sample in subsequent steps and then attached to their respective affinity reagents to form the respective biomarkers within the biological sample.

[0093] In another aspect, a kit for analyzing biological samples is provided, comprising the aforementioned marker and at least one host molecule, said host molecule being configured to form a complex with at least one guest molecule of said marker. In another aspect, the use of the aforementioned marker, or the use of the aforementioned biomarker, or the use of the aforementioned kit, is provided in a proximity assay for analyzing biological samples.

[0094] The marker, the kit, and the method described herein, as well as the use of the method, all have the same advantages as the marker. Furthermore, the marker, the kit, and the method may supplement the features of the marker described herein, specifically the features of the dependent claims of the marker. Attached Figure Description

[0095] The specific implementation scheme is described below with reference to the accompanying drawings, in which:

[0096] Figure 1 It is a schematic diagram of the first and second nucleic acid backbones containing the host molecule and the guest molecule.

[0097] Figure 2 This is a schematic diagram of biomarkers used for analyzing biological samples.

[0098] Figure 3 It is based on Figure 2 A schematic diagram of the logo.

[0099] Figure 4 It is based on Figure 2 A schematic diagram of the logo.

[0100] Figure 5 This is a schematic diagram of the first and second marked parts.

[0101] Figure 6 This is a schematic diagram of the first and second marked portions according to the second embodiment.

[0102] Figure 7 This is a schematic diagram of the marker under the combined conditions according to the second implementation scheme.

[0103] Figure 8 It is based on Figure 7 A schematic diagram of markers under decomposition conditions.

[0104] Figure 9 This is a schematic diagram of several markers under combined and uncombined conditions according to a further implementation scheme.

[0105] Figure 10 This is a schematic diagram of two markers containing secondary affinity reagents.

[0106] Figure 11This is a flowchart of a method for amplifying barcode oligonucleotides used as biomarkers.

[0107] Figure 12 This is a schematic diagram of a marker comprising a tree-like oligonucleotide-based structure configured to bind multiple first and second labeling portions.

[0108] Figure 13 This is a schematic diagram of an hgPHA assay microarray with speckled labels on a solid support under combined conditions.

[0109] Figure 14 It is based on Figure 13 A schematic diagram of the microarray used for hgPHA determination under decomposition conditions.

[0110] Figure 15 This is a schematic diagram of an hgPHA assay that uses a combination of a trapping (micro) array labeled on a solid support under combined conditions.

[0111] Figure 16 It is based on Figure 15 A schematic diagram of the hgPHA determination method under decomposition conditions.

[0112] Figure 17 This is a schematic diagram of a marker attached to a solid substrate.

[0113] Figure 18 This is a schematic diagram of a biomarker containing tertiary antibodies attached to a solid-phase support.

[0114] Figure 19 This is a schematic diagram of a biomarker containing tertiary antibodies attached to a solid-phase carrier according to the second implementation scheme.

[0115] Figure 20 This is a schematic diagram of a biomarker containing tertiary antibodies attached to a solid-phase carrier according to the third implementation scheme.

[0116] Figure 21 This is a schematic diagram of cell samples and markers used to determine the proximity between two cells in a cell sample, and...

[0117] Figure 22 This is a flowchart of a method for analyzing samples with markers. Detailed Implementation

[0118] Figure 1This is a schematic diagram of a first nucleic acid backbone 100 and a second nucleic acid backbone 102, such as a marker used for analyzing biological samples. The first nucleic acid backbone 100 contains a guest molecule 104. Both the first nucleic acid backbone 100 and the second nucleic acid backbone 102 are polynucleotides. The first nucleic acid backbone 100 and the second nucleic acid backbone 102 are at least partially complementary to each other. This enables hybridization of the first nucleic acid backbone 100 and the second nucleic acid backbone 102, such as... Figure 1 As shown on the right.

[0119] One or both of the first nucleic acid backbone 100 and the second nucleic acid backbone 102 may contain nucleic acid analogs, such as xenobiotic nucleic acids (XNA), peptide nucleic acids (PNA), locked nucleic acids (LNA), morpholino (MO), or phosphate-thiolated DNA (PT-DNA). Alternatively, one or both of the first nucleic acid backbone 100 and the second nucleic acid backbone 102 may be naturally occurring nucleic acids. One or both of the first nucleic acid backbone 100 and the second nucleic acid backbone 102 may further contain modified nucleobases.

[0120] Guest molecule 104 is covalently attached to one of the nucleotides (denoted as "N") of the first nucleic acid backbone 100 (and / or the second nucleic acid backbone 102). The coupling of the guest molecule can be as described by Xiao et al. (2022), which "introduced the guest molecules into the 4′-amino position of cytosine and the 6′-amino position of adenine." (See Xiao et al., 2022). Figure 1As shown, this means that the modified bases can still pair. The ratio of the number of nucleotides in guest molecule 104 to the number of nucleotides in the first nucleic acid backbone 100 or the second nucleic acid backbone 102 is preferably selected to allow the first nucleic acid backbone 100 and the second nucleic acid backbone 102 to hybridize with each other. For example, one guest molecule is provided for every 5 to 10 nucleotides of each nucleic acid backbone. The length of the nucleic acid backbone, the number of guest molecules, the type of guest molecules and their affinity for the host molecule, and the buffer composition of the recombination buffer and the decombination buffer (e.g., salt, formamide, dextran, blocking nucleic acid concentration) can each be modified to adjust the melting temperature of the nucleic acid backbone under recombination (Tmc) and decombination (Tmdc) conditions. This can be achieved by allowing the nucleic acid backbone to hybridize only under decombination conditions at room temperature. Alternatively, recombination conditions may include temperatures higher than decombination conditions. For example, the binding of the first and second labeled portions can be carried out at 30°C, 37°C, 50-65°C, or even higher temperatures. The choice of the appropriate temperature for this step will also depend on the specific application and sample type. For example, for immunofluorescence in tissue sections, it is preferable to keep the temperature well below 60°C to avoid the formation of a fluorescence background. In other assays, such as for liquid biopsies or lysates, this may not be a problem, and therefore higher temperatures may be acceptable. In most assays, the first and second nucleic acid backbones of the hybridization can have melting temperatures in the range of 45–65°C and / or 65–90°C.

