Label, marker and method for analysing a biological sample

Abstract

A label (100, 200, 300, 400, 502, 600, 602, 700, 800) for analysing a biological sample is provided. The label (100, 200, 300, 400, 502, 600, 602, 700, 800) comprises a plurality of labelling molecules (104) and a plurality of spacer molecules (102). The label (100, 200, 300, 400, 502, 600, 602, 700, 800) further comprises at least a first nucleic acid backbone (106) and each spacer molecule (102) is attached to the first nucleic acid backbone (106). In further aspects, a marker and a method for analysing a biological sample are provided.

Description

[0001] Label, marker and method for analysing a biological sample

[0002] Technical field

[0003] The invention relates to a label for analysing a biological sample. In further aspects, a marker comprising the label and a method for analysing a biological sample with the marker are provided.

[0004] Background

[0005] The ability to analyse biological samples, such as tissue sections, by proteomic, genomic and transcriptomic techniques has a rapidly progressed in recent years. The ability to provide super-resolution imaging of these samples has equally seen rapid progress.

[0006] For example, providing spatially resolved data for biological samples generally relies on marking the target proteins with optically detectable moieties, e.g. fluorescent molecules. In order to detect a large number of target proteins simultaneously in a given sample, the target proteins are marked with distinguishable fluorescent markers. Further, multiplexing approaches are known in the art, which aim at increasing the number of proteins that may be distinguished by cyclically staining different sets of target proteins with the fluorescent markers.

[0007] However, the use of fluorescent markers inherently limits the number of target analytes that may be detected at any one time even when considering spectral unmixing strategies. Especially the ability to generate a large number of optically detectable labels distinguishable by their optical properties remains a challenge. Further, the repeated staining may degrade the sample and consequently be limiting as well. Thus, it remains an objective to provide robust means for optically distinguishing a large number of target analytes in biological sample at any one time. At the same time, it is desirable to generate bright labels that enable clearly identifying target analytes.

[0008] Summary

[0009] It is an object to provide a bright label and a marker that are robust and a method for analysing a biological sample with the marker.

[0010] The aforementioned object is achieved by the subject-matter of the independent claims. Advantageous embodiments are defined in the dependent claims and the following description.

[0011] In a first aspect, a label for analysing a biological sample is provided. The label comprises a plurality of labelling molecules and a plurality of spacer molecules. The label further comprises at least a first nucleic acid backbone and each spacer molecule is attached to the first nucleic acid backbone.

[0012] The label - and in particular the spacer molecules - enables efficiently controlling the spacing, localisation, or orientation of the labelling molecules to each other, in particular along the first nucleic acid backbone. The labelling molecules preferably are optically detectable. For example, the labelling molecules may be fluorescent molecules, fluorescent dyes, fluorophores, or quantum dots.

[0013] The spacing of labelling molecules, such as fluorophores, fluorescent molecules, or quantum dots, may have a direct effect on optical properties of the labelling molecules. For example, the relationship is described in Benson et al, Chem 6, 1978- 1997, August 6, 2020 (Plug-and-Play Optical Materials from Fluorescent Dyes and Macrocycles) by the following equation: Thus, electronic coupling (J) between neighbouring dyes, in particular their transition dipole moments (p), is influenced by their distance (R) and angle (0). Thus, the label enables the efficient generation of a plurality of labels with differing optical properties based on the spacing and orientation of the labelling molecules.

[0014] The spacer molecules may be macromolecules, for example. The macromolecules may comprise subunits that are covalently and / or non-covalently attached to each other. In particular, the spacer molecules regulate or adjust the spacing of the labelling molecules relative to each other. Thus, the spacer molecules may alternatively be termed spacing molecules. In particular, the spacer molecules may be configured to regulate or adjust the distance and / or angle of adjacent labelling molecules to each other. In particular, the spacer molecules may arrange the labelling molecules relative to each other at a particular distance and / or angle.

