Markers, kits and methods for analyzing biological samples

Abstract

A marker for analyzing a biological sample is provided. The marker comprises an affinity reagent having a backbone and a barcode oligonucleotide attached to the backbone. The backbone is configured to specifically bind to a target analyte. The marker further comprises a marker having a labeled oligonucleotide and at least one labeled moiety. The labeled oligonucleotide and the barcode oligonucleotide of the affinity reagent are configured to hybridize with each other. One of the labeled oligonucleotide and the barcode oligonucleotide is sensitive to the degrading agent, and the other of the labeled oligonucleotide and the barcode oligonucleotide is resistant to the degrading agent. In further aspects, kits and methods of analyzing biological samples using the markers are provided.

Description

Technical Field

[0001] The present invention relates to a marker for analyzing a biological sample, wherein the marker comprises an affinity reagent and a label. In another aspect, a method for analyzing a biological sample using the marker is provided. Background Art

[0002] Existing affinity reagents contain chemically heterogeneous groups, and include, for example, antibodies, nucleic acid probes, and aptamers. Aptamers are typically short nucleic acid sequences folded into three-dimensional structures that can bind target analytes with high affinity and specificity. The use of barcoded affinity reagents, such as antibodies, nanobodies, aptamers, or in situ probes, is an effective strategy for cyclic labeling, and such reagents are connected to oligonucleotide sequences unique to the affinity reagents, and can contain one or more predetermined sequences that can be hybridized by a detectable marker comprising a complementary barcode. Such cyclic labeling is used when biological samples are to be analyzed to determine the presence of a large number of target analytes.

[0003] Fluorescence microscopy allows imaging of biological samples with high spatial resolution, but only involves low amounts of different fluorescent dyes, typically between 1 and 5. Available markers comprising dyes must include markers for identifying the cell type in the same experiment, functional markers such as proteins of interest, and general morphological markers. This means that cell types are only poorly identified in most imaging experiments. This also means that a fairly wide range of multi-cell type colonies are being studied, thereby severely limiting the predictive power and conversion value of the results produced. Although there are modern methods that allow more reliable and more robust identification of cell types, such as analysis based on genetic regulatory networks (GRNs), they need to read out more markers with distinguishable dyes from samples.

[0004] Although the adjacent field of cytometry, mass cytometry and imaging mass cytometry, can distinguish approximately 12 to 30 different markers, they do so at low spatial resolution.

[0005] Spatial profiling techniques based on RNA capture can distinguish a large number of different markers that are several orders of magnitude higher, although with even lower spatial resolution, as they are based on hybridizing oligonucleotides to the sample and then selectively releasing the bound oligonucleotides in a regioselective manner, followed by next-generation sequencing of the released oligonucleotides. Summary of the invention

[0006] One object of the present application is to provide a barcoded marker that can effectively and repeatedly stain a biological sample, and a method for analyzing a biological sample using the marker.

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

[0008] In one aspect, a marker for analyzing a biological sample is provided. The marker comprises an affinity reagent having a skeleton and a barcode oligonucleotide (Barcode Oligonucleotide) attached to the skeleton. The skeleton is configured to specifically bind to a target analyte. The marker further comprises a marker having a label oligonucleotide (Label Oligonucleotide) and at least one labeling portion. The barcode oligonucleotide of the label oligonucleotide and the affinity reagent are configured to hybridize with each other. One of the label oligonucleotide and the barcode oligonucleotide is sensitive to a degradation agent (Degradation Agent), and the other of the label oligonucleotide and the barcode oligonucleotide is resistant to the degradation agent. This can preferably make the sensitive one of the label oligonucleotide and the barcode oligonucleotide degraded, and therefore remove it from the affinity reagent (particularly the affinity reagent skeleton) by adding a degradation agent. Preferably, the affinity reagent (particularly the affinity reagent skeleton) remains intact in the presence of a degradation agent. Specifically, the affinity reagent skeleton can continue to be configured to specifically bind to a target analyte in the presence of a degradation agent. Thus, preferably, the affinity agent backbone is resistant to degradation agents.

[0009] The degradation agent only causes the degradation of the sensitive one of the barcode oligonucleotide and the labeled oligonucleotide, and in particular, the degradation agent can digest the sensitive oligonucleotide or fragment the sensitive oligonucleotide. This degradation usually leads to the loss of the oligonucleotide function, and in particular, the sensitive oligonucleotide no longer binds or can no longer bind to the resistant oligonucleotide. In other words, the sensitive oligonucleotide is configured to be degraded by the degradation agent (specifically). Preferably, the degradation of the sensitive oligonucleotide is irreversible. Therefore, the degradation agent causes the marker (particularly the labeled oligonucleotide) to be removed from the barcode oligonucleotide, and makes the barcode oligonucleotide accessible to another marker in another round of labeling.

[0010] The barcode oligonucleotide is preferably covalently attached to the backbone of the affinity reagent. The term "backbone of the affinity reagent" is not limited to a specific part or element. For example, in the case where the backbone of the affinity reagent is substantially composed of nucleic acids, the backbone of the affinity reagent does not specifically refer to the phosphate backbone of the nucleic acid. Therefore, the barcode oligonucleotide can be attached to any of the phosphate backbone, pentose or nucleobase of the nucleic acid of the backbone of the affinity reagent.

[0011] In another example, in the case where the backbone of the affinity agent is substantially composed of amino acids (i.e., polypeptides), the backbone of the affinity agent does not specifically refer to the polypeptide backbone (i.e., peptide chain) of a polypeptide, protein, antibody, nanobody, or aptamer (Aptabody). Therefore, the barcode oligonucleotide can be attached to any of a polypeptide, an amino acid, or an amino acid residue, or can be a modification of an amino acid residue, such as glycosylation, prenylation, or other post-translational modifications of the backbone of the affinity agent.

[0012] For example, the same applies in the case where the backbone of the affinity agent consists essentially of a non-naturally occurring polymer. Likewise, in this case, the backbone does not exclusively refer to the backbone of the polymer, i.e. the repeating polymeric units, but also to the residues and any modifications of the residues.

[0013] Documents WO 2022 / 242849 A1 and WO 2022 / 242887 A1 (the entire contents of which are incorporated herein by reference) propose markers and methods for increasing the number of distinguishable markers that can be used in single fluorescence microscopy experiments. Each marker includes a unique combination of dyes that form a code, and the code identifies the corresponding marker in principle. However, there is still some ambiguity, especially when a large number of markers are very close to each other. In order to resolve these ambiguities, it is necessary to remove the marker and repeat image acquisition with different groups of markers. In particular, in the document WO 2022 / 242887 A1, a method is disclosed that can use microscopy and encoding / decoding processes for high-complexity analysis (High-Plex Analysis), which is based on cyclic imaging. The core aspect of this method is the so-called "Code Swapping", which refers to labeling analytes with the nth code in the nth round and labeling analytes A with the nth+1th code in the nth+1th round. The present invention provides a particularly preferred method to exchange codes in an efficient manner in real biological samples, namely combinatorial labels comprising multiple colors or comprising a combination of different label parts.