[0121] In this regard, it is clear that after primary affinity reagents bind to their targets (e.g., primary antibodies and their respective target analytes), the affinity reagents may crosslink with the analytes or other proteins in the sample. Alternatively or additionally, the sample may be infused with a polymeric component such as acrylamide, and a gel may form in the sample. Such a gel may have reactive groups that allow crosslinking and may also promote sample swelling. Those skilled in the art will understand this technique from the field of expansion microscopy and may readily extend and / or modify the methods provided herein without departing from the invention.

[0122] Figure 1The left side provides the complexation conditions, specifically a high concentration of host molecule 106. Guest molecule 104 and host molecule 106 are configured to form guest-host complex 108. For guests with very high affinity such as adamantane (AD) or adamantane derivatives, this guest-host complex 108 lowers the melting temperature, preferably to / from about 10 °C for each incorporated guest molecule. For ferrocene (FC), Xiao et al. (2022) reported that for 15nt oligonucleotides with a single modified nt, ΔTm was about -9.6 °C when the CB[7] concentration ranged from 1 to 300 μM. For ferrocene (FC), ΔTm was 0 to -8.5 °C for the same sequence in the range of 1 to 300 μM. Another 19nt oligonucleotide containing two modified guest bases has Tm values ​​of 55.2 (AD) and 55.5 (FC) in the absence of CB[7], which decrease to 30.5 °C (AD) and 37 °C (FC) at 300 μM, respectively. This example well illustrates the useful length of the first and second nucleic acid backbones, the useful number of guest molecules per unit length (nt), and the expected difference in melting temperature under recombination and derecombination conditions.

[0123] The presence of host molecule 106 leads to the dissociation of the first nucleic acid backbone 100 and the second nucleic acid backbone 102, or prevents the initial hybridization of the first nucleic acid backbone 100 and the second nucleic acid backbone 102.

[0124] exist Figure 1 On the right side, the conditions for recombination are provided, specifically a low concentration of the host molecule 106. As described above, this allows hybridization of the first nucleic acid backbone 100 and the second nucleic acid backbone 102.

[0125] The interaction between guest and host molecules is a fundamental concept in supramolecular chemistry, where the host molecule provides the structural framework for encapsulating or binding the guest molecule through non-covalent interactions. These interactions can include hydrogen bonds, van der Waals forces, electrostatic interactions, and hydrophobic effects. The specificity and strength of the interaction depend on the complementarity between the host and guest molecules in terms of shape, size, and functional groups. For example, cyclodextrins can act as host molecules capable of forming inclusion complexes with a variety of guest molecules, such as aromatic compounds and fatty acids.

[0126] Another example is cucurbit[n]urea, a class of macrocyclic molecules that can encapsulate guests ranging from small metal ions to large organic molecules such as drugs and dyes to form highly stable complexes. Examples of small organic molecules that form stable complexes with cucurbit[7]urea include adamantane and adamantane derivatives (AD) and ferrocene and ferrocene derivatives (FC). Examples of aromatic compounds that form stable complexes with cucurbit[7]urea include methyl viologen and 1,4-dimethoxybenzene. Specific examples of suitable guest molecules include 1-adamantane methylamine (AD), ferrocene methylamine (FC), 1,4-phenylenediamine (BA), and 4-tert-butylbenzylamine (TB), as reported by Xiao et al. 2022. Importantly, it is noted that the affinity of these guest molecules may change when coupled with DNA, as further reported by Xiao et al. 2022, by isothermal titration calorimetry (ITC) analysis, “CB[7] still binds to AD (K) in the context of DNA strands.” a =1.31×10 7 M -1 ) and FC(K a =2.95×10 6 M -1 "It binds specifically to the BA and TB groups, but exhibits weaker affinity for them." The effect on the stability of the duplex varies strongly with affinity, as further described by Xiao et al., and summarized in Table 1 therein. More suitable guest molecules can be found by using modern molecular docking methods.

[0127] By applying recombination or de-recombination conditions to the host molecule and guest molecules 104 and 106, the binding or dissociation of the host molecule and guest molecules 104 and 106 can be achieved. Subsequently, this enables the first nucleic acid backbone and the second nucleic acid backbone 100 and 102 to hybridize and form a double strand, or to melt and dissociate.