[0015] The spacer molecule may in particular be a host molecule configured to form a supramolecular complex with a guest molecule. In particular, the spacer molecule is a host molecule that is configured to complexate with a labelling molecule guest molecule. Suitable host molecules that can be used as spacer molecules include but are not limited to cucurbit[n]urils, cyclodextrin, cyanostar.

[0016] Preferably, the spacer molecules of the plurality of spacer molecules are attached to the first nucleic acid backbone in predetermined intervals, in particular at regular intervals. This enables efficiently generating the label with uniform optical properties.

[0017] Preferably, each spacer molecule is configured to interact with at least one of the labelling molecules of the plurality of labelling molecules. This enables efficiently arranging the labelling molecules at a particular distance and / or angle relative to each other. The interaction may, in particular, be based on intermolecular forces such as ionic bonds, hydrogen bonds, and / or van der Waals forces. ln a particularly preferred embodiment, the interaction between the spacer molecules and the labelling molecules may be a guest / host interaction. For example, the spacer molecule may be a cucurbituril such as CB[7]. The cucurbituril may be configured to specifically interact with the labelling molecules. Thus, each cucurbituril may bind one of the labelling molecules and arrange the labelling molecules at a particular distance and / or angle relative to each other. Alternatively or in addition, the labelling molecules may be configured to specifically interact with the cucurbituril.

[0018] In a particularly preferred embodiment, the spacer molecule may be cyclodextrin, for example, a-, b-, or g-cyclodextrin. Particularly preferred combinations of spacer molecules and labelling molecules include alpha- and / or beta-cyclodextrin with labelling molecules such as ATTO488, ATTO 647N, ATTO 390, and ATTO 425, for example.

[0019] Preferably, each spacer molecule is configured to electronically decouple at least two adjacent labelling molecules of the plurality of labelling molecules. This enables generating labels with labelling molecules that have the same optical properties as the native labelling molecules. In particular, labels may be generated that comprise the labelling molecules at a high density enabling bright labels with consistent optical properties. In order to electronically decouple adjacent labelling molecules, the spacer molecules may electronically shield the adjacent labelling molecules or the spacer molecules may arrange the labelling molecules at a distance at which the adjacent labelling molecules are electronically decoupled, for example. In particular, the spacer molecules may be configured to arrange the plurality of labelling molecules at a predetermined distance from each adjacent labelling molecule.

[0020] Preferably, each spacer molecule is configured to arrange at least two adjacent labelling molecules of the plurality of labelling molecules at a distance at which the at least two adjacent labelling molecules are electronically coupled. In particular, this enables selectively changing the optical properties of the label, in particular of the labelling molecules. For example, adjacent labelling molecules may form a FRET pair. In this case, the spacer molecules may be configured to arrange the adjacent labelling molecules at a distance at which FRET occurs. This distance may generally be predetermined and / or in a range of 1 to 10 nm.

[0021] Preferably, the labelling molecules are interleaved with the spacer molecules. This enables efficiently securing the labelling molecules with the spacer molecules and a robust label. In particular, the labelling molecules are interleaved between the spacer molecules. For example, the labelling molecules may be regularly arranged between the spacer molecules attached to the first nucleic acid backbone.

[0022] Preferably, each labelling molecule is attached to the first nucleic acid backbone, in particular, at regular intervals. This enables a robust label.

[0023] Nucleic acids may be functionalised position-selectively. The positional resolution in this case is limited to the size of a nucleotide. This has been exploited in the prior art to generate fluorescent standards, wherein fluorescent dyes are connected to precisely located bands on a DNA origami. These standards are known as "nanoruler" and are used for the calibration of imaging systems like confocal or super resolution microscopes (e.g. STED), for example, as disclosed by US2014 / 0057805 Al. Similarly, the labelling molecules and / or the spacer molecules may be attached to the nucleic acid backbone, for example.

[0024] Preferably, each labelling molecule is attached to a second nucleic acid backbone, in particular, at regular intervals. This enables a robust label.

