Affinity reagents, markers and methods for analyzing biological samples

Abstract

The present invention provides an affinity reagent (102) for analyzing a biological sample. The affinity reagent (102) comprises a backbone (106) and a barcode oligonucleotide (108) attached to the backbone (106). The backbone (106) consists essentially of nucleic acid analogs. The backbone (106) is configured to specifically bind to a target analyte (114) by its complex structure. The barcode oligonucleotides (108) consist essentially of nucleic acid analogs. In another aspect, there is provided a marker (100, 206) comprising an affinity reagent (102) and a marker (104, 202). Furthermore, a method for analyzing a biological sample having a marker (100, 206) is provided.

Description

Technical Field

[0001] The present invention relates to an affinity reagent for analyzing a biological sample, the affinity reagent comprising a backbone and a barcode oligonucleotide. In another aspect, a marker comprising an affinity reagent and a method for analyzing a biological sample having the marker are provided. Background Art

[0002] Affinity reagents contain chemically heterogeneous groups and include, for example, antibodies, nanobodies, nucleic acid probes and aptamers. Affinity reagents are commonly used in life science research and diagnostic applications to detect target molecules, also known as analytes. To this end, a variety of detectable labels are used, which include at least one labeling moiety.

[0003] Aptamers are usually 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, which is connected to an oligonucleotide sequence unique to the affinity reagent and can contain one or more predetermined sequences that can be hybridized by a detectable marker containing 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.

[0004] 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 cell types in the same experiment, functional markers such as proteins of interest, and general morphological markers. This means that the cell types in most imaging experiments are only poorly identified. 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.

[0005] 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.

[0006] 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

[0007] One object is to provide barcoded affinity reagents and markers that enable efficient and reproducible staining of biological samples, and methods for analyzing biological samples having said markers.

[0008] 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.

[0009] An affinity reagent for analyzing a biological sample is provided. The affinity reagent comprises a backbone and a barcode oligonucleotide attached to the backbone. The backbone consists essentially of nucleic acid analogs. The backbone is configured to specifically bind to the target analyte through its complex structure. The barcode oligonucleotide consists essentially of nucleic acid analogs.

[0010] Specifically, the barcode oligonucleotide is configured as a labeling oligonucleotide of a hybrid detectable marker, so that the marker is attached to the affinity reagent. The complex structure of the skeleton of the affinity reagent can be a secondary, tertiary or quaternary structure of a skeleton nucleic acid analog. Therefore, the skeleton of the affinity reagent has a secondary, tertiary or quaternary structure in particular. Specifically, due to the complex structure of the skeleton of the affinity reagent, the affinity reagent specifically binds to the target analyte. Specifically, a single discontinuous nucleotide of the nucleic acid analog of the skeleton can be bound to the target analyte, rather than the continuous nucleotides of the sequence of the nucleic acid analog and the target analyte, particularly the complementary sequence hybridization of the target analyte. This enables affinity reagent to be combined with high specificity. Specifically, by paratope-epitope interactions rather than by hybridization of complementary nucleic acid sequences, the skeleton of the affinity reagent, such as an aptamer, can be bound to its target.

[0011] Oligonucleotide refers specifically to nucleotide sequences of any length and nucleic acid-based structures that may be monopartite or multipartite in nature.

[0012] Generally, nucleic acid analogs are compounds that are structurally similar to naturally occurring RNA and DNA. Nucleic acid is a nucleotide chain, which is composed of three parts: a phosphate backbone, a pentose (ribose or deoxyribose) and one of four nuclear bases. Analogs can have any one of these changed. Therefore, specifically, nucleic acid analogs can be non-naturally occurring, modified, artificial or heterologous nucleic acids (XNA). Although natural nucleic acids or naturally occurring nucleic acids (DNA and RNA) are usually sensitive to natural degradation agents, such as nucleases, nucleic acid analogs are usually resistant to these degradation agents, such as nucleases. Therefore, this can provide an affinity reagent that is resistant to degradation by natural degradation agents (such as nucleases), and therefore a specific robust affinity reagent can be achieved. Specifically, this can utilize a degradation agent based on an affinity reagent to repeatedly stain a biological sample without causing denaturation or degradation of the affinity reagent. For example, a biological sample can be a tissue section. Other examples of the biological sample include cells, cell cultures or body fluid samples.

[0013] In particular, the barcode oligonucleotide is 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, the backbone of the affinity reagent does not refer only to the phosphate backbone of the nucleic acid analog. Thus, the barcode oligonucleotide can be attached to any of the phosphate backbone, pentose, or nucleobase of the nucleic acid analog of the backbone of the affinity reagent or their corresponding analogs.