[0128] An example of recombining conditions could be the addition of a competing guest molecule, thereby causing the host molecule 106, which has complexed with guest molecule 104, to instead form a complex with the competing guest molecule. The competing guest molecule can be of the same molecular species (in its free form only) or of different molecular species. In this regard, FC could be used as the guest molecule to modify the first and / or second nucleic acid backbone, which might require a relatively high concentration of the host molecule as detailed above under recombining conditions, e.g., ~100-300 μM, followed by AD as the competing guest molecule.

[0129] Other examples of parameters that could lead to recombination and decomposition or may affect the affinity of guest molecules and host molecules 104, 106 are salt concentration, pH, and temperature.

[0130] Figure 2This is a schematic diagram of a biomarker 200 used for analyzing biological samples. Biomarker 200 includes a first biomarker portion 202 and a second biomarker portion 204. The first biomarker portion 202 may include a first labeling portion having a first nucleic acid backbone 206, a plurality of first labeling portions 208, and a plurality of guest molecules 104. The first labeling portions 208 and guest molecules 104 may attach to the first nucleic acid backbone 206. In addition, a plurality of host molecules 106 are provided. Each guest molecule 104 forms a complex 108 with one of the host molecules 106.

[0131] The second marker portion 204 includes a second labeling portion having a second nucleic acid backbone 210, and a plurality of second labeling portions 212 attached to the second nucleic acid backbone 210.

[0132] The first nucleic acid backbone 206 and the second nucleic acid backbone 210 are at least partially complementary to each other. This enables hybridization of the first nucleic acid backbone 206 and the second nucleic acid backbone 210. Figure 2 The image shows marker 200 together with host molecule 106. The resulting host-guest complex 108 prevents hybridization between the first nucleic acid backbone 206 and the second nucleic acid backbone 210.

[0133] The first biomarker portion 202 further includes a first affinity reagent 214, which is configured to specifically bind to the first target analyte 216. Similarly, the second biomarker portion 204 further includes a second affinity reagent 218, which is configured to specifically bind to the second target analyte 220.

[0134] The first label portion 202 is attached to the first affinity reagent 214 via a barcoded oligonucleotide 222. Similarly, the second label portion 204 is attached to the second affinity reagent 218 via a barcoded oligonucleotide 224. Each barcoded oligonucleotide 222, 224 is preferably specific to its respective affinity reagent 214, 218 and / or its respective label portion 202, 204. Specifically, the first and second affinity reagents 214, 218 are antibodies.

[0135] The first marker portion 202 binds to the first target analyte 216 via the first affinity reagent 214. Similarly, the second marker portion 204 binds to the second target analyte 220 via the second affinity reagent 218.

[0136] exist Figure 2In an exemplary embodiment, target analytes 216 and 220 come into contact, for example, due to affinity interactions between them. This results in marker portions 202 and 204 being in proximity once bound to target analytes 216 and 220. As described above, despite the proximity of the first nucleic acid backbone 206 and the second nucleic acid backbone 210, they do not hybridize due to the host-guest complex 108. This enables easy handling of marker 200, specifically its marker portions 202 and 204, during analysis of biological samples containing target analytes 216 and 218. For example, if marker portions 202 and 204 are not bound to target analytes 216 and 218, the host-guest complex 108 prevents undesirable, uncontrolled hybridization of the first nucleic acid backbone 206 and the second nucleic acid backbone 210.

[0137] Alternatively or additionally, guest molecule 104 may attach to the second nucleic acid backbone 210.

[0138] The first labeled portion 208 and the second labeled portion 212 can be fluorophores that act as FRET donors or FRET acceptors, respectively. Therefore, each of the first labeled portions 208 can form an FRET pair with one of the second labeled portions 212. Because the first nucleic acid backbone 206 and the second nucleic acid backbone 210 are in... Figure 2 In the state shown, there is no hybridization, therefore the first nucleic acid backbone 206 and the second nucleic acid backbone 210 are not adjacent to each other. Specifically, the first labeled portion 208 and the second labeled portion 212 are not within the distance that allows FRET to occur.

[0139] Figure 3 This is a schematic diagram of marker 200 in the absence of the main molecule 106. Therefore, in Figure 3 In the state shown, no subject-object complex 108 is formed. For simplicity, Figure 3 Only two exemplary guest molecules 104 are shown in the figure.

[0140] In the absence of host molecule 106, the first nucleic acid backbone 206 and the second nucleic acid backbone 210 can hybridize, such as Figure 3 As shown. Hybridization of the first nucleic acid backbone 206 and the second nucleic acid backbone 210 brings the first labeled portion 208 and the second labeled portion 212 into proximity, thereby allowing FRET to occur between the first labeled portion 208 and the second labeled portion 212. Therefore, the proximity of the target analytes 216 and 220 can be determined by the occurrence of FRET between the first labeled portion 208 and the second labeled portion 212, for example, by fluorescence microscopy. Figure 3 In this context, FRET may occur between all first marked portions 208 and second marked portions 212.

[0141] like Figure 2 As shown, in a method for analyzing biological samples, marker 200, specifically marker portions 202 and 204, can initially be introduced into the biological sample under composite conditions, forming a guest-host complex 108 with the present host molecule 106. After waiting for a suitable period of time for the marker portions 202 and 204 to bind, any marker portions 202 and 204 that have not bound to their respective target analytes 216 and 218 can be removed, for example, by washing the biological sample. These steps are preferably performed under composite conditions.