[0025] Preferably, the first nucleic acid backbone and the second nucleic acid backbone are configured to hybridise. This enables a robust label. In particular, the first nucleic acid backbone and the second nucleic acid backbone may be at least partially complementary and form a double stranded nucleic acid. Preferably, the adjacent labelling molecules are at an angle to each other around the longitudinal axis of the first nucleic acid backbone or the second nucleic acid backbone. This enables efficiently generating labels with different optical properties depending on the angle. In particular, the labelling molecules are attached to the first nucleic acid backbone or the second nucleic acid backbone at the angle.

[0026] Preferably the label comprises a third nucleic acid backbone, wherein the first nucleic acid backbone and / or the second nucleic acid backbone are attached, in particular covalently, to the third nucleic acid backbone. This enables a robust label. In particular, the third nucleic acid backbone may crosslink the first nucleic acid backbone and / or the second nucleic acid backbone in order to stabilise the label.

[0027] In a particularly preferred embodiment, the label may comprise several first nucleic acid backbones and / or second nucleic acid backbones. In this case, the third nucleic acid backbone may similarly crosslink the first nucleic acid backbones and / or second nucleic acid backbones.

[0028] Preferably, at least one of the first nucleic acid backbone, the second nucleic acid backbone and the third nucleic acid backbone is a DNA origami, DNA brick or other DNA nanostructure. This enables a robust label that is easy to assemble. Further, this enables predetermining a three-dimensional structure of the assembled label.

[0029] Preferably, adjacent labelling molecules are at a distance from each other in a range of 0.33 nm to 50 nm, preferably in a range of 10 nm to 30 nm, more preferably in a range of 15 nm to 30 nm. This enables a label with a high density of labelling molecules that do not influence each other, in particulartheir optical properties or characteristics with regard to their emission of fluorescent light. In particular, the spacer molecules are attached to the first nucleic acid at that distance. Thus, the spacer molecules may preferably be configured to arrange the labelling molecules at that distance. Alternatively, the labelling molecules may be attached to the second nucleic acid backbone at that distance.

[0030] In another aspect, a marker for analysing a biological sample is provided. The marker comprises at least one label as described above and an affinity reagent configured to bind specifically to a target analyte of the biological sample. In particular, the label is attached to the affinity reagent.

[0031] In a further aspect, a labelling kit for analysing a biological sample is provided. The labelling kit comprises a first plurality of labels as described above, and a second plurality of labels as described above. The first plurality of labels and the second plurality of labels differ from each other by a distance of adjacent labelling molecules to each other and / or by an angle of adjacent labelling molecules to each other around the longitudinal axis of the first nucleic acid backbone or the second nucleic acid backbone. In particular, this enables a labelling kit with labels that have different optical properties despite comprising the same labelling molecules. For example, the first plurality of labels has labelling molecules arranged at a first distance / angle and the second plurality of labels has labelling molecules arranged at a different second distance / angle. In particular, this may result in different emission characteristics of fluorescent light being emitted by the first plurality of labels compared to the second plurality of labels.

[0032] In a further aspect, a method for analysing a biological sample is provided, comprising the steps: introducing a plurality of markers as described above into the biological sample or introducing a plurality of labels from the labelling kit as described above, and generating an optical readout of the biological sample with the markers.

[0033] Generally, a plurality of markers may be introduced into the biological sample, the markers being specific to respective target analytes, in order to identify a large number of target analytes at the same time. Preferably, the target analyte is identified and / or localised within the biological sample based on the labels of the markers, in particular the labelling molecules, associated with the target analyte in the optical readout.

[0034] Short Description of the Figures

[0035] Hereinafter, specific embodiments are described referring to the drawings, wherein:

[0036] Figure 1 is a schematic view of a label with labelling molecules and spacer molecules,

[0037] Figure 2 is a schematic view of a further label,

[0038] Figure 3 is a schematic view of a label with a plurality of cucurbiturils,

[0039] Figure 4 is a schematic view of a label with guest-molecule modified labelling molecules,

[0040] Figure 5 is a schematic view of a label comprising a plurality of cucurbiturils,

[0041] Figure 6 is a schematic view of two labels comprising the same labelling molecules,

[0042] Figure 7 is a schematic view of a label with labelling molecules attached to cucurbiturils via a flexible linker, and

[0043] Figure 8 is a schematic view of a label comprising a plurality of cucurbiturils with lid molecules.