[0014] Preferably, the skeleton comprises 10 to 100 nucleotides. This realizes a particularly compact affinity reagent, which is easily and quickly introduced into a biological sample, but still has high specificity to the corresponding target analyte. Specifically, these nucleotides can be nucleotide analogs. Preferably, the skeleton comprises 30 to 80 nucleotides. In particular and in a related metric, the size of the skeleton can be 10 to 30 kDa. In a particular embodiment, the skeleton can be polymerized and comprise several copies of the skeleton, and the skeleton contains 10 to 100 nucleotides.

[0015] Preferably, the nucleic acid analog of the backbone includes at least one of the following: locked nucleic acid (LNA), morpholino, a nucleic acid backbone containing thiophosphate (PT) modification, or may include a complete PT-DNA backbone, a peptide nucleic acid (PNA), a bridged nucleic acid (BNA), 2'-O-methyl substituted RNA, glycol nucleic acid (GNA), hexitol nucleic acid (HNA), altritol nucleic acid (ANA), threose nucleic acid (TNA), cyclohexene nucleic acid (CeNA), fluoroarabinose nucleic acid (FANA), L-deoxyribonucleic acid. Specifically, L-deoxyribonucleic acid contains an L-pentose backbone. This enables a robust backbone of the affinity reagent.

[0016] Preferably, the nucleic acid analog of the barcode oligonucleotide includes at least one of the following: locked nucleic acid (LNA), morpholino, a nucleic acid backbone containing thiophosphate (PT) modification, or may include a complete PT-DNA backbone, peptide nucleic acid (PNA), bridged nucleic acid (BNA), 2'-O-methyl substituted RNA, glycol nucleic acid (GNA), hexitol nucleic acid (HNA), altritol nucleic acid (ANA), threose nucleic acid (TNA), cyclohexene nucleic acid (CeNA), fluoroarabinose nucleic acid (FANA), L-deoxyribonucleic acid. Specifically, the L-deoxyribonucleic acid contains an L-pentose backbone. This enables the robust barcode oligonucleotide of the affinity reagent to be achieved.

[0017] The use of LNA, PT-DNA, morpholino or PNA for the backbone of the affinity reagent or the barcode oligonucleotide may be particularly preferred, since these XNAs are not only nuclease resistant, which improves their stability, but also bind to complementary DNA with higher affinity.

[0018] Particularly preferably, the framework is an aptamer. This enables efficient generation of a framework of the affinity reagent that is specific to a series of target analytes (such as various proteins). Aptamers can be generated in various ways, with SELEX being the most typical approach. In addition, preferably, the nucleic acid analog framework of the aptamer can include modifications, such as glycosylation or a nuclear base modification with an amino acid or a chemical moiety similar to an amino acid side chain. Such modifications have been introduced, for example, on slow OFF-rate Modified Aptamers (SOMAmer) or Aptabody, and improve target binding. Specifically, SOMAmer exhibits a slower dissociation rate than most aptamers.

[0019] Aptamers are next generation affinity reagents, which provide many advantages compared to antibodies, including lower production costs, higher batch-to-batch consistency, digital storage, on-demand synthesis (i.e., oligonucleotide synthesis), ease of functionalization, low immunogenicity, free selection of required epitopes, and short development time. Aptamers are usually found by using SELEX. Traditional or classical SELEX has been described in US 5,475,096 and US 5,279,163. At the same time, a variety of SELEX methods have been developed, which have improved some aspects of the original method and allow the development of aptamers with improved efficiency, better dissociation rate, higher affinity or further functionalization, such as covalently locked to the aptamer of its target (e.g., Chemi-SELEX; ​​Photo-SELEX). Large libraries of aptamers or modified aptamers (such as aptamers (SOMAmers) modified with slow dissociation rate) are commercially available from SOMALogic (Boulder, USA) or other aptamer companies. In the case of microscopy, the small size of the aptamer is a specific advantage as it allows better penetration into thicker samples. Likewise, in previous applications, additional advantages of using aptamers in microscopy applications or proteomics using microscope readouts have been disclosed in EP applications with corresponding application numbers 23178065.1 and 23183879.8, the entire corresponding contents of which are incorporated herein by reference.

[0020] Preferably, the barcode oligonucleotide is adapted to be linked to a marker via a label oligonucleotide, the label comprising the label oligonucleotide and at least one labeling moiety, wherein preferably the backbone and the barcode oligonucleotide are resistant to a degradation agent, while the label oligonucleotide is sensitive to the degradation agent. This enables easy addition of a label to the affinity reagent in order to generate a marker.