[0142] Subsequently, decomposition conditions can be applied, thereby causing the decomposition of the guest-host complex 108. Specifically, this results in the removal of the host molecule 106 from the marker 200. The decomposition conditions may include a temperature change for the decomposition of the guest-host complex 108, and subsequent removal of the host molecule 106 by, for example, washing the biological sample. Alternatively or additionally, a competing guest molecule may be introduced into the biological sample. Figure 3 The marker 200 after removing the main molecule 106 is shown.

[0143] Removal of the host molecule 106 enables hybridization of the first nucleic acid backbone 206 with the second nucleic acid backbone 210. Subsequently, an optical reading of the biological sample containing the marker 200 can be generated. The optical reading can be an image, such as an image generated by a fluorescence microscope.

[0144] Optical readout enables the determination of whether target analytes 216 and 220 are adjacent to each other. Specifically, by observing the changes in the optical properties of the first labeled portion 208 and the second labeled portion 212 due to FRET, optical readout can be used to determine whether FRET has occurred between them. Furthermore, at least one suitable optical readout can be used to determine whether unbound FRET pairs in the biological sample (i.e., the first labeled portion 208 and the second labeled portion 212 are not adjacent) have been bound to the target analyte. For example, this can be achieved by exciting the labeled portions acting as donors with suitable excitation light and determining whether these labeled portions excite intrinsic fluorescence according to their inherent characteristics. Another readout method is to excite the labeled portions acting as acceptors with appropriate excitation light and determine whether these labeled portions excite intrinsic fluorescence according to their inherent characteristics.

[0145] Figure 4 This is a schematic diagram of marker 200, in which... Figure 2 and Figure 3 In comparison, target analytes 216 and 220 are further apart. Figure 3Similarly, since the host molecule 106 is absent, the first nucleic acid backbone 206 and the second nucleic acid backbone 210 can hybridize. However, the large distance between the target analytes 216 and 220 results in only partial hybridization between the first nucleic acid backbone 206 and the second nucleic acid backbone 210, specifically with... Figure 3 Compared to the states shown, target analytes 216 and 220 are in contact with each other, indicating a lower degree of hybridization. For simplicity, Figure 4 The diagram shows only two exemplary guest molecules 104. The marker 200 may contain more guest molecules 104.

[0146] Partial hybridization of the first nucleic acid backbone 206 and the second nucleic acid backbone 210 results in some of the first labeled portions 208 and the second labeled portions 212 being at a greater distance from each other. Specifically, these first labeled portions 208 and the second labeled portions 212 may be at a distance from each other at which FRET does not occur between them. Therefore, with Figure 3 Compared to the state shown, the overall observable FRET efficiency is reduced. This reduced FRET efficiency can be observed when generating optical readout of biological samples using marker 200.

[0147] In summary, and for specific reference Figure 3 and Figure 4 It can be seen that the distance between target analytes 216 and 220 directly affects the degree of hybridization between the first nucleic acid backbone 206 and the second nucleic acid backbone 210, and therefore also directly affects the overall FRET efficiency between the first labeled portion 208 and the second labeled portion 212. Therefore, the FRET efficiency between the first labeled portion 208 and the second labeled portion 212 is proportional to the distance between target analytes 216 and 220, and can be used to determine the distance or proximity between target analytes 216 and 220.

[0148] Therefore, the spatial strictness of this assay depends on the size of the affinity reagent, specifically the distance between complementary sites: epitope interfaces, or more generally, the distance between the site where the target analyte binds to the affinity reagent and the site where the first or second labeled moiety is coupled (coupling site). Furthermore, spatial strictness also depends on the length of the linker and / or barcode oligonucleotide, as well as the length of the first and second labeled moiety. Thus, the spatial strictness of the hgPHA assay is tunable over a wide range, for example, from a few nanometers to several hundred nanometers. For assays with extremely low spatial strictness (which can be used to detect cell proximity in tissues), the first and second labeled moiety may further include non-nucleic acid linkers, such as PEG linkers.

[0149] Figure 5This is a schematic diagram of a first labeling portion 500 and a second labeling portion 502 that can be used in a FRET-based hgPHA assay. Each of the first and second labeling portions 500 and 502 contains at least partially complementary nucleic acid backbones 504 and 506. Figure 5 This is shown as dotted line 504 and dashed line 506. For the FRET-based hgPHA assay, the first labeling portion 500 is coupled to one of the FRET donor 508 and FRET receptor 510, while the second labeling portion 502 is coupled to the other of the FRET donor 508 and FRET receptor 510. Suitable FRET pairs are described in the section on FRET above.

[0150] The first labeled portion 500 further includes a guest molecule 104. In the configuration shown by reference numeral 500a, the first labeled portion 500, specifically the guest molecule 104, decomposite from the host molecule 106. In the configuration shown by reference numeral 500b, the first labeled portion 500, specifically the guest molecule 104, recombines with the host molecule 106.

[0151] In the following figure, the first marking portion 500 and the second marking portion 502 can be used Figure 5 The respective semicircles shown are used to illustrate the concept.

[0152] As an alternative or supplementary method to FRET, which allows for the detection of hybridization, the hgPHA assay can also be established based on the dequenching of strongly quenched markers.