[0014] While the present invention is well suited for multiplexed and high complexity analysis using, for example, a microscope or a plate reader, it can also be used in many other applications that could benefit from a rapid, robust and cost-effective way to label analytes in a cyclical manner.

[0015] Barcoding Affinity Reagents is an effective strategy that allows various modes of cyclic imaging processes, which are referred to in this document as marker cycles, label cycles, and barcode cycles.

[0016] Preferably, the backbone of the affinity reagent consists essentially of amino acids. For example, the backbone of the affinity reagent may be a protein. This enables the affinity reagent to be resistant to oligonucleotide-specific degradation agents. In particular, the protein may carry post-translational modifications. Thus, for example, the amino acids of the backbone may be glycosylated.

[0017] It is particularly preferred that the backbone of the affinity reagent is an antibody or antibody fragment. This enables the affinity reagent to have a specific specificity for a particular target analyte (such as a protein).

[0018] In an alternative, the backbone of the affinity reagent is preferably substantially composed of nucleic acids. For example, this enables the affinity reagent to specifically bind to a target analyte (such as mRNA). Preferably, the nucleic acids of the affinity reagent backbone are resistant to the degradation agent.

[0019] Preferably, the backbone of the affinity reagent is configured to specifically bind to the target analyte by hybridizing a continuous nucleotide sequence with a complementary continuous target nucleotide sequence. For example, this enables the affinity reagent to specifically bind to a target analyte (such as mRNA), similar to a FISH probe. In this case, the backbone is configured to specifically bind to the target analyte by hybridizing a continuous nucleotide sequence with a complementary continuous target nucleotide sequence.

[0020] Preferably, the framework of the affinity reagent has a composite structure, and the framework is configured to specifically bind to the target analyte through its composite structure. The composite structure can preferably be a secondary structure, a tertiary structure or a quaternary structure of the framework. For example, this enables the affinity reagent to specifically bind to the target analyte (such as a protein). In the case where the framework has a composite structure, the discontinuous nucleotides of the nucleic acid sequence or the extensions of the nucleic acid sequence interact with the target analyte and / or specifically bind to the target analyte.

[0021] Particularly preferably, the skeleton of the affinity reagent is an aptamer. This can effectively produce the skeleton of the affinity reagent, wherein the skeleton has specificity to a series of target analytes (such as various proteins). Aptamers can be produced in various ways, wherein SELEX is the most typical approach. Although most aptamers comprise nucleic acid sequences (RNA, DNA, XNA), they can also be produced by other polymers (such as, for example, peptides). In addition, aptamers have been recently proposed, which are basically aptamers with nucleic acid skeletons, and the aptamers comprise modifications (for example, glycosylation) naturally present in the protein.

[0022] Preferably, one of the marker oligonucleotide and the barcode oligonucleotide is substantially composed of a nucleic acid (e.g., a natural nucleic acid) that is sensitive to a degradation agent, and the other of the marker oligonucleotide and the barcode oligonucleotide is substantially composed of a nucleic acid analog that is resistant to the degradation agent. This can effectively produce the marker oligonucleotide and the barcode oligonucleotide that are sensitive or resistant to the degradation agent. Generally, nucleic acid analogs are compounds that are structurally similar to naturally occurring RNA and DNA. Nucleic acids are chains of nucleotides that consist of three parts: a phosphate backbone, a pentose (ribose or deoxyribose) and one of four nuclear bases. Analogs may change any of these. The nucleic acid analogs may be artificial nucleic acids or heterologous nucleic acids. Natural nucleic acids or naturally occurring nucleic acids (DNA and RNA) are generally sensitive to degradation agents (such as nucleases), while nucleic acid analogs are generally resistant to degradation agents (such as nucleases). In addition, in the case where the affinity reagent backbone is substantially composed of nucleic acids, these nucleic acids are preferably nucleic acid analogs that are resistant to the degradation agent.

[0023] Preferably, the marker oligonucleotide is essentially composed of natural nucleic acid, and the barcode oligonucleotide is essentially composed of nucleic acid analogs. This can effectively produce an affinity reagent resistant to the degradation agent and a marker that can be effectively degraded and thus selectively removed from the affinity reagent. In this case, the affinity reagent backbone is preferably essentially composed of nucleic acid analogs. Therefore, the barcode oligonucleotide and the backbone are preferably resistant to the degradation agent.

[0024] It is particularly preferred that the nucleic acid analogs include at least one of 1,5-anhydrohexitol nucleic acid, cyclohexene nucleic acid, threose nucleic acid, ethylene glycol nucleic acid, locked nucleic acid, peptide nucleic acid, fluoroarabino nucleic acid, bridged nucleic acid, morpholino, phosphothioate backbone, L-pentose backbone, and nucleic acid containing at least one modified nucleoside. This allows for high resistance to the degradation agent.

[0025] Preferably, the at least one labeling moiety is optically detectable. This enables the production of a marker that is easily detectable, for example by microscopy.

[0026] Preferably, the degrading agent is a nuclease, which can easily degrade the sensitive barcode oligonucleotide or marker oligonucleotide.

[0027] In a particularly preferred combination, the sensitive barcode oligonucleotide or marker oligonucleotide is sensitive to a nuclease degrading agent such as DNase I, and the resistant barcode oligonucleotide or marker oligonucleotide is resistant to the nuclease degrading agent such as DNase I. In this case, the resistant barcode oligonucleotide or marker oligonucleotide preferably consists essentially of nucleic acid analogs such as non-naturally occurring nucleic acids or XNAs.

[0028] Preferably, the degrading agent and the sensitive marker oligonucleotide or sensitive barcode oligonucleotide are configured such that application of the degrading agent releases the barcode oligonucleotide from the marker oligonucleotide-barcode oligonucleotide duplex, whereby the barcode oligonucleotide is able to hybridize with another marker oligonucleotide.

[0029] Preferably, the degrading agent is an artificially engineered peptidase or protease, which is configured to degrade peptide nucleic acids or xenogeneic nucleic acids.

[0030] Preferably, the degradation agent is silver, iodine or mercury, and the sensitive nucleic acid analog is a phosphorothioate-modified nucleic acid.

[0031] Preferably, the degradation agent is based on an inorganic or organic chemical that degrades the sensitive nucleic acid analog.

[0032] In a further aspect, a kit is provided, the kit comprising at least one marker and the degrader.

[0033] Additionally, a kit is provided, comprising at least one barcode oligonucleotide and chemical components necessary to couple the barcode oligonucleotide to the affinity reagent backbone to generate the marker.

[0034] In addition, a kit is provided, comprising the degrading agent and at least one marker, wherein the at least one marker comprises a labeled oligonucleotide to produce the marker.

[0035] In another aspect, a method for analyzing a biological sample containing at least a target analyte is provided. The method comprises introducing at least a first marker or a component thereof into the biological sample. The marker comprises an affinity reagent having a backbone specific for the target analyte and a first marker having at least a first labeling portion. The method further comprises generating a first reading of the biological sample having at least one marker, and applying the degradation agent to the biological sample to degrade the marker oligonucleotide of the marker. The method further comprises: introducing at least a second marker having at least a second labeling portion into the biological sample to produce a second marker, the second marker comprising a second marker oligonucleotide configured to hybridize with a barcode oligonucleotide of the affinity reagent; and generating a second reading of the biological sample having the second marker. The method is capable of iteratively labeling target analytes using several markers. This is particularly beneficial for procedures intended to detect a large number of different target analytes in a biological sample. In this case, repeated imaging of the labeled biological sample using varying markers can help to correctly identify the corresponding target analyte. Preferably, the marker comprises a barcode oligonucleotide resistant to the degradation agent and a marker oligonucleotide sensitive to the degradation agent.