[0021] For example, the labeling portion may be one of the following: a fluorescent dye, a fluorescent structure, a Tau tag (TauTag) disclosed in PCT application No. PCT / EP2022 / 060035 (the entire content of the PCT application is incorporated herein by reference), WO 2022 / 242849 A1 and WO 2022 / 242887 A1 combines the combined (fluorescent) markers disclosed in PCT application number PCT / EP2023 / 075470 (the entire corresponding contents of these documents are incorporated herein by reference), the responsive markers disclosed in EP application number 23192184.2 (the entire content of this EP application is incorporated herein by reference), oligonucleotide markers configured for sequencing-based readings, affinity tags configured for affinity purification (e.g., FLAG tags, StrepII tags, Myc tags), polystyrene beads, metal tags, enzymes (e.g., horseradish peroxidase), radioactive tags, bioluminescent tags, FRET tags, Raman tags, polyalkyne-based tags, and SRS tags.

[0022] On the other hand, a marker for analyzing a biological sample is provided. The marker comprises: an affinity reagent, and a marker comprising a labeled oligonucleotide and at least one labeling portion. The barcode oligonucleotide of the labeled oligonucleotide and the affinity reagent are complementary to each other, and based on the complementary base pairing between the labeled oligonucleotide and the barcode oligonucleotide, the marker hybridizes with the affinity reagent. Specifically, the continuous sequence of the labeled oligonucleotide is bound to the complementary continuous sequence of the barcode oligonucleotide. One of the labeled oligonucleotide and the barcode oligonucleotide is sensitive to a degradation agent, while the other of the labeled oligonucleotide and the barcode oligonucleotide is resistant to the degradation agent. Therefore, the sensitive oligonucleotide is configured to be degraded by the degradation agent. The resistant oligonucleotide is configured not to be degraded by the degradation agent. This enables selective degradation of the labeled oligonucleotide or the barcode oligonucleotide.

[0023] In particular, this enables the selective degradation of the sensitive one of the marker oligonucleotide and the barcode oligonucleotide, and thus, by adding the degradation agent, the label is removed from the affinity reagent, in particular from the affinity reagent backbone. Preferably, the affinity reagent, in particular the affinity reagent backbone, remains intact in the presence of the degradation agent. In particular, the affinity reagent backbone can continue to be configured to specifically bind to the target analyte in the presence of the degradation agent. Therefore, preferably, the affinity reagent backbone is resistant to the degradation agent.

[0024] The degradation agent only causes a sensitive degradation of the labeled oligonucleotide and the barcode oligonucleotide, specifically, the degradation agent can digest or fragment the sensitive oligonucleotide. This degradation usually leads to the loss of the oligonucleotide function, particularly 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 (specifically) degraded by the degradation agent. Preferably, the degradation of the sensitive oligonucleotide is irreversible. Therefore, the degradation agent preferably removes the marker, particularly the labeled oligonucleotide from the barcode oligonucleotide, and makes the barcode oligonucleotide accessible to another marker in another round of labeling. The marker is specifically removed from the affinity reagent while keeping the affinity reagent intact, so that it is easy to repeat adding and removing markers to the affinity reagent in a cyclic dyeing method.

[0025] In the sense of this document, the virtual assignment or mapping of an affinity reagent to a detectable label or the physical association of the two can represent a marker. Markers in the sense of this document can be particularly used to label analytes for image-based readings (e.g., microscopy, plate readings), colorimetric readings, blood cell counting readings, densitometer readings, or visual inspections. Such markers are used in various applications, including but not limited to Western blots, ELISA assays, ELISpot assays, FluroSpot assays, LUMINEX TM Assays, AVEXIS assays, immunofluorescence, immunocytochemistry, immunohistochemistry, proximity ligation assays, proximity extension assays, protein purification, protein quantification, proteomic analysis, biomarker analysis, pathogen detection and identification, and cytometry. Currently, most markers used in life science research and protein detection diagnostics are antibody based.

[0026] The sequences of the barcode oligonucleotide and the label oligonucleotide may preferably be specific for the corresponding affinity reagent and / or the corresponding labeling moiety.

[0027] Preferably, the labelled oligonucleotide is sensitive to the degradation agent, while the affinity reagent is resistant to the degradation agent. Thus, preferably, the barcode oligonucleotide and the backbone of the affinity reagent are resistant to the degradation agent. This makes the robust affinity reagent particularly suitable for cyclic staining methods.

[0028] Preferably, the labeled oligonucleotide is essentially composed of natural nucleic acids. Therefore, the labeled oligonucleotide is preferably not a nucleic acid analog. This makes the labeled oligonucleotide particularly sensitive to the degradation agent. The natural nucleic acid is generally sensitive to naturally occurring degradation agents.

[0029] Preferably, the at least one labeling moiety is optically detectable. This enables easy detection of the marker. For example, the labeling moiety is a fluorophore or a combination of multiple fluorophores. The optically detectable labeling moiety can be detected by fluorescence microscopy, or other suitable optical readouts.

[0030] Alternatively, the labeling moiety may be an alkyne detectable, for example, by Raman spectroscopy.