[0153] For example, Figure 6This is a schematic diagram of a first labeling portion 600 and a second labeling portion 602. Each of the first and second labeling portions 600 and 602 contains at least partially complementary nucleic acid backbones 504 and 506. The first labeling portion 600 does not contain a labeling portion. However, the second labeling portion 602 is coupled to a strongly (self-)quenching labeling portion 604, such as ATTO647N or other hydrophobic dyes. These hydrophobic dyes freely aggregate and quench each other when coupled to the highly flexible single-stranded DNA backbone 506. This quenching strongly reduces the emission of the labeling portion 604 of the second labeling portion 602. For example, a label based on a ~120 nt ssDNA oligonucleotide containing 10 ATTO647N dye molecules will exhibit approximately the same or lower emission as a single ATTO647N dye molecule. This single-stranded labeling portion 602 forms a double strand with the complementary strand 504 of the first labeling portion 600, resulting in a strong increase in fluorescence emission that is easily detected. This is further described in European Patent Application No. 24177155.9, the entire contents of which are incorporated herein by reference. Examples commonly used for analyzing hydrophobic labeled portions of biological samples include ATTO 390, ATTO 425, ATTO 550, ATTO Rho12, ATTO 633, and ATTO647N. Specifically, the above dyes may exhibit undesirable aggregation when aggregated on a single-stranded DNA backbone with a dye spacing of 8 nt.

[0154] The first labeled portion 600 further includes a guest molecule 104. In the configuration shown by reference numeral 600a, the first labeled portion 600, specifically the guest molecule 104, decomposite from the host molecule 106. In the configuration shown by reference numeral 600b, the first labeled portion 600, specifically the guest molecule 104, recombines with the host molecule 106.

[0155] Similar to markers 500 and 502, markers 600 and 602 can be used in subsequent figures. Figure 6 The respective semi-circular shapes are schematically shown in the diagram.

[0156] Figure 7This is a schematic diagram of a dequenching-based hgPHA assay according to another embodiment including marker 700. Marker 700 comprises first and second labeling portions 600, 602, which are adjacent to each other when affinity reagents 214, 218 bind to their respective interacting target analytes 216, 220. Under recombination conditions, the first labeling portion 600, comprising guest molecule 104, is fully or at least substantially recombinated with host molecule 106, such as CB[7], thereby inhibiting hybridization with the complementary second labeling portion 602. The second labeling portion 602 comprises a variety of hydrophobic or moderately hydrophobic dye molecules 604, such as ATTO 390, ATTO 425, ATTO 550, ATTO Rho12, ATTO 633, ATTO 647N, ATTO 680, and ATTO 700. Such hydrophobic or moderately hydrophilic dye molecules tend to aggregate and self-quench when polymerized on a nucleic acid backbone 506, such as the second labeling moiety 602. As described in European Patent Application No. 24177155.9, this self-quenching on the ssDNA backbone can be avoided by forming a double strand or a double-stranded nucleic acid complex. This behavior is used as a sensing mechanism for dequenching-based hgPHA assays. A double strand can only be formed in the presence of the first labeling moiety 600 and the second labeling moiety 602, i.e., only when they are present and adjacent, and only under deconjugation conditions, resulting in a decrease in the flexibility of the nucleic acid backbone double strand or an increase in its persistent length.

[0157] Figure 8 A marker 700 is shown, wherein a double strand 800 is formed between the labeled portions 600 and 602. The corresponding increase in persistence length reduces the ability of dye-dye interactions and self-quenching. In a particularly preferred embodiment, the first labeled portion 600 is approximately 100-120 nt in length and contains 5-20 guest molecules 104, while the second labeled portion 602 is approximately 100-120 nt in length and contains 5-20 dye molecules 604. For example, the dye molecules can be arranged at a distance of 5-15 nt, more preferably 8 nt. Using dequenching as a detection mechanism for hybridization or double strand formation between the first labeled portion 600 and the second labeled portion 602 provides the particular advantage of requiring only one labeled portion, such as the second labeled portion 602, to be coupled to the dye molecule 604. This is advantageous for setting up hgPHA multiplexing assays where multiple colors are used, for example, different second labeled portions 602 can contain different fluorescent dyes. A range of dyes, from hydrophobic to moderately hydrophilic, are well-suited for setting up dequenching-based hgPHA assays. This also reduces the cost of the assay. Notably, FRET and dequenching can also be combined in a single assay.

[0158] Figure 9 This is a schematic diagram of various hgPHA forms. The left side shows exemplary markers 900a, 900b, 900c, and 900d bound to the target analyte under recombination conditions. The right side shows the respective markers 902a, 902b, 902c, and 902d under decoupling conditions. The marking portions of markers 900a, 900b, 900c, 900d, 902a, 902b, 902c, and 902d are... Figure 5 and Figure 6 The semi-circular symbol shown is illustrated.

[0159] The first row shows markers 900a and 902a, which are suitable for detecting protein-protein proximity as a measure of protein-protein interactions. In this example, two antibodies are shown, but other affinity reagents that bind to proteins, such as affimers, aptamers, nanobodies, and single-domain antibodies, are equally suitable. Similarly, in addition to proteins, the target analyte can also be, for example, a metabolite or any other class of analytes.

[0160] The second row shows biomarkers 900b and 902b, which contain one or more pairs of antibodies for detecting a single target analyte. This can be used to improve specificity or to probe post-translational modifications, alternative splicing, or proteolytic processing / degradation.