[0044] Detailed Description Figure 1 is a schematic view of a label 100. The label 100 comprises a plurality of spacer molecules 102 and a plurality of labelling molecules 104. The spacer molecules 102 are attached to a first nucleic acid backbone 106 and the labelling molecules 104 are attached to a second nucleic acid backbone 108. The spacer molecules 102 and the labelling molecules 104 may interact with each other. In particular, the spacer molecules 102 may be configured to interact with at least one of the labelling molecules 104. At least a part of the first nucleic acid backbone 106 and / or the second nucleic acid backbone 108 may have an essentially linear or elongated structure or shape.

[0045] The spacer molecules 102 are spaced along and attached to the first nucleic acid backbone 106, in particular at regular intervals. Similarly, the labelling molecules 104 are spaced along and attached to the second nucleic acid backbone 108, in particular at the same intervals as the spacer molecules 102. Thus, the spacer molecules 102 are kept at a particular distance from each other. Similarly, the labelling molecules 104 are kept at a particular distance from each other.

[0046] When the spacer molecules 102 are brought into contact with the labelling molecules 104, the spacer molecules 102 interact with the labelling molecules 104 such that the labelling molecules 104 interleave between the spacer molecules 102. This generates the label 100, as exemplarily shown on the right side of the arrow in Figure 1.

[0047] In an alternative embodiment, the labelling molecules 104 are not attached to the second nucleic acid backbone 108 and the label 100 does not comprise the second nucleic acid backbone 108 (not shown in Figure 1). In this case, the labelling molecules 104 may be kept at a particular distance from each other only by the spacer molecules 102. Generally, the spacer molecules 102 may enable efficiently adjusting the spacing of the labelling molecules 104 to each other. For example, the spacer molecules 102 may ensure that adjacent labelling molecules 104 are kept at a minimum distance from each other. In particular, this may ensure that the adjacent labelling molecules 104 are electronically decoupled from each other.

[0048] The spacer molecules 102 enable providing a label with a high density of the labelling molecules 104 whilst ensuring that the labelling molecules 104 do not interfere with each other. For example, the spacer molecules 102 may be attached to the first nucleic acid backbone 106 such that adjacent labelling molecules 104 are at a distance in a range of 10 nm to 50 nm, preferably in a range of 15 nm to 30 nm. Alternatively or in addition, the spacer molecules 102 may be of a size such that the labelling molecules 104 are at that distance.

[0049] Alternatively, the spacer molecules 102 may be configured to arrange at least two adjacent labelling molecules 104 at a distance at which the two adjacent labelling molecules 104 are electronically coupled. In particular, this enables selectively changing the optical properties of the adjacent labelling molecules 104 compared to the optical properties in their electronically decoupled states. In this case, the distance between adjacent labelling molecules 104 may be in a range of 1 to 10 nm.

[0050] Thus, adjusting the distance between adjacent labelling molecules 104 enables generating several labels 100 based on the same labelling molecules 104, but with different optical properties.

[0051] The first nucleic acid backbone 106 and the second nucleic backbone 108 may optionally be at least partially complementary. This causes the first nucleic acid backbone 106 and the second nucleic backbone 108 to form a double stranded nucleic acid molecule. This enables a particular stable and robust label 100. Figure 2 is a schematic view of a label 200. The label 200 comprises the spacer molecules 102 and the labelling molecules 104. The label 200 further comprises several first nucleic acid backbones 106 and the spacer molecules 102 and the labelling molecules 104 are attached to one of the first nucleic acid backbones 106. By providing several parallel first nucleic acid backbones 106 the label 200 comprises the labelling molecules 104 at a high density.

[0052] Optional ly, a third nucleic acid backbone may be provided, to which the first nucleic acid backbones 106 are attached. This ensures that the spacing of labelling molecules 104 of adjacent first nucleic acid backbone 106 is kept constant.