[0036] Preferably, in the first reading and the second reading, the target analyte is identified based on the first marker and the second marker associated with the target analyte. This can increase the probability of correctly identifying or locating the target analyte in the biological sample.

[0037] Preferably, in the step of introducing the at least one first marker, an affinity reagent for the at least one first marker is introduced before the corresponding label for the first marker, and wherein the affinity reagent is immobilized in the biological sample before the label for the first marker with the first labeling portion is introduced into the biological sample. This enables a particularly robust method, since the marker, in particular the affinity reagent, remains in close proximity to the corresponding target analyte even when a large number of reads are generated.

[0038] Preferably, the first labeling portion and the second labeling portion have different detectable properties. This can increase the probability of correctly identifying or locating the target analyte in the biological sample based on the sequence of properties of the labeling portions detected in the reading. For example, in the case where the detectable portion is a fluorophore, the detectable property can be fluorescence emission, or excitation wavelength, or emission lifetime.

[0039] Preferably, after the step of generating the second reading, the steps of applying the degradation agent to degrade the labeled oligonucleotide, adding at least one additional label having an additional labeling portion, and generating additional readings of the biological sample are iteratively repeated. This can increase the probability of correctly identifying or locating the target analyte in the biological sample based on a large number of readings. Preferably, for each iteration, a different label is used.

[0040] It is particularly preferred that the readout generated is an optical readout, for example by microscopy. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] In the following, specific embodiments are described with reference to the accompanying drawings, in which: Figure 1 A set of examples of markers that bind to a target analyte are schematically shown, Figure 2 Schematically shows how a detectable label can comprise a plurality of different labeling moieties, Figure 3 Schematically showing a marker having multiple labeling parts, Figure 4 The use of the barcoded affinity reagent for cycle labeling of the present invention is schematically shown. Figure 5 A workflow is shown in which in a first step barcoded affinity reagents are introduced into a biological sample and allowed to bind to their targets. Figure 6 Shows Figure 5 An optional extension of the workflow depicted in , in which a second degrader is used, Fig. 7A A marker having a label is shown, wherein the label has a sensitive barcode oligonucleotide, Figure 7B Schematically illustrates how the method can be used to produce markers, Figure 7C Schematically illustrates how the method can be used to produce markers, Figure 8 Schematically illustrates how the method can be used to label the same analyte with different labels in multiple rounds (code swapping). Fig. 9Schematic diagram of the in situ assembly of markers. Fig.10 Schematic diagram of the use in affinity purification and affinity capture based assays, Fig.11A A marker with a barcode oligonucleotide is shown, wherein the barcode oligonucleotide comprises three barcodes (predetermined sequences) and a single-color fluorescent label, Fig. 11B A marker with a barcode oligonucleotide is shown, wherein the barcode oligonucleotide comprises three barcodes (predetermined sequences) and a multicolor fluorescent label (combinatorial label or combined fluorescent coding, CFC), Fig. 11C A marker having a barcode oligonucleotide comprising a barcode (predetermined sequence) and a polyalkyne tag, and Fig.11D Shown is a marker with a barcode oligonucleotide comprising 1 barcode (predetermined sequence) and a polyalkyne-fluorescent combination label. DETAILED DESCRIPTION

[0042] Figure 1 A set of examples of markers 102a, 102b bound to their respective target analytes 100a, 100b are shown. The markers 102a, 102b, 102c can include an affinity reagent having a backbone, such as an antibody 104a, an oligonucleotide probe 104b (which can include a naturally occurring nucleic acid or a non-naturally occurring nucleic acid (XNA)), or an aptamer, an aptamer, or other modified aptamer 104c. In addition, the affinity reagent backbone can be based on another non-naturally occurring polymer and non-naturally occurring residues and / or modifications.

[0043] Marker 102a, 102b, 102c comprises corresponding affinity reagent skeleton 104a, 104b, 104c. Marker 102a, 102b, 102c further comprises a marker with a labeling portion (e.g., an optically detectable fluorophore 106). The marker comprises an oligonucleotide with at least a labeled oligonucleotide 112. Similarly, the affinity reagent comprises an oligonucleotide with at least a barcode oligonucleotide 110, and this combination can be referred to as a barcoded affinity reagent. The labeled oligonucleotide 112 and the barcode oligonucleotide 110 are configured to specifically hybridize with each other. Therefore, the marker can be specifically attached to the corresponding affinity reagent with antibody 104a, 104b, 104c to produce marker 102a, 102b, 102c based on the specificity of the corresponding labeled oligonucleotide 112 and the barcode oligonucleotide 110. The labeled oligonucleotide 112 can be covalently attached to the fluorophore 106 of the label. Similarly, the barcode oligonucleotide 110 can be covalently attached to the backbone of the affinity reagent.

[0044] In the sense of this document, marker 102a, 102b, 102c refers to the physical association (Physical Association) of affinity reagent and detectable marker, or refers to the virtual allocation or mapping of affinity reagent to detectable marker. Marker 102a, 102b, 102c can be physically constituted before, during or after being introduced into biological sample. Similarly, detectable marker can be physically constituted before, during or after being introduced into biological sample. Biological sample can be any one of the following: cell, cell culture (monolayer, suspension, 3D cell culture, spheroid, organoid, class tissue), tissue section (e.g., FFPE, frozen section), solid biopsy, liquid biopsy, body fluid sample (e.g., blood, serum, plasma, sperm, saliva, liquid, lymph, interstitial fluid). This biological sample can include at least one target analyte of interest. This target analyte 100 can be, for example, protein 100a or mRNA 100b.

[0045] For the methods disclosed in this document, a barcoded affinity reagent, i.e., an antibody, nanobody, ISH probe, aptamer, comprising a barcode oligonucleotide 110 is used, which comprises at least one unique sequence that is uniquely assigned to the affinity reagent. The barcode oligonucleotide 110 may comprise a plurality of barcode sequences, which may be addressed by a plurality of detectable markers (particularly marker oligonucleotides 112) simultaneously or in a chronological manner. In the sense of this document, an affinity reagent may also be any molecule that exhibits high affinity for a target analyte and binds to the analyte in a substantially specific manner. In the sense of this document, drugs, drug-like molecules, small molecules or toxins may also be used as affinity reagents and coupled to the barcode oligonucleotide 110.

[0046] Therefore, the detectable marker comprises a labeling oligonucleotide 112, which comprises at least one barcode sequence, which is configured to be substantially complementary and is therefore capable of specifically binding to the barcode sequence of the barcode oligonucleotide 110 of the assigned affinity reagent to allow flexible connection (Flexible Connection) of the affinity reagent and the detectable marker, i.e., marker formation.