[0031] Preferably, the marker comprises a plurality of labeling parts. The plurality of labeling parts may all have the same detectable properties, or they may have different properties. This makes the marker particularly easy to detect or the markers particularly easy to distinguish from each other.

[0032] Preferably, the degradation agent is a nuclease. For example, the degradation agent may be DNase I. This makes the sensitive oligonucleotide, especially the naturally occurring nucleic acid, easily degraded, and in particular, the resistant oligonucleotide may be a nucleic acid analog.

[0033] Alternatively, the degradation agent can be, for example, iodine. In this case, the sensitive oligonucleotide can be a phosphorothioate-modified nucleic acid analog that is sensitive to iodine, and the resistant oligonucleotide can be a naturally occurring nucleic acid (e.g., D-DNA) or a nucleic acid analog that is resistant to iodine, such as L-DNA.

[0034] Particularly preferred combinations of marker oligonucleotides, barcode oligonucleotides, the affinity reagent backbone and the degradation agent are: a backbone of an affinity reagent consisting essentially of L-deoxyribonucleic acid, a barcode oligonucleotide consisting essentially of locked nucleic acid, a marker oligonucleotide consisting essentially of D-deoxyribonucleic acid and a nuclease (such as DNase I) as a degradation agent. In this case, the D-DNA marker oligonucleotide is sensitive to the degradation agent and is easily digested by the nuclease when it is added. The remaining nucleic acid is resistant to the nuclease degradation agent. Alternatively, a backbone of an affinity reagent consisting essentially of L-deoxyribonucleic acid, a barcode oligonucleotide consisting essentially of thiophosphate-based nucleic acid, a marker oligonucleotide consisting essentially of D-deoxyribonucleic acid and a nuclease such as DNase I as a degradation agent. In this case, the D-DNA marker oligonucleotide is sensitive to the degradation agent and is easily digested by the nuclease when it is added. The remaining nucleic acid is resistant to the nuclease degradation agent. Further alternatively, the backbone of the affinity reagent consisting essentially of D-deoxyribonucleic acid, the barcode oligonucleotide consisting essentially of D-deoxyribonucleic acid, the labeling oligonucleotide consisting essentially of nucleic acid based on thiophosphate, and silver, iodine or mercury as a degradation agent. In this case, the labeling oligonucleotide based on nucleic acid of thiophosphate is sensitive to the degradation agent and is easily degraded by the degradation agent when it is added. The remaining nucleic acid is resistant to the degradation agent. Further alternatively, the backbone of the affinity reagent consisting essentially of L-deoxyribonucleic acid, the barcode oligonucleotide consisting essentially of locked nucleic acid, the labeling oligonucleotide consisting essentially of nucleic acid based on thiophosphate, and silver, iodine or mercury as a degradation agent. In this case, the labeling oligonucleotide based on nucleic acid of thiophosphate is sensitive to the degradation agent and is easily degraded by the degradation agent when it is added. The remaining nucleic acid is resistant to the degradation agent.

[0035] On the other hand, a method for analyzing a biological sample containing at least a target analyte is provided. The method comprises introducing at least a first marker, in particular a component thereof, into the biological sample. The first marker comprises an affinity reagent having a skeleton specific for the target analyte and a first marker having at least a first labeling portion. Specifically, by introducing the marker, the marker can be bound to the corresponding target analyte in the biological sample. The method also includes generating a first reading of the biological sample having at least one first marker. This step may include directing excitation light onto the biological sample to visualize the optically detectable marker, and generating a reading from light emitted by the marker, for example, by a microscope.

[0036] The method also includes applying a degradation agent to the biological sample to remove the first marker of the first marker. Specifically, this can degrade and release the labeled oligonucleotide from the marker, particularly from the affinity reagent of the marker. The step may optionally include washing the biological sample to remove the released marker. The method also includes the following steps: introducing at least one second marker having at least a second labeling portion into the biological sample to generate a second marker, the second marker comprising a second labeled oligonucleotide configured to hybridize with the barcode oligonucleotide of the affinity reagent (or with the barcode oligonucleotide of the affinity reagent), and generating a second reading of the biological sample with the second marker. This enables robust analysis of the biological sample, particularly the target analyte. Using the marker with a sensitive oligonucleotide and a resistant oligonucleotide can easily degrade the specific elements of the marker so that the marker associated with the affinity reagent can circulate. Preferably, the labeled oligonucleotide is sensitive to the degradation agent.

[0037] The second labeling oligonucleotide can be the same as the labeling oligonucleotide of the first label. Therefore, the first labeling oligonucleotide and the second labeling oligonucleotide can be bound to the same barcode oligonucleotide. Alternatively, the affinity reagent can include several barcode oligonucleotides whose nucleotide sequences are different. In this case, the first labeling oligonucleotide and the second oligonucleotide can have a sequence complementary to a specific one of the several barcode oligonucleotides.