[0053] Labelling molecules 304 may be hosted inside cucurbituril 302 (CB[n]) cavities or cavities of other macrocycles (e.g. cyclodextrin, calix[n]arenes), which act as spacer molecules, as schematically depicted in Figure 3 for a label 300. In particular, two CB[n] molecules 302 are attached to the first nucleic acid backbone 106 at a suitable distance to accommodate ends of the labelling molecule 304, which preferably has an elongated shape. The cucurbituril molecules 302 may be attached to the first nucleic acid backbone 106 at specific positions along the first nucleic acid backbone 106, which enables determining or modifying the spacing of the labelling molecules 304 from each other.

[0054] The CB[n] 302 may be loaded with labelling molecules 304 at a high concentration and subsequently the CB[n] 302 may optionally be covalently linked together. Suitable modifications of the rim of the CB[n] molecules 302 can be made to allow this linking together. For example, a first modified CB[n] may comprise a first clickable group such as an azide moiety and the second CB[n] may comprise a second clickable group like for example an alkyne, which will react in the presence of copper to form a covalent link 306. Using this strategy, a number of different labelling molecules 304 can be loaded onto a plurality of labels 300 build from a common nucleic acid backbone comprising said first and second CB[n] variants. Figure 4 schematically shows a label 400 comprising the first nucleic acid backbone 106 comprising CB[n] 302 or other macrocycles (e.g. cyclodextrin, calix[n]arenes) which host labelling molecules 402 comprising adamantane, ferrocene or other high affinity guest modifications 404, which bind with very high affinity to CB[n] 302. For cucurbit[n]urils high affinity guests are known with affinities exceeding the streptavidin-biotin interaction. An example, are adamantane and ferrocene derivatives. An adamatanylated-labelling molecule would attach firmly to the CB[n] molecule 302, for example. Similarly to the embodiment according to Fig. 3, the specific attachment of the cucurbituril molecules 302 of the label 400 along the first nucleic acid backbone 106 determines the distance between the labelling molecules 402.

[0055] When labelling molecules are multimerized on a nucleic acid backbone of a label, they may aggregate and quench strongly. This is in particular the case, when said dye molecules are hydrophobic and the label is in an aqueous solution, in which case the persistence length of the nucleic acid backbone is small. An exemplary aggregated label is indicated by the reference sign 500. Labels that comprise multiple hydrophobic dyes that exhibit strong quenching may be "rescued" by addition of CB[n] or other suitable macrocyles (e.g. cyclodextrin, calix[n]arenes), which serve as a host molecules for the hydrophobic dyes thereby breaking up dye-dye-interactions. This is schematically shown Figure 5. In particular, a label 502 may comprise the first nucleic acid backbone 106 to which labelling molecules 104 are attached. The label 502 further comprises cucurbituril molecules 504, which hinder or reduce intermolecular forces between the labelling molecules 104, such that the label 502, in particular its labelling molecules 104, does not aggregate.

[0056] Figure 6 schematically shows labels 600, 602 comprising a first nucleic acid backbone 604a, 604b and the second nucleic acid backbone 108. The first nucleic acid backbones 604a, 604b are at least partially complementary to the second nucleic acid backbone 108. The first nucleic acid backbone 604b of the label 602 comprises a plurality of cucurbituril molecules 302, which are attached to the first nucleic acid backbone 604b. The first nucleic acid backbone 604a of the label 600 only has the nucleic acid. A plurality of labelling molecules 104, in particular fluorescent molecules, are attached to the second nucleic acid backbone 108.

[0057] In case of the label 600, the labelling molecules 104 are not hosted inside a host cucurbituril molecules 302. This results in a fluorescence lifetime of the labelling molecules 104 of ~1 ns. In case of the label 602, the labelling molecules 104 are hosted inside the host cucurbituril molecules 302 of the first nucleic acid backbone 604b. This has an effect on the fluorescence lifetime of the labelling molecules 104, resulting in a fluorescence lifetime of ~2 ns. In this way two labels 600, 602 can be generated that comprise the same labelling molecules 104 but which are nevertheless distinguishable by their fluorescence lifetime. Further modification of the dye molecules or the host molecule may then be used to increase the number of discernible labels comprising the same dye even further.