[0047] In the sense of the present document, the labeled oligonucleotide 112 can be a short dye-coupled oligonucleotide, a long flexible oligonucleotide (comprising, for example, a barcode sequence for attaching a third marker-coupled oligonucleotide), or can even comprise a 2D- / 3D-DNA based structure, such as a DNA origami or DNA bricks based structure.

[0048] The barcode oligonucleotide 110 is preferably configured such that it is resistant to the degradation agent 116 in its entirety or at least with respect to the mediating portion of the interaction (ie, the barcode mediating the linkage between the affinity agent and the detectable label).

[0049] The marker oligonucleotide 112 is preferably configured such that it is sensitive in its entirety or at least with respect to the interaction-mediating portion (i.e., the barcode that mediates the connection between the affinity agent and the detectable label) to the degradation agent 116. Sensitive means that the degradation agent 116 can cause a change in the marker oligonucleotide 112 sufficient to remove the label. This can be a nicking of the barcode in the center, thereby triggering the remaining half to dissociate from the barcode oligonucleotide 110. This can be partial digestion of the barcode sequence, complete digestion of the barcode sequence, or even partial or complete degradation of other components including the detectable label.

[0050] For example, the barcode oligonucleotide 110 may comprise PT-modified DNA, or LNA, or PNA, or morpholino, or another DNase I-resistant XNA. Figure 1 Such barcode oligonucleotides 110 can be easily coupled to antibodies, for example by using commercially available coupling kits such as the oligonucleotide coupling kit (ab218260) from Abeam (Cambridge, UK).

[0051] Thus, the degradation sensitive label oligonucleotide 112 (depicted in solid lines) can be a naturally occurring nucleic acid (such as DNA) that is sensitive to degradation by the DNase I degradation agent 116. The degradation resistant barcode oligonucleotide 110 (depicted in dashed lines) can be a nucleic acid analog, such as a non-naturally occurring nucleic acid (XNA) such as L-DNA, that is resistant to degradation by the DNase I degradation agent 116. In the case of a nucleic acid based affinity reagent backbone 104b, the backbone 104b is preferably equivalent to a nucleic acid analog, such as a non-naturally occurring nucleic acid (such as degradation resistant L-DNA).

[0052] Figure 2 Schematically shows how detectable markers 200, 202 in the sense of the present document can contain various different labeling moieties for various applications, including optical detection or non-optical detection, and imaging-based or non-imaging-based readings. The labeling moiety can be conveniently attached to the second oligonucleotide or the third oligonucleotide. In Figure 2In, the third (or fourth ...) oligonucleotide is covalently coupled to the labeling part. Click chemistry allows the effective generation of such conjugates, but other chemistries are also suitable (e.g., NHS coupling). In addition, via high-affinity interactions (biotin-streptavidin; cucurbit uryls [CB], such as CB [7] or CB [n] homologues and high-affinity guests (e.g., PubChem ID 101402794), Lambert et al. 2019 described a kind of expanded cubane (Expanded Cubane)) of the labeling part based on affinity-interaction coupling is a suitable strategy for connecting the labeling part to the skeleton of the detectable label. Then, such a skeleton can further include a structure based on DNA / XNA or a non-DNA particle, such as polystyrene beads 204.

[0053] Figure 3 A particularly preferred embodiment of a marker 300 having a label is shown, which comprises a long oligonucleotide 302 used as an adaptor, to which are hybridized a plurality of label portions 304 with oligonucleotides 306. The long oligonucleotide 302 comprises a labeling oligonucleotide 112 for specific hybridization with a barcode oligonucleotide 110 attached to an antibody 104a. Similarly, a nucleic acid-based marker 308 may comprise a long oligonucleotide 302. Fig. 9 It is further shown how this embodiment can also be used for in situ assembly of detectable labels.In situ assembly offers many advantages, for example in terms of penetration into the sample or reduced production costs.

[0054] Figure 4 The use of the invention for cycle labeling using a barcoded affinity reagent is schematically shown. The invention is particularly preferred for use in combination with DNase I as the degradation agent 116, since DNase I is very stable, allows rapid (ie, catalytic) removal, and is easy and inexpensive to produce. Figure 4 The top view of FIG. 4 shows a marker 400 having a label with a label portion 106, which is removed from the barcode oligonucleotide 110 by degrading the label oligonucleotide 112 with a degradation agent 116. In a next step, an additional label comprising a label portion 402 different from the label portion 106 can be attached to the barcode oligonucleotide 110.

[0055] This is similar to Figure 4 1 , in contrast to the alternative illustrated in the bottom view of , in which both the oligonucleotides to which the label and the affinity agent are attached are susceptible to the degradation agent 116. In this case, upon application of the degradation agent 116, both oligonucleotides are degraded, and no additional label can be attached to the affinity agent.

[0056] Figure 5 The workflow is shown in which, in a first step (indicated by "1"), a plurality of barcoded affinity reagents 500 are introduced into a biological sample and allowed to bind to their corresponding target analytes. For simplicity, this is described in detail in FIG. Figure 5 500 is exemplarily shown for antibodies, but is meant to be understood as being generally applicable to any affinity reagent. For example, the affinity reagent may comprise an in situ hybridization probe, an aptamer, an aptamer, or a nanobody. In addition, the plurality of affinity reagents 500 may comprise any of the above categories, i.e., a mixed composition in terms of the type or category of the affinity reagent backbone.

[0057] Again, for simplicity, Figure 5 Target is not shown. After optional steps 2 and 3 (represented by "2" and "3"), in which the sample may undergo further post-fixation, expansion (Expansion) and / or cross-linking, a first group of multiple detectable markers 502 with sensitive labeled oligonucleotides are introduced into the biological sample and are specifically bound to the resistant barcode oligonucleotides (represented by "4") of the corresponding affinity reagent. Once one of the markers 502 hybridizes with one of the affinity reagents 500, a corresponding marker is produced. Optionally, any unbound marker 502 is subsequently washed away. Then, the bound marker is read out by a readout device, for example, by fluorescence microscopy when the labeled portion of the marker 502 is a fluorophore. Therefore, by detecting the associated marker of the marker, particularly the labeled portion of the marker, the marker can identify and locate the target analyte to which the corresponding marker is bound. In the subsequent step 5 (indicated by "5"), a degradation agent 116 is applied, the labeled oligonucleotide of the marker is degraded and removed from the affinity reagent 500, and the remaining components of the marker are removed from the biological sample by washing. Then, steps 4 to 5 (including the corresponding readouts) are repeated until the barcoded affinity reagents of the introduced plurality of affinity reagents 500 (step 1) have been labeled and read out at least once. Therefore, when steps 4 and 5 are repeated, not all of the plurality of affinity reagents 500 need to be labeled with one of the introduced markers 502 in a particular step 4 and 5. Instead, the number of distinguishable markers 502 that can be generated determines the number of markers that can be generated from the affinity reagents 500 and the markers 502 in any one step 4.