[0038] It is particularly preferred that the reading generated is an optical reading, such as a reading generated by a microscope.

[0039] Typically, a plurality of markers specific for corresponding target analytes may be introduced into the biological sample in order to identify a large number of target analytes simultaneously.

[0040] Preferably, the target analyte is identified and / or localized within the biological sample based on the first and second markers, in particular the labeling moieties, associated with the target analyte in the first and second readings. This enables identification and / or localization of the target analyte with improved accuracy. Since the affinity reagent is bound to the target analyte, the first and second markers introduced into the biological sample are associated with the target analyte via the affinity reagent.

[0041] Preferably, in the step of introducing the at least one first marker, in particular separately, before the first marker of the first marker, an affinity reagent of the at least one first marker is introduced, and before the first marker with the first labeling part is introduced into the biological sample, the affinity reagent is fixed in the biological sample. This enables the marker to be introduced into the biological sample separately as a smaller single element that can more easily penetrate the biological sample, and then the marker is generated in situ. The affinity reagent can be fixed by cross-linking it with the biological sample or part thereof. Preferably, the affinity reagent is fixed after being bound to the target analyte. This keeps the affinity reagent in place, which may be particularly beneficial in the case of many rounds of cyclic labeling subsequently. The marker is preferably introduced after the affinity reagent is cross-linked.

[0042] Preferably, the first labeling part and the second labeling part have different detectable properties. This makes it possible to easily distinguish the labeling parts. For example, the labeling part can be a fluorophore with different, distinguishable emission wavelengths, excitation wavelengths or emission durations (such as, for example, fluorescence lifetimes).

[0043] Preferably, after the step of generating the second reading, the following steps are repeated at least once iteratively: applying the degradation agent to degrade the second labeled oligonucleotide, adding at least one other marker with another labeling portion, and generating another reading of the biological sample. This enables robust analysis of the biological sample, particularly the target analyte. Specifically, this enables identification and / or positioning of the target analyte with improved accuracy. In particular, in the case of analyzing a biological sample with a large number of target analytes, each target analyte can be labeled with a specific marker to identify and / or position each target analyte with improved accuracy. By iteratively removing different markers and attaching different markers to corresponding affinity reagents, the target analyte can be identified and / or positioned with improved accuracy. The other marker preferably has a detectable property different from the first marker and the second marker (particularly their respective labeling portions) to distinguish these markers.

[0044] In another aspect, a kit comprising at least one marker and a degradation agent is provided. Alternatively, the kit may include an affinity agent, a marker and a degradation agent. The marker comprises a labeled oligonucleotide and at least one labeling portion. The marker is also configured to constitute a marker when the marker is connected to the affinity agent. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] In the following, specific embodiments are described with reference to the accompanying drawings, in which:

[0046] Figure 1is a schematic diagram of the affinity reagent and label of the marker, and

[0047] Figure 2 is a schematic diagram of the steps of a method for analyzing a biological sample for markers. DETAILED DESCRIPTION

[0048] Figure 1 1 is a schematic diagram of an affinity reagent 102 and a label 104 of a marker 100. The affinity reagent 102 comprises a backbone 106 and a barcode oligonucleotide 108. The backbone 106 consists essentially of nucleic acid analogs. Similarly, the barcode oligonucleotide 108 consists essentially of nucleic acid analogs.

[0049] Generally, nucleic acid analogs are compounds similar in structure to naturally occurring RNA and DNA. Nucleic acid is a nucleotide chain, which is composed of three parts: a phosphate backbone, a pentose (ribose or deoxyribose) and one of four nuclear bases. Analogs can have any one of these changed. Nucleic acid analogs can be artificial nucleic acids or heterologous nucleic acids. Specific examples of nucleic acid analogs include: nucleic acids with modified sugars, such as L-DNA containing L-pentose, or modifications of ribose modified by, for example, 2'-amino, 2'-O-methyl, 2'-fluoro at the 2' position. In addition, nucleic acids have modified phosphate backbones, such as phosphorothioate (PT) or boronated phosphate (BP) modifications. These nucleic acid analogs are generally resistant to naturally occurring nucleases. This provides a particularly robust affinity reagent, which is particularly suitable for applications in biological samples that generally contain naturally occurring nucleases, such as staining.