[0161] The third row shows biomarkers 900c and 902c (one containing a protein-binding affinity reagent (e.g., an antibody), and another containing an affinity reagent (e.g., an oligonucleotide probe) that binds to a nucleic acid target. These biomarkers can be used to detect, for example, RNA-protein or DNA-protein interactions, and to study transcription factor binding and its effects on gene regulation, histone binding or modification at specific loci, and to assess integration sites of gene modifications. The latter is particularly relevant to the development and quality control of cell and gene therapies. Furthermore, this format makes it suitable for use with CRISPR and similar systems to assess target binding.

[0162] The fourth row shows biomarkers 900d and 902d for detecting nucleic acid targets, which can be used to detect mRNA with high specificity.

[0163] Figure 10This is a schematic diagram illustrating two biomarkers 1000 and 1002 based on a modified indirect immunofluorescence assay. Biomarkers 1000 and 1002 comprise labeled portions 202 and 204, paired primary antibodies 214 and 218 (configured to bind to target analytes 216 and 220 in biological samples), and secondary antibodies 1004 and 1006. Secondary antibodies 1004 and 1006 can specifically bind to one of the primary antibodies 214 and 218. In the case of biomarker 1000, labeled portions 202 and 204 can be directly conjugated to secondary antibodies 1004 and 1006. Alternatively, in the case of biomarker 1002, labeled portions 202 and 204 can be indirectly conjugated to secondary antibodies 1004 and 1006, for example, by hybridization with their respective barcode oligonucleotides 222 and 224. The use of barcoded affinity reagents is desirable in many cases because it enables antibody (one or both of primary and secondary antibodies) binding under decoupling conditions, which can be buffers typically used in immunofluorescence protocols for washing, antibody binding, or blocking. In this case, the first and second labeling portions 202, 204 can be applied to the sample in separate steps under decoupling conditions.

[0164] In addition, such as Figure 11 As illustrated in the schematic workflow, the use of primary and secondary affinity reagents 214 and 218 for barcode amplification allows for the amplification of barcodes 222 and 224 via enzymatic methods (including loop-mediated isothermal amplification (LAMP), rolling circle amplification (RCA), polymerase chain reaction (PCR)) or non-enzymatic reactions (e.g., hybridization chain reaction (HCR), primer exchange reaction (PER)), or by using additional oligonucleotides (such as “L-adaptor” 1200) (see [link to documentation]). Figure 12 This allows for amplification by generating a dendritic structure 1202, or by attaching DNA nanostructures (such as DNA origami), or by generating DNA nanostructures in situ (such as DNA brick-based structures or similar structures). Therefore, barcode oligonucleotides 222 and 224 can be amplified in situ to generate multiple attachment sites for hybridization marker motifs (particularly their respective backbones).

[0165] The workflow may include the following steps:

[0166] Step 1100: Introduce oligonucleotide-barcoded affinity reagents (ARs) and allow them to bind to the target analyte. Then wash away any unbound ARs.

[0167] Step 1102: Optionally, the barcode sequence is amplified using, for example, LAMP or RCA; optionally, amplification can be achieved by constructing a dendritic DNA-based structure or by hybridization chain reaction (HCR).

[0168] Step 1104: Hybridize the first and second marker portions with the amplified barcode sequence, avoiding hybridization between the first and second marker portions by applying a recombination condition. Optionally, perform “baseline” readout / imaging.

[0169] Step 1106: By applying a dissociation condition, hybridization of the first and second nucleic acid backbones of the first and second labeled portions is allowed. Unbound labeled portions are washed away. Readout / imaging is then performed.

[0170] Optionally, after step 1106, the marked portion can be removed by applying an inactivating agent or a removing agent (e.g., DNase I), and the process can be repeated starting from step 1100.

[0171] Figure 12 An example illustrating how a tree structure 1202 can be constructed using the "L-adaptor" oligonucleotide 1200 is shown. For this purpose, Figure 12 The marker 1204 shown includes a first marker portion having a first attaching oligonucleotide 1206, which contains a plurality of attaching sequences, and the backbone 206 of the first marker portion 202 is partially complementary to these attaching sequences. Similarly, the marker 1204 includes a second marker portion having a second attaching oligonucleotide 1208, which contains a plurality of attaching sequences, and the backbone 210 of the second marker portion 204 is partially complementary to these attaching sequences. Therefore, the plurality of first and second marker portions 202, 204 can hybridize with their respective attaching oligonucleotides 1206, 1208.