[0058] Figure 6 Shows Figure 5 Optional extensions of the workflow depicted in . In particular, Figure 6 The steps shown can be followed for Figure 5 Step 5 described above. Specifically, Figure 6The second degradation agent 600 is used in the affinity reagent to remove all barcode oligonucleotides. Thus, the barcode oligonucleotides of the affinity reagent may be sensitive to the second degradation agent 600, but not to the first degradation agent 116. Such resetting of the biological sample may be desirable if only a limited number of physical barcodes are available. For example, if only 50 barcodes are physically present and the user wants to analyze 200 markers, this allows the method to be performed in 4 cycles, where each cycle contains 50 markers and the number of markers is calculated based on the number of markers. Figure 5 The sub-cycles are performed using a given number of sub-cycles. For example, after each sub-cycle, the barcode oligonucleotides of the markers 500 used in the sub-cycle are removed by adding a second degradation agent 600, and the next plurality of 50 markers are used to initiate the process according to Figure 5 Another sub-cycle of .

[0059] FIG. 7A to FIG. 7C Different label removal mechanisms are schematically depicted in combination with various types of labels (e.g., single color, multicolor (combination), Raman (e.g., polyalkynes)) and hybridization labels comprising more than one modality (e.g., fluorescent and Raman labeling moieties).

[0060] Fig. 7A A label 700 is shown with a sensitive marker oligonucleotide 112 and a sensitive long oligonucleotide 302, as well as a sensitive oligonucleotide-dye conjugate 702. In this case, the degradation agent 116 results in complete degradation of the detectable label 700.

[0061] Figure 7B and 7A The difference is that the dye-oligonucleotide conjugate 704 of resistance here connects the dye to sensitive long oligonucleotide 302 and sensitive labeling oligonucleotide 112. Therefore, the decomposition by degradation agent 116 is incomplete. However, the dye-oligonucleotide conjugate 704 of resistance is still very small and can be effectively removed from the sample. In this example, PNA-dye conjugate can mediate the strong connection with the sensitive long oligonucleotide 302 such as comprising DNA, and the sensitive long oligonucleotide 302 is connected to the barcode oligonucleotide 110 of the DNA modified by PNA or PT- via labeling oligonucleotide. This is particularly advantageous because DNA / PNA interacts more specifically and has a better signal-to-background ratio (Signal-to-Background) due to lower cross hybridization. In addition, only a part of detectable markers, i.e. labeling oligonucleotide 112 can be sensitive, i.e., only labeling oligonucleotide 112 comprises DNA, and the rest comprises such as PNA.

[0062] Alternatively, if Figure 7CAs depicted, both the barcode oligonucleotide 110 and the marker oligonucleotide 112 may comprise the same backbone DNA, RNA or XNA, but the marker oligonucleotide 112 comprises a point modification / mutation 706 that is recognized by a structure-specific enzyme as a degrading agent that introduces a nick at that position. The introduction of the nick results in degradation of the marker oligonucleotide 112. Alternatively or in addition, CRISPR Cas9 or a derivative, such as Cas9 or Cas9 H840A nickase, may be used to cleave the sensitive marker oligonucleotide 112, in which case a suitable guide RNA or guide LNA would be used to target the CRISPR / Cas9 to the corresponding sequence.

[0063] Figure 8 Schematically shows how the present invention can be used to use the nth marker 106e (in the nth round) Figure 8 In the example of the top row of n=1), the target analyte 100a is labeled, a read is performed, the marker 106e is removed, and the second marker 106f is re-labeled on the same barcode oligonucleotide 110 or on another barcode oligonucleotide 110, wherein the second marker 106f is bound to the same affinity reagent with the antibody 104a. In this way, the target analyte 100a is labeled with corresponding markers 106e, 106f, and 106g in the first round, the second round, and the third round. This method of labeling the same target analyte 100a with multiple markers in multiple rounds in a time-sequential manner using barcoding can be referred to as a barcode cycle. In the sense of the published application WO 2022 / 242887 A1 (which discloses a method for high-complexity analysis), this is a particularly preferred mode for performing "code exchange" using the present invention.

[0064] Fig. 9 The formation of marker 300 and the formation of associated detectable labels in situ (ie, in a sample) are schematically depicted. Assembly can be performed by introducing all components of marker 300 simultaneously, or by Fig. 9 The sequence shown is introduced for assembly. The marker can be further compacted using the method disclosed in the European patent application with application number EP 23174843, the entire content of which is incorporated herein by reference. For this reason, a staple strand 902 is added to the long oligonucleotide 302, and a short strand 902 is hybridized with the corresponding binding site of the long oligonucleotide 302 so that the long oligonucleotide 302 is folded into a compact shape.

[0065] Fig.10A schematic diagram of affinity purification using a column matrix, such as polystyrene beads 1000, attached to a barcoded capture affinity reagent is shown. In this case, the method can be used to remove bound affinity reagent:target complexes by removal of the labeled oligonucleotide or barcode oligonucleotide. This can also be used to regenerate the column matrix.

[0066] FIG. 11A to FIG. 11D Various exemplary embodiments of markers are schematically shown. For example, Fig.11A A marker 1100 comprising three barcode oligonucleotide sequences 1102a, 1102b, 1102c and a single color fluorescent label 1104 for attaching labels is shown. Fig. 11B A marker 1106 comprising three barcode oligonucleotide sequences 1102a, 1102b, 1102c and a multicolor fluorescent label 1108 (combinatorial label or combined fluorescent coding, CFC) is shown. Fig. 11C A marker 1110 is shown having a barcode oligonucleotide 110 and a label 1112 comprising a polyalkyne tag moiety. Fig.11D A marker 1114 with a barcode oligonucleotide 110 comprising a polyalkyne-fluorescent combination label 1116 is shown.

[0067] Barcoded affinity reagent skeleton, such as antibody, nano antibody, aptamer, target DNA or mRNA in situ probe, is connected to the oligonucleotide comprising at least one sequence, and this at least one sequence is also referred to as the barcode unique to the affinity reagent. The affinity reagent of this oligonucleotide labeling can be referred to as barcoded affinity reagent. The barcode compilation of affinity reagent has many advantages compared with the direct coupling of for example fluorescent dye, because it allows to flexibly use barcoded antibodies such as with various detectable markers in different applications or in different rounds of the cyclic process of the same application. Detectable marker can refer to optical detectable markers and non-optical detectable markers used for microscopy, cytometry or space biology applications, which can be used for affinity purification, cyclic protein blot (Cyclical Western Blotting), ELISA determination and ELISPOT determination, high complexity proteomics (for example, plasma proteomics). Therefore, detectable marker comprises at least one labeling part, which can be for example fluorescent dye, affinity tag, metal tag or enzyme. Detectable marker can further comprise at least one marker oligonucleotide, and this at least one marker oligonucleotide comprises the sequence of the complementary barcode oligonucleotide that is configured to be bonded to the barcoding affinity reagent of distribution.Therefore, marker oligonucleotide can be the simple short oligonucleotide that is attached to marker, such as the oligonucleotide of dye coupling, or the structure of more delicate single part or multipart.The latter can be such as long flexible oligonucleotide skeleton, the skeleton based on dendritic nucleic acid or the skeleton based on rigid DNA with 2 dimension / 3 dimension structure (for example, based on DNA origami, based on DNA brick), or the skeleton that is made up of nucleic acid component and non-nucleic acid component, such as the oligonucleotide that is attached to the polystyrene beads of dye coupling.Barcode oligonucleotide and marker oligonucleotide can comprise one or more predetermined sequences or barcoding sequences.