[0050] For example, the backbone 106 of the affinity reagent 102 can be an aptamer based on a nucleic acid analog. The barcode oligonucleotide 108 can be covalently attached to the backbone 106 of the affinity reagent 102. The barcode oligonucleotide 108 can be attached to any of the phosphate backbone, pentose, or nucleobase of the nucleic acid analog of the backbone 106 of the affinity reagent 102, or their corresponding analogs. For example, the attachment of the barcode oligonucleotide 108 can be performed by azide / alkyne click chemistry. Alternatively, the attachment of the barcode oligonucleotide 108 can be performed by a near covalent interaction between streptavidin-biotin. To this end, the barcode oligonucleotide 108 can be covalently attached to one of (monovalent) streptavidin and biotin, and the backbone 106 of the affinity reagent 102 can be covalently attached to the other of (monovalent) streptavidin and biotin. When the affinity reagent 102 is assembled, streptavidin binds to the biotin, resulting in the attachment of the barcode oligonucleotide 108 to the backbone 106. This may be particularly advantageous when a large library of aptamers needs to be individually ligated to barcodes.

[0051] The barcode oligonucleotide 108 can be attached to the backbone 106 during solid phase synthesis of the backbone.

[0052] The barcode oligonucleotide 108 can be attached to the backbone 106 after backbone synthesis using, for example, click chemistry such as azide / alkyne modification to covalently link the barcode oligonucleotide 108 and the backbone 106 .

[0053] The barcode oligonucleotide 108 may be attached to the backbone 106 via an affinity interaction, for example the backbone 106 may be biotinylated and the barcode oligonucleotide may be conjugated to (monovalent) streptavidin.

[0054] The barcode oligonucleotide 108 can be attached to the backbone 106 using a linker oligonucleotide that is configured to hybridize to a portion of the barcode oligonucleotide and a portion of the backbone to form a triplex structure in which the barcode oligonucleotide 108 and the backbone 106 are directly opposite. In this case, T4 DNA ligase or a similar DNA ligase can be used to close the open "nick", i.e., the missing covalent link in the backbone of the barcode oligonucleotide 108 and the backbone 106. When large libraries must be ligated to corresponding barcodes, suitable universal adapter sequences on the barcode oligonucleotide 108 and the backbone 106 can be used for this purpose.

[0055] The skeleton 106 of affinity reagent 102 is configured to specifically bind to a specific target analyte 114, such as a protein of a biological sample. Specifically, the skeleton 106 can form or fold into a complex structure or conformation. The complex structure of the skeleton 106 of affinity reagent 102 can be a secondary, tertiary and / or quaternary structure of the skeleton 106 nucleic acid analog. Therefore, the skeleton 106 of affinity reagent 102 has a secondary, tertiary or quaternary structure in particular. Due to the complex structure of the skeleton 106 of affinity reagent 102, affinity reagent 102 specifically binds to target analyte 114. Specifically, a single discontinuous nucleotide of the nucleic acid analog of the skeleton 106 binds to the target analyte 114. This combination can include intermolecular forces, such as hydrogen bonds, dipole-dipole interactions and van der Waals forces. This is in contrast to nucleic acid analog hybridization, which is characterized by the hybridization of a continuous nucleotide sequence of a nucleic acid analog with a target analyte, particularly a complementary nucleotide sequence of a target analyte.

[0056] Based on the complex structure of the backbone 106 , the binding of the backbone 106 of the affinity reagent 102 to the target analyte 114 achieves particularly high affinity binding and specificity for the target analyte 114 .

[0057] The nucleic acid analogs of the backbone 106 may include multiple individual sequences of nucleic acid analogs. These individual sequences may bind to each other and form a quaternary structure.

[0058] The marker 104 comprises a marker oligonucleotide 110 and a marker portion 112. The marker oligonucleotide 110 and the barcode oligonucleotide 108 are complementary to each other so that they can hybridize with each other. Preferably, the barcode oligonucleotide 108 sequence is specific to the backbone 106 of the affinity reagent 102. Therefore, the identity of the target analyte 114 that is specific to the backbone 106 of the affinity reagent 102 can be encoded in the barcode oligonucleotide 108. Similarly, the detectable properties of the marker portion 112 can be encoded in the barcode oligonucleotide 108. Therefore, these properties or identities can be similarly encoded in the complementary marker oligonucleotide 110.

[0059] The labeling part 112 can be detected by a specific detection instrument. For example, the labeling part 112 can be a fluorophore detectable by a fluorescence microscope.

[0060] The marker 100 is generated by attaching a label 104 to an affinity reagent 102 based on complementary barcode oligonucleotides 108 and labeling oligonucleotides 110 .

[0061] One of the marker oligonucleotide 110 and the barcode oligonucleotide 108 is sensitive to a degradation agent, while the other of the marker oligonucleotide 110 and the barcode oligonucleotide 108 is resistant to the degradation agent. The backbone 106 of the affinity reagent 102 is preferably resistant to the degradation agent. This enables efficient removal of the marker 104 from the affinity reagent 102 by adding a degradation agent. For example, in the case where the marker oligonucleotide 110 is sensitive to a degradation agent and the barcode oligonucleotide 108 is resistant to the degradation agent, the marker oligonucleotide 110 is degraded by the degradation agent, thereby causing the marker 104 to be removed or dissociated from the affinity reagent 102. For this reason, the marker oligonucleotide 110 is preferably substantially composed of a natural nucleic acid such as D-DNA.