[0058] The shielding of labelling molecules 104 inside CB[n] 302 cavities may also protect the labelling molecules 104 from oxidative damage, reduce bleaching, and may also be used to tune the emission or excitation or lifetime characteristics to desired points. For example in the presence of ImM CB[7], TMR, Cy5, PyY, and PyB show a marked lifetime elongation by a factor of ~2. This is important, when considering that in practical terms a confocal microscope such as STELLARIS 8 FALCON (Leica Microsystems, Mannheim) can effectively differentiate labelling molecules that exhibit ~2x difference in fluorescence lifetime even if the spectral properties are identical. In some cases however it may be undesirable to add ImM CB[7] to the imaging buffer as it may influence other labelling molecules in the panel in unintended ways. The strategy proposed in this document, i.e. to provide a high local concentration of CB[7] by attaching it to the label 602 itself solves this problem and enables building bright fluorescent labels through multiplication of labelling molecules on nucleic acid backbones, while also allowing to build two versions of many of such labels, one that has no CB[7] (short lifetime) and another that comprises CB[7] (elongated lifetime due to host-guest interaction). This enables driving plexity, which has many applications in the areas of life science research e.g. microscopy, spatial biology, proteomics, cytometry.

[0059] A further embodiment that realises the abovementioned advantages is exemplarily shown in Figure 7 for a label 700. The label 700 comprises the first nucleic acid backbone 106, to which cucurbituril molecules 302 are attached. The label 700 further comprises labelling molecules 104, which are each connected to one of the CB molecules 302 via a short flexible linker such as for example a PEG[n] linker or similar. This offers the labelling moieties 104, especially a hydrophobic labelling molecule a hydrophobic cavity in close proximity.

[0060] Figure 8 schematically shows a label 800 comprising cucurbituril molecules 802 having a lid molecule 804. The lid molecules 804 are designed to exhibit a high affinity towards the rim of the cavity of the cucurbituril molecule 802. Such lid molecules 804 can be used to trap labelling molecules 104 inside, which is important as most labelling molecules 104 are expected to have low to moderate affinity to CB[7] and would otherwise leach out from a cucurbituril molecule. The capping of the cucrbituril molecule 802 solves this problem and opens an additional avenue to modify the photophysical properties by leveraging lid-dye interactions.

[0061] In order to generate a marker, the labels 100, 200, 300, 400, 502, 600, 602, 700, 800 may be attached to an affinity reagent 202, for example, via an attachment oligonucleotide 204 (shown in Fig. 2). In particular, at least one of the first nucleic acid backbone 106, the second nucleic acid backbone 108, and the third nucleic acid backbone may be complementary to the attachment oligonucleotide in order to attach the label 100, 200, 300, 400, 502, 600, 602, 700, 800 to the affinity reagent 202. The marker comprising the affinity reagent 202 and the label 100, 200, 300, 400, 502, 600, 602, 700, 800 may be used in a method for analysing a biological sample. The marker, in particular the affinity reagent 202, is specific to a target analyte of the biological sample. When introducing the marker into the biological sample, the marker binds specifically to the target analyte. Subsequently an optical readout may be generated of the biological sample, for example, by means of a microscope. The label 100, 200, 300, 400, 502, 600, 602, 700, 800of the marker may be detected in the optical readout and the presence or location of the target analyte in the biological sample may be determined.

[0062] Identical or similarly acting elements are designated with the same reference signs in all Figures. As used herein the term "and / or" includes any and all combinations of one or more of the associated listed items and may be abbreviated as

[0063] Although some aspects have been described in the context of an apparatus, it is clear that these aspects also represent a description of the corresponding method, where a block or device corresponds to a method step or a feature of a method step. Analogously, aspects described in the context of a method step also represent a description of a corresponding block or item or feature of a corresponding apparatus.