[0068] Cycle dyeing

[0069] Barcoding of affinity reagents and detectable labels is an effective strategy for cyclic labeling, which can be used, for example, for multiplexed biomarkers, high-complexity RNA imaging, or high-complexity spatial or non-spatial proteomics applications. Likewise, it is also a prerequisite for high-complexity spatial interactomics or interrogating protein modifications at high complexity.

[0070] Marker Cycle

[0071] In the sense of cyclic staining, barcoded affinity reagents can be used in different modes: for example marker cycling, where markers are introduced into the biological sample in each round and allowed to bind to their targets during cyclic staining, imaging, inactivation. In this case, the markers are pre-assembled and each round includes a marker introduction step, which is time consuming.

[0072] Marker Cycle

[0073] In addition, the barcoded affinity reagents can be used for label circulation, which is a particularly preferred operating mode in the sense of the present invention. In this mode, in a first step, all barcoded affinity reagents from a plurality of barcoded affinity reagents are introduced into the sample, and then, in a subsequent step, a subset of the assigned barcoded detectable labels is introduced into the sample, bound to the assigned barcoded affinity reagents, read out and inactivated or removed. Then, the process is repeated in a cyclic manner until, for example, all barcoded affinity reagents from the plurality of barcoded affinity reagents are read out. In contrast to the marker circulation, the marker circulation does not require the repeated introduction of affinity reagents, and therefore requires substantially less time, because the introduction of affinity reagents and their binding to targets may generally take 1h-24h. This strategy provides the advantage that, for example, all antibodies can be bound to their analytes in a single step, followed by post-fixation, and then these antibodies can be labeled in multiple rounds using barcoded labels in cyclic in situ hybridization. Since binding of antibodies to their targets takes about 1-3 h when tissue sections are stained, and label hybridization may take about 10 min, this allows for increased throughput. To remove bound labels, several strategies can be employed, such as cleavage with chemical reagents such as TCEP, which requires disulfide bridge-modified oligonucleotide barcode labels, melting, or strand displacement.

[0074] Barcode loop

[0075] Another strategy that can be used for barcoding affinity reagents is various label cycles, which we refer to as barcode cycling in this document. In this case, the same barcode is addressed multiple times with different detectable labels, which can be achieved by selecting a configuration in which the affinity reagent and the barcode of the detectable label are configured so that they are only transiently associated, as is the case with DNA-PAINT, for example, or in a cycling process in which binding is controlled by buffer conditions (e.g., DNA exchange imaging), temperature (e.g., melting), absence / presence of displaced strands, or strand invasion of guest-host complexes.

[0076] Marker removal mechanism

[0077] Degradation agents can be used to selectively cause oligonucleotides to be configured to remove oligonucleotides that are sensitive to degradation agents, thereby allowing the detectable markers carrying sensitive labeled oligonucleotides to dissociate from the marker. This requires that degradation agents do not necessarily have to completely degrade sensitive oligonucleotide parts, but degrade in a manner sufficient to allow detectable markers to dissociate from the barcoded affinity reagents of combination. Degradation agents and sensitive oligonucleotides are configured to make degradation agents effectively remove resistance barcodes from the sensitive complementary barcodes of combination, so that the resistance barcodes of the barcoded affinity reagents are freely re-hybridized with different detectable markers in a subsequent round.

[0078] When using nucleic acid-based labels comprising, for example, a nucleic acid-based backbone, the degrading agent can additionally allow for the removal of the detectable label, thereby resulting in degradation of the label, which can promote rapid inactivation and washing of degraded components of the detectable label from the biological sample.

[0079] Therefore, degraders can have the following functions: Introducing a nick into the sensitive oligonucleotide, resulting in dissociation of the hybridized marker and barcode oligonucleotides; · substantially degrade a sensitive oligonucleotide or substantially degrade a sensitive moiety; Partially degrading the sensitive oligonucleotide or partially degrading the sensitive portion in a manner suitable to release the resistance barcode and allow removal of the label; • Partially or substantially degrading a detectable marker moiety including, for example, a label moiety and / or a dye-conjugated oligonucleotide.

[0080] Suitable degradation agents

[0081] The core idea is to use a degrading agent and at least two different nucleic acids, a marker oligonucleotide and a barcode oligonucleotide, selected from naturally occurring nucleic acids or nucleic acid analogs, such as artificial / xenogeneic nucleic acids (XNA), wherein the pairing comprises at least one sensitive oligonucleotide and one resistant oligonucleotide. Therefore, it is important to note that sensitivity and resistance are properties associated in any case with the selected degrading agent, which can be selected from one of the following: Endonuclease Restriction endonucleases / restriction enzymes AP endonuclease DNA repair enzymes and structure-specific endonucleases Endonuclease III (Nth), Endonuclease IV ·APE 1 RNase HII (e.g., nicked ribonucleotides incorporated into DNA duplexes) RNA endonucleases ·RNase A, RNase T1 and RNase H Drosha and Dicer Exonuclease ·ExoI, ExoIII, ExoV, λ exonuclease, T5 Exo, ExoV, ExoVII, ExoT, T7Exo RNA exonucleases RNase D, RNase T · Nuclease P1, mung bean nuclease, RecJfr, DNase I, micrococcal nuclease · CRISPR and related enzymes Artificial enzymes Engineered artificial peptidases or proteases that degrade the PNA backbone Organic or inorganic chemical components that can detect at least one sensitive nucleic acid and at least one resistant nucleic acid Iodine, silver ions (e.g., cleaves phosphorothioate-modified DNA)

[0082] In this sense, it is clear that many combinations of enzymatic or non-enzymatic degraders with sensitive and resistant nucleic acids can be found that can be used to implement embodiments of the present invention. In addition, it is clear that a nucleic acid (e.g., a thiophosphate-modified DNA) is resistant to a first degradation agent (e.g., DNase I) but sensitive to a second degradation agent (e.g., silver or iodide ions). Another example of an inorganic chemical is an oligonucleotide modified with a thiophosphate (PT) that is cleaved with, for example, iodine or silver. Multiple PT cleavage sites can be incorporated into the barcode or backbone of the marker, and iodine and / or silver can be used as a removal agent.

[0083] This enables a configuration in which the sensitivity and resistance of the barcode oligonucleotides and the labeled oligonucleotides can be reversed in an experiment or process by changing from a first degrading agent to a second degrading agent. In the sense of a cyclic imaging process, this can be used, for example, to perform label cycling with label removal as described above, where in the first stage of the process, the labeled oligonucleotides of the detectable label are sensitive to the first degrading agent, and then in the second stage all barcode oligonucleotides of the barcoded affinity reagent are removed by the second degrading agent. This may be useful in situations where the total number of available barcode sequences is limited, for example when there are only 50 physically available barcodes and 200 markers need to be analyzed, this can be performed in 4 stages of labeling cycles, where after each stage, the barcode of the affinity reagent is removed.

[0084] Likewise, both the marker oligonucleotide and the barcode oligonucleotide may be sensitive to a second degrading agent.