[0062] In a particularly preferred embodiment, the backbone 106 consists essentially of L-DNA, and the barcode oligonucleotide 108 consists essentially of a PT-modified or BP-modified nucleic acid analog. In this case, the labeling oligonucleotide 110 can be D-DNA, and the degradation agent can be a naturally occurring nuclease, such as DNase I. The use of L-DNA and PT- or BT-modified nucleic acid analogs for the backbone 106 and barcode oligonucleotide 108, respectively, has the following advantages: (A) both are highly resistant to nucleases and will be stable during cyclic staining, and (B) the modified barcode oligonucleotide 108 comprising D-DNA will not hybridize with the aptamer backbone comprising L-DNA.

[0063] In an alternative embodiment, the labeling oligonucleotide 110 can be a PT-modified nucleic acid analog that is sensitive to iodine degradation agents. The backbone 106 and barcode oligonucleotide 108 can be iodine-resistant nucleic acid analogs, such as L-DNA.

[0064] Figure 2 1 is a schematic diagram of the steps of the method for analyzing a biological sample (not shown in the figure) with a marker 100. Initially, the marker 100 is introduced into the biological sample and is bound to the target analyte 114. Similarly, a variety of (different) markers can be introduced into the biological sample, wherein the various markers have markers that are specific to different target analytes. The markers of the various markers can be different according to their specificity to a specific target analyte. Then, a reading of the biological sample can be generated, such as an optical reading. For example, in the case where the labeling portion 112 is a fluorophore, the reading can be an optical reading generated by a fluorescence microscope. In the reading, a detectable marker can be identified.

[0065] In the next step, a degradation agent 200 may be applied to the biological sample. For example, the degradation agent 200 may be a nuclease, such as DNase I. The marker oligonucleotide 110 is sensitive to the degradation agent 200, for example, because the marker oligonucleotide 110 is D-DNA. The resistant barcode oligonucleotide 108 and the backbone 106 of the affinity reagent may be L-DNA resistant to the nuclease degradation agent 200. Digestion of the marker oligonucleotide 110 by the nuclease results in the removal of the marker 104 from the affinity reagent 102 of the marker 100. Optionally, the elements 110, 112 of the marker 104 remaining in the biological sample may be washed away.

[0066] In the next step, a second marker 202 is introduced into the biological sample, the second marker comprising a marker oligonucleotide 110 and a second marker portion 204. The marker oligonucleotide 110 is easily hybridized with the barcode oligonucleotide 108 of the affinity reagent 102. This generates a second marker 206 in the biological sample attached to the target analyte 114. Preferably, the second marker portion 204 has different detectable properties so as to distinguish between the first marker 102 and the second marker 202 that are initially attached to the target analyte 114 by the affinity reagent 102. In the case of a fluorophore labeling portion, the detectable properties may include an excitation wavelength, an emission wavelength, and an emission duration. A second reading of the biological sample having the second marker 206 attached to the target analyte 114 can be generated. Based on the first reading and the second reading, the target analyte 114 can be identified and / or located in the biological sample.

[0067] The method can be repeated by removing the second marker 202 with the degradation agent 200 and iteratively adding at least another marker to the biological sample and removing at least another marker from the biological sample. This enables the use of multiple markers comprising different markers to reliably identify and / or locate a large number of target analytes. In each iteration of the method, a marker with a specific detectable property can be assigned to a specific affinity reagent and, therefore, to a target analyte. Based on this assignment, the target analyte can be identified and / or located in the generated readings.

[0068] 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 " / ".

[0069] 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.

[0070] Reference numerals 100, 206 markers 102 Affinity reagent 104, 202 markers 106 Affinity Reagent Backbone 108 Barcoded oligonucleotides 110 Labeled oligonucleotide 112, 204 Marking part 114 Target Analytes 200 Degradation Agent

Claims

1. An affinity reagent (102) for analyzing a biological sample, comprising: skeleton (106), and a barcode oligonucleotide (108) attached to said backbone (106), wherein the backbone (106) consists essentially of nucleic acid analogs, the backbone (106) being configured to specifically bind to a target analyte (114) via its complex structure, and Wherein the barcode oligonucleotide (108) consists essentially of nucleic acid analogs.

2. The affinity reagent according to claim 1, wherein the backbone (106) comprises 10 to 100 nucleotides.

3. The affinity reagent according to claim 1 or 2, wherein the nucleic acid analog of the backbone (106) comprises at least one of the following: locked nucleic acid, morpholino, nucleic acid backbone comprising thiophosphate modification, peptide nucleic acid, bridged nucleic acid, 2'-O-methyl substituted RNA, ethylene glycol nucleic acid, hexitol nucleic acid, altritol nucleic acid, threose nucleic acid, cyclohexene nucleic acid, fluoroarabinose nucleic acid, L-DNA.