[0172] Figures 13 to 22 This is a schematic diagram illustrating further embodiments of the disclosed markings and the uses of the markers. Specifically, Figures 13 to 17 Markers 1300 and 1500 are shown attached to solid supports 1304 and 1700. For this purpose, markers 1300 and 1500 comprise anchor oligonucleotides 1302, one end of which may be attached (covalently) to an antibody 214 of one of the marker moieties of markers 1300 and 1500. The other end may be attached to the respective solid supports 1304 and 1700, such as the bottom of a flow cell or a glass slide 1304 having multiple microarray spots 1306. Each spot 1306 may contain multiple markers 1300. Markers 1300 may adhere to spots 1306, for example, by simply drying the marker moieties onto a glass slide coated with poly-L-lysine, or by connecting to the spots through biotin-streptavidin or any other pairwise high-affinity interaction suitable for substantially stably coupling the marker moieties to the glass slide. The marker 1300 may include marking portions 600, 602, and is configured for use as follows: Figure 13 and 14The dequenched hgPHA shown is illustrated, and / or can be configured for FRET-based hgPHA (not shown). Because the first biomarker portion is immobilized, this configuration can be used to capture the target analyte 1308 onto the first affinity reagent 214, for example, a first antibody to capture the analyte, which may be a surface protein expressed on a bacterial pathogen (e.g., a pathogenic E. coli strain) or a virus (such as the S-protein), or may be a component of an exosome. Therefore, this embodiment is particularly suitable for diagnostic purposes. Figure 13 and Figure 14 The hgPHA microarray shown can contact one or more biological samples in different ways depending on the user's intent. In one embodiment, the user may wish to measure multiple proteins from a single biological sample with, for example, very high specificity, in which case each spot may contain a different biomarker. In other cases, it may be desirable to test a large number of biological samples against the same target analyte 1308. For example, for viral infections (such as avian influenza, COVID-19) or certain bacterial pathogens (such as EHEC), when testing many test objects, the entire chip may be applied by immersing the hgPHA microarray 1304 or by attaching a flow cell and creating a hermetically sealed (microfluidic) assembly. The latter is particularly useful because it is easy to automate and requires only a small amount of sample and reagents. Alternatively, a microarray may contain spots with different first and / or second biomarker portions.

[0173] Figure 13 The marker 1300 is shown under complexation conditions, wherein the host molecule 106 forms a complex with the guest molecule 104 of the labeled portion 600. Figure 14 The marker 1300 is shown under decomposition conditions, where the host molecule 106 has been removed from the marker 1300. Similarly, Figure 15 and Figure 16 The marker 1500, which includes marking portions 500 and 502, is shown under both composite and decomposition conditions.

[0174] Figure 17 A marker 1500 is shown attached to a microbead solid support 1700. The microbeads 1700 make the marker 1500 easy to handle, especially in the case of an attached target analyte 1702, for example, during flow cytometry, sample sorting, or as part of a stationary phase in liquid chromatography.

[0175] exist Figure 18 , Figure 19 and Figure 20In the implementation scheme, marker 200 is used to bind target analyte 1308. A tertiary affinity reagent 1800 (such as an antibody) attached to solid supports 1304, 1700, and 2000 is used to capture target analyte 1308. Solid support 2000 is a nucleic acid-based carrier, specifically a DNA origami-based carrier, as described, for example, in European patent applications WO2022 / 207832A1 and / or application number 24216812.8, the entire contents of which are incorporated herein by reference. For example, tertiary affinity reagent 1800 may be initially attached to solid supports 1304, 1700, and 2000 to capture or bind target analyte 1308. Subsequently, the presence of target analyte 1308 can be determined by adding marker 200 and generating a solid support 1304, 1700, and 2000 with target analyte 1308 and marker 200 through optical readout.

[0176] exist Figure 21 In one embodiment, the marker 2100, including marker portions 500, 502, is used, for example, to determine the proximity of two cellular target analytes 2102, 2104 in the cell sample 2106. The marker portion of the marker 2100 may be specific to either of the cellular target analytes 2102, 2104. Therefore, when the optical signal of the marker 2100 can be determined, it is determined that the target analytes 2102, 2104 are adjacent within the sample 2106.

[0177] According to Figure 22 In the implementation scheme, in the initial step 2210, the analyte 2200 of the sample is immobilized on a solid support 2202. In the next step 2212, the first and second marker portions, respectively comprising first and second labeling portions 500, 600, 502, and 602, are contacted with the immobilized analyte 2200 to bind the marker portions to their respective target analytes 2204. Specifically, step 2212 is performed under recombination conditions. In the next step 2214, any unbound marker portions can be washed away from the solid support 2202. In the next step 2216, derecognition conditions are applied to remove the host molecule 106 from the labeled portions 500 and 600. This enables hybridization between the labeled portions 500, 600, 502, and 602 and detects the presence of analyte 2204 in optical readout. If the abundance of target analyte 2204 in the analyzed sample is low, then... Figure 22 The method described may be particularly applicable.

[0178] In all figures, elements that function the same or similarly are indicated by the same reference numerals. As used herein, the term "and / or" includes any and all combinations of one or more of the relevant listed items and may be abbreviated as " / ".

[0179] Although some aspects are already described in the context of the device, it is clear that these aspects also represent a description of the corresponding method, where a module or apparatus corresponds to a method step or a feature of a method step. Similarly, aspects described in the context of a method step also represent a description of a corresponding module, item, or feature of the corresponding device.

[0180] Figure Labels 100, 206, 504 First nucleic acid backbone 102, 210, 506 Second nucleic acid backbone 104 object molecules 106 main molecules 108 Guest-Subject Complex 200, 700, 800, 900a, 900b, 900c, Markers 900d, 902a, 902b, 902c, 902d, 1000, 1002, 1204, 1300, 1500, 2100 202 First Marker Part 204 Second Marker Part 208, 508, 604 First marked part 212, 510 Second marked part 214 First Affinity Reagent 218 Second Affinity Reagent 216, 2102 First target analytes 220, 2104 Second target analytes 222 and 224 barcode oligonucleotides 500, 600 First Marking Part 502, 602 Second Marking Part Decomposition conditions for the 500a and 600a marked portions Composite conditions for 500b and 600b markings 1004 and 1006 secondary affinity reagents Methods for amplifying barcoded oligonucleotides at 1100, 1102, 1104, and 1106 Legal steps 1200 L-adaptor oligonucleotides 1202 Tree structure 1206 First-attachment oligonucleotide 1208 Second Attachment Oligonucleotide 1302 Anchor Oligonucleotides 1304, 1700, 2000, 2202 solid support 1306 spots Target analytes 1308, 1702, and 2204 1800 Third Affinity Reagent 2106 cell samples 2200 analytes 2210, 2212, 2214, and 2216 analyze samples containing biomarkers. Steps of the method