[0064] Reference signs

[0065] 100, 200, 300, 400, 502, 600, Label

[0066] 602, 700, 800

[0067] 102 Spacer molecule

[0068] 104, 304, 402 Labelling molecule

[0069] 106, 604a, 604b First nucleic acid backbone

[0070] 108 Second nucleic acid backbone

[0071] 202 Affinity reagent

[0072] 204 Attachment oligonucleotide

[0073] 302, 504, 802 Cucurbituril

[0074] 306 Covalent link

[0075] 404 Guest modification molecule

[0076] 500 Aggregated label

[0077] 804 Lid molecule

Claims

Claims1. A label (100., 200, 300, 400, 502, 600, 602, 700, 800) for analysing a biological sample comprising a plurality of labelling molecules (104), a plurality of spacer molecules (102), and at least a first nucleic acid backbone (106), and wherein each spacer molecule (102) is attached to the first nucleic acid backbone (106).

2. The label according to claim 1, wherein the spacer molecules (102) of the plurality of spacer molecules (102) are attached to the first nucleic acid backbone (106) in predetermined intervals.

3. The label according to one of the preceding claims, wherein each spacer molecule (102) is configured to interact with at least one of the labelling molecules (104) of the plurality of labelling molecules (104).

4. The label according to one of the preceding claims, wherein each spacer molecule (102) is configured to electronically decouple at least two adjacent labelling molecules (104) of the plurality of labelling molecules (104).

5. The label according to one of the preceding claims 1 to 3, wherein each spacer molecule (102) is configured to keep at least two adjacent labelling moleculesleast two adjacent labelling molecules (104) are electronically coupled.

6. The label according to one of the preceding claims, wherein the labelling molecules (104) are interleaved with the spacer molecules (102).

7. The label according to one of the preceding claims, wherein each labelling molecule (104) is attached to the first nucleic acid backbone (106).

8. The label according to one of the preceding claims, wherein each labelling molecule (104) is attached to a second nucleic acid backbone (108).

9. The label according to claim 8, wherein the first nucleic acid backbone (106) and the second nucleic acid backbone (108) are configured to hybridise.

10. The label according to one of the preceding claims 7 to 9, wherein adjacent labelling molecules (104) are at an angle to each other around the longitudinal axis of the first nucleic acid backbone (106) or the second nucleic acid backbone (108).

11. The label according to one of the preceding claims, comprising a third nucleic acid backbone, wherein the first nucleic acid backbone (106) and / or the second nucleic acid backbone (108) are attached to the third nucleic acid backbone.

12. The label according to one of the preceding claims, wherein at least one of the first nucleic acid backbone (106), the second nucleic acid backbone (108) and the third nucleic acid backbone is a DNA origami.

13. The label according to one of the preceding claims, wherein adjacent labelling molecules (104) are at a distance from each other in a range of 10 nm to 50 nm.

14. A marker for analysing a biological sample comprising: at least one label (100., 200, 300, 400, 502, 600, 602, 700, 800) according to one of the preceding claims, and an affinity reagent (202) configured to bind specifically to a target analyte of the biological sample.

15. A labelling kit for analysing a biological sample comprising: a first plurality of labels (100, 200, 300, 400, 502, 600, 602, 700, 800) according to one of the preceding claims 1 to 13, and a second plurality of labels (100, 200, 300, 400, 502, 600, 602, 700, 800) according to one of the preceding claims 1 to 13, and wherein the first plurality of labels (100, 200, 300, 400, 502, 600, 602, 700, 800) and the second plurality of labels (100, 200, 300, 400, 502, 600, 602, 700, 800) differs from each other by a distance of adjacent labelling molecules (104) to each other and / or by an angle of adjacent labelling molecules (104) to each other around the longitudinal axis of the first nucleic acid backbone (106) or the second nucleic acid backbone (108).

16. A method for analysing a biological sample comprising the steps: introducing a plurality of markers according to claim 14 into the biological sample, andregenerating an optical readout of the biological sample with the markers.

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