[0085] Suitable Nucleic Acids and Xenogeneic Nucleic Acids (XNA)

[0086] Suitable nucleic acids or xenogeneic nucleic acids (XNA) from which pairs of sensitive and resistant oligonucleotides or nucleic acids can be selected: Locked Nucleic Acid (LNA), Morpholinyl, Phosphorothioate-modified nucleic acids, Peptide nucleic acid (PNA), Bridging Nucleic Acids (BNA), 2'O-methyl substituted RNA, Glycol Nucleic Acid (GNA), Hexitol nucleic acid (HNA), Altritol Nucleic Acid (ANA), Threonose nucleic acid (TNA) Cyclohexene nucleic acid (CeNA), Fluoroarabinoic acid (FANA) D-DNA ·L-DNA RNA - Duplexes of any combination of DNA, RNA, XNA, wherein a portion of the duplex contains at least one modified or modified base that is recognized by a structure-specific enzyme.

[0087] Sensitive barcode oligonucleotides and / or sensitive marker oligonucleotides may include: DNA, RNA, any XNA, which is substantially sensitive to removal agents, i.e., is cut at multiple sites, which causes the corresponding oligonucleotide to degrade in the presence of the removal agent or the corresponding marker oligonucleotide of the marker, thereby substantially degrading and releasing the marker from the affinity reagent.

[0088] In a particularly preferred embodiment, the barcoded affinity reagent comprises a barcode oligonucleotide 110 that is resistant to a degradation agent 116 , and the detectable label comprises a marker oligonucleotide 112 that is sensitive to the degradation agent 116 .

[0089] In a particularly preferred embodiment, the degrading agent 116 is DNase I, and the barcoded affinity reagent comprises a barcode oligonucleotide 110 that is resistant to a degrading agent 116 comprising, for example, PNA, LNA, PT-modified DNA, GNA, FANA, or a backbone, and the detectable label comprises a labeled oligonucleotide 112 that is sensitive to a degrading agent 116 comprising, for example, a DNA backbone.

[0090] In a particularly preferred embodiment of the present invention, DNase I is used as the degradation agent 116 and the barcoded affinity reagent comprises a barcode oligonucleotide 110 which is resistant to the degradation agent.

[0091] In a particularly preferred embodiment of the invention, DNase I is used for efficient removal of the label while keeping the barcode on the affinity reagent intact. In this case, the resistance barcode can comprise any XNA that is resistant to DNase I, such as PNA or phosphorothioate-modified (PT) DNA. The benefit is a rapid, robust and cost-effective method for label removal using a barcoded affinity reagent.

[0092] In addition to enabling a fast and cost-effective removal method (which is based on DNase I, combined with label cycling via hybridization), the present method provides another advantage. When barcoded affinity reagents are used to dye analytes in biological samples and detectable labels are bound to them via hybridization, cross-hybridization of the label with the OFF barcode can occur and produce false positive results. Therefore, it is necessary to improve the specificity of binding, which can be achieved by increasing the temperature at which hybridization is performed. However, this may lead to the formation of background in the sample. The use of hybridization configurations such as XNA:DNA or first XNA:second XNA proposed in this document can also significantly improve the specificity of barcode recognition under conditions commonly used for dyeing (e.g., temperature, ionic strength, pH).

[0093] For example, it is known that PNA:DNA hybridization is more stringent than DNA:DNA hybridization.PNA is very stable and resistant to both proteases and nucleases.Similarly, it is known that morpholino or LNA can bind to their targets with large specificity.PNA probes have been successfully used in single molecule fluorescence in situ hybridization and have been shown to provide higher specificity than DNA probes.This is because PNA oligonucleotides have higher affinity relative to DNA oligonucleotides of the same length.

[0094] Therefore, in a particularly preferred embodiment of the present invention, the detectable label comprises a PNA-oligonucleotide, which comprises a covalently coupled labeling moiety, such as, for example, a fluorescent dye, a polyalkyne or other Raman labeling. The PNA probe for smFISH or a PNA oligonucleotide with a dye-coupled dye molecule of 1, 2, 4 or n directly coupled can be commercially available from, for example, (Biomers.net GmbH, Ulm, Germany), wherein the direct coupling can be at the 2' position of the sugar backbone. The coupling of the labeling moiety can be easily performed using click chemistry. For example, using the Click-FISH PNA oligonucleotide from (Biomers.net GmbH, Ulm, Germany), the labeling moiety can be coupled via azide-alkyne click chemistry. Therefore, various labeling moieties can be quickly and easily coupled to the PNA oligonucleotide. The labeling moiety can be: an optically detectable label, such as a fluorescent dye, PDot, QDot, SMILE, an optically readable semiconductor particle, or a Raman label (e.g., polyalkynes), or Sequenceable tags, such as oligonucleotide barcodes (i.e., compatible with next generation sequencing and / or amplification, PCR, digital PCR), or Affinity tags, such as biotin, or high affinity guest molecules, such as cucurbit-7-urea based or other cucurbit-N-urea based host molecules, or FLAG tags, Myc tags, 3HA tags, His tags commonly used for affinity purification, or they can be, Enzyme tags, such as horseradish peroxidase, luciferase Electron microscopy can detect labels such as gold particles Radioactive labels XNA enzyme tags are tags containing catalytically active XNA Aptamer tag Electrical Impedance Spectroscopy Tags

[0095] In the sense of the present invention, the detectable marker may be physically fully constituted before being introduced into the biological sample. In some cases, it may also be already linked to an affinity reagent before being introduced into the biological sample.

[0096] However, in particularly preferred embodiments of the present invention, the detectable marker is formed by self-assembly following introduction of its components into a biological sample, wherein all components may be introduced simultaneously or in a sequential manner.

[0097] The detectable marker may further comprise a backbone, which may be a nucleic acid backbone, such as a flexible oligonucleotide, or a rigid 2D, 3D structure formed by, for example, DNA origami or DNA bricks technology. Likewise, the backbone may be a dendritic structure.

[0098] The availability of Click-FISH PNA probes (Biomers.net GmbH, Ulm, Germany) or similar click-chemistry functionalized PNA oligonucleotides allows for particularly cost-effective generation of fluorescently detectable markers by clicking azide-coupled dyes onto PNA oligonucleotides and hybridizing them to, for example, a DNA backbone containing the labeled oligonucleotide. Azide-coupled fluorescent dyes can be readily commercially obtained, for example, from ATTO-Tec GmbH (Siegen, Germany).

[0099] Using the same principle, detectable markers can also be functionalized with biotin tags for affinity purification of said detectable markers, which may be of interest when more complex detectable markers need to be made. This may be the case, for example, when using DNA origami-based or DNA brick-based backbones.

[0100] Affinity reagent is coupled to the first barcode containing oligonucleotide

[0101] In a particularly preferred embodiment of the invention, the resistant barcode oligonucleotide is coupled to the affinity reagent via click chemistry. For example, if an antibody is to be barcoded, this can be done by site-specific coupling of the barcode oligonucleotide to the IgG using azide-alkyne click chemistry, for which there are commercially available kits, such as the GlyCLICK Azide Activation Kit from Genovis AB (Lund, Sweden, #L1-AZ1-100), which produces a degree of labeling of 2 by site-specific coupling.

[0102] In all figures, the same or similar acting elements 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 associated listed items and may be abbreviated as " / ".