Claims

1. A marker for analyzing biological samples, comprising: The first marker portion containing the first nucleic acid backbone (100, 206), and The second marker portion contains the second nucleic acid backbone (102, 210); and Each of the first nucleic acid backbone (100, 206) and the second nucleic acid backbone (102, 210) is configured to at least partially hybridize with the other of its kind. The marker further comprises at least one first marking portion (208, 212) and at least one second marking portion (212), and The marker further comprises at least one guest molecule (104) configured to form a complex (108) with the host molecule (106).

2. The marker according to claim 1, wherein the at least one guest molecule (104) is configured to form the complex (108) with the host molecule (106) under recombination conditions.

3. The marker according to any one of the preceding claims, wherein the complex (108) is configured to invade the duplex of the first nucleic acid backbone (100, 206) and the second nucleic acid backbone (102, 210) and reduce the melting temperature of the duplex.

4. The marker according to any one of the preceding claims, wherein each of the first nucleic acid backbone (100, 206) and the second nucleic acid backbone (102, 210) comprises at least one of a natural nucleic acid and a nucleic acid analog.

5. The marker according to any one of the preceding claims, wherein the at least one first marking portion (208) and / or the at least one second marking portion (212) are optically detectable.

6. The marker according to any one of the preceding claims, wherein the at least one first marking portion (208) and / or the at least one second marking portion (212) preferably comprises the same fluorescent dye, or The at least one first labeled portion (208) contains at least one first fluorescent dye, and the at least one second labeled portion (212) contains at least one second fluorescent dye, wherein the at least one first fluorescent dye and the at least one second fluorescent dye have different characteristics.

7. The marker according to any one of the preceding claims, wherein the at least one first marking portion (208) and the at least one second marking portion (212) are configured to perform nonradiative energy transfer between them.

8. The marker according to any one of the preceding claims, wherein the at least one first labeled portion (208) and the at least one second labeled portion (212) are attached to, preferably covalently attached to, the first nucleic acid backbone (100, 206) or the second nucleic acid backbone (102, 210).

9. The marker according to any one of claims 1 to 7, wherein the at least one first labeled portion (208) is attached to, preferably covalently attached to, the first nucleic acid backbone (100, 206), and the at least one second labeled portion (212) is attached to, preferably covalently attached to, the second nucleic acid backbone (102, 210).

10. The marker according to any one of the preceding claims, wherein the first nucleic acid backbone (100, 206) extends along a first direction and a plurality of the first labeled portions (208) are arranged along the first direction on the first nucleic acid backbone (100, 206), and / or the second nucleic acid backbone (102, 210) extends along a second direction and a plurality of the second labeled portions (212) are arranged along the second direction on the second nucleic acid backbone (102, 210).

11. The marker according to any one of the preceding claims, wherein the spacing between each marked portion (208, 212) and any adjacent marked portion (208, 212) is substantially equal.

12. The marker according to any one of the preceding claims comprises a plurality of guest molecules (104), wherein the guest molecules (104) are uniformly spaced along the first nucleic acid backbone (100, 206) and / or the second nucleic acid backbone (102, 210).

13. The marker according to any one of the preceding claims, wherein the at least one guest molecule (104) is one of 1-adamantanemethylamine, ferrocenemethylamine, 1,4-phenylenediamine and 4-tert-butylbenzylamine.

14. The marker according to any one of the preceding claims, comprising at least one of the main molecules (106).

15. The marker according to any one of the preceding claims, wherein the host molecule (106) is cucurbituril.

16. A biomarker (200, 500, 600) for analyzing biological samples containing multiple target analytes (216, 504, 220), comprising: The marker according to any one of the preceding claims comprises a first marker portion and a second marker portion. The first marker portion (202, 502) comprises a first affinity reagent (214, 503) and the first marker portion, and The second marker portion (204, 602) comprises the second affinity reagent (218, 604) and the second marker portion, and Each of the first affinity reagent (214, 503) and the second affinity reagent (218, 604) is configured to specifically bind to one of the target analytes (216, 504, 220) of the biological sample.

17. The marker of claim 16, comprising an anchor oligonucleotide (1302) configured to anchor the marker (1500) to a solid support (1304, 1700, 2000).

18. A method for analyzing biological samples, the method comprising the following steps: Introducing at least one biomarker (200, 500, 600) according to claim 16 or 17 into the biological sample. The decomposition conditions were applied to the biological sample, and Optical readout of the biological sample having the markers (200, 500, 600) is generated.

19. A kit for analyzing biological samples, comprising a biomarker (200, 500, 600) according to claim 16 or 17 and at least one host molecule (106), said host molecule being configured to form a complex with said at least one guest molecule (104) of said biomarker (200, 500, 600).

20. Use of the marker according to any one of claims 1 to 15, or the marker (200, 500, 600) according to claim 16 or 17, or the kit according to claim 18, in a proximity assay for analyzing biological samples.

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