4. The affinity reagent according to any one of claims 1-3, wherein the nucleic acid analog of the barcode oligonucleotide (108) comprises at least one of the following: locked nucleic acid, morpholino, a nucleic acid backbone comprising thiophosphate modification, a peptide nucleic acid, a bridged nucleic acid, 2'-O-methylated RNA, ethylene glycol nucleic acid, hexitol nucleic acid, altritol nucleic acid, threose nucleic acid, cyclohexene nucleic acid, fluoroarabinose nucleic acid, L-DNA.

5. The affinity reagent according to any one of claims 1 to 4, wherein the scaffold (106) is an aptamer.

6. An affinity reagent according to any one of claims 1-5, wherein the barcode oligonucleotide (108) attached to the backbone (106) is suitable for being connected to a marker (104, 202) via a labeling oligonucleotide (110), the marker (104, 202) comprising the labeling oligonucleotide (110) and at least one labeling portion (112, 204), wherein the backbone (106) and the barcode oligonucleotide (108) are resistant to a degradation agent (200), and the labeling oligonucleotide (110) is sensitive to the degradation agent (200).

7. A marker (100, 206) for analyzing a biological sample, comprising: The affinity reagent (102) according to any one of claims 1 to 6, and a label (104, 202) comprising a labeled oligonucleotide (110) and at least one labeling moiety (112), wherein the labeling oligonucleotide (110) and the barcode oligonucleotide (108) of the affinity reagent (102) are complementary to each other, and based on the complementary base pairing between the labeling oligonucleotide (110) and the barcode oligonucleotide (108), the label (104, 202) hybridizes with the affinity reagent (102), and One of the labeled oligonucleotide (110) and the barcode oligonucleotide (108) is sensitive to a degradation agent (200), while the other of the labeled oligonucleotide (110) and the barcode oligonucleotide (108) is resistant to the degradation agent (200).

8. The marker according to claim 7, wherein the labeled oligonucleotide (110) is sensitive to the degradation agent (200), and the affinity reagent (102) is resistant to the degradation agent (200).

9. The marker according to claim 7 or 8, wherein the labeling oligonucleotide (110) consists essentially of natural nucleic acid.

10. The marker according to any one of claims 7-9, wherein the at least one labeling portion (112, 204) is optically detectable.

11. The marker according to any one of claims 7 to 10, wherein the marker (104, 202) comprises a plurality of labeling parts (112, 204).

12. The marker according to any one of claims 7-11, wherein the degrading agent (200) is a nuclease.

13. A method for analyzing a biological sample comprising at least a target analyte (114), the method comprising the steps of: introducing into the biological sample at least a first marker (100) according to any one of claims 7 to 12, the at least first marker (100) comprising an affinity reagent (102) having a backbone (106) specific for the target analyte (114) and a first marker (104) having at least a first labeling portion (112), generating a first reading of the biological sample having the at least one first marker (100), applying a degradation agent (200) to the biological sample so as to remove the first marker (104) of the first marker (100), introducing at least one second label (202) having at least a second labeling portion (204) into the biological sample to generate a second marker (206), the second label (202) comprising a second labeling oligonucleotide (110) configured to hybridize with the barcode oligonucleotide (108) of the affinity reagent (102), and A second readout of the biological sample having the second marker (206) is generated.

14. The method of claim 13, wherein the target analyte (114) is identified based on the first marker (104) and the second marker (202) associated with the target analyte (114) in the first reading and the second reading.

15. A method according to claim 13 or 14, wherein in the step of introducing the at least one first marker (100), the affinity reagent (102) of the at least one first marker (100) is introduced before the first marker (104) of the first marker (100), and wherein the affinity reagent (102) is fixed in the biological sample before the first marker (104) having the first labeling part (112) is introduced into the biological sample.

16. The method according to any one of claims 13-15, wherein the first labeling moiety (112) and the second labeling moiety (204) have different detectable properties.

17. A method according to any one of claims 13-16, wherein after the step of generating the second reading, the following steps are iteratively repeated: applying the degradation agent (200) to degrade the second labeled oligonucleotide (110), adding at least one additional marker having an additional labeling portion, and generating an additional reading of the biological sample.

18. A kit comprising at least one marker (100, 206) and a degradation agent (200) according to any one of claims 7 to 12, or The kit comprises an affinity agent (102) according to any one of claims 1-6, a label (104, 202) and a degrading agent (200), wherein the label (104, 202) comprises a labeled oligonucleotide (110) and at least one labeling part (112, 204), and wherein the label (104, 202) is configured to constitute a marker (100, 206) according to any one of claims 7-12 when the label (104, 202) is linked to the affinity agent (102).

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