[0103] Although some aspects have been described in the context of an apparatus, it is clear that these aspects also represent a description of a corresponding method, where a block or device 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 block or item or feature of a corresponding apparatus.

[0104] Reference numerals 100a, 100b Target analyte 102a, 102b, 102c, 300, 308, markers 400, 1100, 1106, 1110, 1114 104a, 104b, 104c Affinity reagent backbone 106, 304, 402 marking part 110, 1102a, 1102b, 1102c Barcode oligonucleotides for affinity reagents 112 Labeled oligonucleotides 116 Degradation Agent 200, 202, 700, 106e, 106f, markers 106g, 1104, 1108, 1112, 1116 302 long oligonucleotides 306 oligonucleotides of the labeling part Over 500 Affinity Reagents 502 various markers 702 Sensitive Oligonucleotide-Dye Conjugate 704 Antibody Oligonucleotide-Dye Conjugate 706 Site modification of labeled oligonucleotides 902 Staple Chain 1000 Polystyrene Beads

Claims

1. A marker (102a, 102b, 102c, 300, 308) for analyzing a biological sample, comprising: an affinity reagent having a backbone (104a, 104b, 104c) and a barcode oligonucleotide (110) attached to the backbone (104a, 104b, 104c), wherein the backbone (104a, 104b, 104c) is configured to specifically bind to a target analyte (100a, 100b), and a label (200, 202) comprising a labeling oligonucleotide (112) and at least one labeling moiety (106, 304), wherein the labeling oligonucleotide (112) and the barcode oligonucleotide (110) of the affinity reagent are configured to hybridize with each other, One of the labeled oligonucleotide (112) and the barcode oligonucleotide (110) is sensitive to a degradation agent (116), and the other of the labeled oligonucleotide (112) and the barcode oligonucleotide (110) is resistant to the degradation agent (116).

2. The marker according to claim 1, wherein the backbone (104a, 104b, 104c) of the affinity reagent consists essentially of amino acids.

3. The marker according to claim 2, wherein the backbone (104a, 104b, 104c) of the affinity reagent is an antibody or an antibody fragment.

4. The marker according to claim 1, wherein the backbone (104a, 104b, 104c) of the affinity reagent consists essentially of nucleic acids.

5. The marker according to claim 4, wherein the backbone (104a, 104b, 104c) of the affinity reagent is configured to specifically bind to the target analyte (100a, 100b) through hybridization of a continuous nucleotide sequence with a complementary continuous target nucleotide sequence.

6. The marker according to claim 5, wherein the backbone (104a, 104b, 104c) of the affinity reagent has a composite structure, and the backbone (104a, 104b, 104c) is configured to specifically bind to the target analyte through its composite structure.

7. The marker according to claim 6, wherein the backbone (104a, 104b, 104c) of the affinity reagent is an aptamer.

8. The marker according to any one of claims 1-7, wherein one of the labeling oligonucleotide (112) and the barcode oligonucleotide (110) consists essentially of a nucleic acid that is sensitive to a degradation agent (116), and the other of the labeling oligonucleotide (112) and the barcode oligonucleotide (110) consists essentially of a nucleic acid analog that is resistant to the degradation agent (116).

9. The marker according to claim 8, wherein the labeling oligonucleotide (112) consists essentially of natural nucleic acid and the barcode oligonucleotide (110) consists essentially of nucleic acid analogs.

10. The marker of claim 9, wherein the nucleic acid analog comprises at least one of 1,5-anhydrohexitol nucleic acid, cyclohexene nucleic acid, threose nucleic acid, ethylene glycol nucleic acid, locked nucleic acid, peptide nucleic acid, fluoroarabino nucleic acid, bridged nucleic acid, morpholino, phosphorothioate backbone, L-pentose backbone, and nucleic acid comprising at least one modified nucleoside.

11. The marker according to any one of claims 1 to 9, wherein the at least one labeling portion (106, 304) is optically detectable.

12. The marker according to any one of claims 1-11, wherein the degrading agent (116) is a nuclease.

13. A marker according to any one of claims 1-12, wherein the degrading agent (116) and the sensitive labeled oligonucleotide (112) or sensitive barcode oligonucleotide (110) are configured such that application of the degrading agent (116) releases the barcode oligonucleotide (110) from the labeled oligonucleotide-barcode oligonucleotide duplex, thereby enabling the barcode oligonucleotide (110) to hybridize with another labeled oligonucleotide (112).

14. The marker according to any one of claims 1-13, wherein the degrading agent (116) is an artificially engineered peptidase or protease configured to degrade peptide nucleic acids or xenogeneic nucleic acids.

15. The marker according to any one of claims 1 to 14, wherein the degradation agent (116) is silver, iodine or mercury, and the sensitive nucleic acid analog is a phosphorothioate-modified nucleic acid.

16. The marker according to any one of claims 1 to 15, wherein the degradation agent (116) is based on an inorganic or organic chemical that degrades the sensitive nucleic acid analog.

17. A kit comprising at least one marker (102a, 102b, 102c, 300, 308) according to any one of claims 1 to 16 and the degradation agent (116).

18. A kit comprising at least one barcode oligonucleotide and chemical components necessary to couple the barcode oligonucleotide to the affinity reagent backbone to produce the marker according to any one of claims 1-16.

19. A kit comprising at least one label and a degrading agent, wherein the at least one label comprises a labeled oligonucleotide to produce a marker according to any one of claims 1 to 16.

20. A method for analyzing a biological sample comprising at least a target analyte (100a, 100b), the method comprising the following steps: introducing into the biological sample at least a first marker (102a, 102b, 102c, 300, 308) according to any one of the preceding claims 1 to 16, the at least first marker comprising an affinity reagent having a backbone (104a, 104b, 104c) specific for the target analyte and a first marker (200, 202) having at least a first labeling portion (106, 304), generating a first reading of the biological sample having the at least one marker (102a, 102b, 102c, 300, 308), applying the degradation agent to the biological sample to degrade the labeled oligonucleotide (112) of the marker (102a, 102b, 102c, 300, 308), introducing at least one second label (200, 202) having at least a second label portion into the biological sample to produce a second marker (102a, 102b, 102c, 300, 308), the second label comprising a second labeling oligonucleotide configured to hybridize with the barcode oligonucleotide of the affinity reagent, and A second reading of the biological sample having the second marker (102a, 102b, 102c, 300, 308) is generated.

21. The method of claim 20, wherein the target analyte is identified based on the first marker and the second marker associated with the target analyte in the first and second readings.

22. A method according to claim 20 or 21, wherein in the step of introducing the at least one first marker, the affinity reagent for the at least one first marker is introduced before the corresponding labeling of the first marker, and wherein the affinity reagent is fixed in the biological sample before the label of the first marker having the first labeling part is introduced into the biological sample.

23. The method of any one of the preceding claims 20-22, wherein the first labeling moiety and the second labeling moiety have different detectable properties.

24. A method according to any one of the preceding claims 20-23, wherein after the step of generating the second reading, the following steps are iteratively repeated: applying the degradation agent (116) to degrade the labeled oligonucleotide (112), adding at least one additional label having an additional label portion, and generating an additional reading of the biological sample.

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