Patent Description:
In order to address key problems in the field of life sciences it is vital to precisely identify and to locate certain structures or target molecules within biological samples, e.g. tissue samples or cell cultures. This can be done by introducing markers into the sample that only bind to specific structures, e.g. specific biomolecules. These markers typically comprise an affinity reagent that only attaches to the structure in question and one or more fluorescent dyes or labels that are either directly conjugated to the affinity reagent or attached to the affinity reagent by other means, for example a secondary affinity reagent.

Fluorescence microscopy for example allows for imaging the sample with high spatial resolution but generally involves only a low number of different fluorescent dyes, typically between <NUM> and <NUM>. The available dyes have to be distributed to all markers that are used to identify cell types, functional markers like protein-of-interest and general morphological markers in the same experiment. This means that only a limited number of structures can be marker by unique fluorescent dyes and thus identified at the same time. This further means that different cell types can only be poorly identified in most imaging experiments. While modern approaches that allow for a much more reliable and robust identification of cell types, e.g. based on the analysis of genetic regulatory networks (GRNs), exist, they require a much higher number of different markers to be read-out from the sample. While documents <CIT> and <CIT> disclose methods for analysing biological samples with a large number of fluorescent molecules at the same time, generating a diverse set of markers with different affinities and labels with varying fluorescent properties remains time consuming and expensive.

The documents <CIT> and <CIT> disclose probes for binding to target molecules in a biological sample. The probes comprise an affinity reagent to bind to the target molecule and connected to a backbone, the backbone configured to bind to a label by means of affinity interactors.

It is an object to provide a connector, a marker and a method for analysing biological samples that allow for generating markers and analysing the samples at a low cost and time expenditure.

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.

A connector is provided for analysing biological samples comprising at least one first affinity reagent configured to bind directly or indirectly to a target molecule; a backbone connected to the first affinity reagent and comprising at least one first affinity interactor; wherein the first affinity interactor is configured to specifically bind to a second affinity interactor comprising a label in order to bind the label to the backbone; wherein the backbone comprises a cleavage site for irreversibly separating the first affinity reagent and the first affinity interactor with the label. The label may, in particular, be optically detectable. The target molecule is a particular structure of the biological sample, for example, a protein of the biological sample, a small molecule (e.g. lactic acid), or a mRNA molecule of the biological sample. Further examples are peptides, small molecules, metabolites, hormones, neurotransmitters, metal ions. The biological sample may be, for example, tissue, bodily fluids, a solid biopsy, a liquid biopsy, embryos (e.g. zebrafish, Drosophila), model organisms (e.g. zebrafish larvae, Drosophila embryos, C. elegans), cells (e.g. prokaryotes, eukaryotes, archaea), multicellular organisms (e.g. Volvox), suspension cell cultures, monolayer cell cultures, 3D cell cultures (e.g. spheroids, tumoroids, organoids derived from various organs such as intestine, brain, heart, liver), a lysate of any of the aforementioned, or a virus.

Thus, the connector comprises in order: the first affinity reagent, the backbone with the cleavage site, and the first affinity interactor.

The binding between the first affinity reagent and the target molecule may be specific, in particular. That means, that the first affinity reagent only binds to the target molecule. However, when the binding is indirect, the first affinity reagent may only bind to a further entity, which in turn binds only to the target molecule. Thus, in this case, the first affinity reagent binds indirectly only to the target molecule.

The first affinity interactor may only bind to the second affinity interactor. This binding may be irreversible at physiological conditions.

In particular, this may further overcome disadvantages with current solutions, for example: In particular, the present invention enables generating connectors using monovalent (first) affinity reagents such as for example a nanobody, a single-chain antibody, an aptamer, an oligonucleotide, or a small molecule compound, which is combined with a backbone that comprises a cleavage site for dye inactivation and an affinity interactor, which is used to mediate the high affinity interaction or coupling of the connector to the label. This design has many advantages, for example it is possible to construct fully peptide-based or fully-nucleotide based connectors, which allows their synthesis in one go or their expression from a single expression cassette. This makes manufacturing particularly easy, cost-efficient and reproducible. Owing to this design scheme, only the label needs to be coupled to the second affinity interactor using standard coupling chemistries. This can, however, be done by a manufacturer in larger scale. For the (end-)user, the design has a key advantage in multiplexing applications, in which users commonly need to perform coupling of labels to the primary antibodies that they intend to use in a study, which can easily be ><NUM> antibodies. Performing ><NUM> coupling reactions is a tedious, manual process connected to significant batch-to-batch variability. Using connectors described in this document, users may simply pre-incubate a suitable connector with a second affinity reagent (e.g. the primary antibody) to connect the second affinity reagent to the connector. This can be performed by simply mixing connectors and antibodies in a multi-well plate, which is easily automated using standard laboratory automation and liquid handling equipment such as pipetting robots or dispensers.

Preferably, the first affinity reagent is a nanobody, a single domain antibody, an aptamer, a small-molecule, or an oligonucleotide. This enables particularly easy assembly of the connector and binding to the target molecule with high affinity and specificity.

More preferably, when the first affinity reagent is configured to bind indirectly to the target molecule, the connector comprises a second affinity reagent bound to the first affinity reagent, and the second affinity reagent is configured to (directly) bind to the target molecule. This enables particularly flexible assembly and use of the connector, for example, in combination with several antibodies of the same isotype.

Preferably, the second affinity reagent is an antibody and the first affinity reagent is configured to bind to a fragment crystallisable region (Fc region) of the antibody. This enables binding to the target molecule with particularly high affinity and specificity. In particular, the first affinity reagent may be a nanobody.

It is preferred, that the backbone comprises an oligonucleotide or a peptide. For example, the backbone may be DNA-origami-based or a nanoruler. This enables easy assembly and efficient production of the backbone.

Preferably, the cleavage site is a photocleavable cleavage site. This enables easy cleaving of the cleavage site.

In a preferred embodiment the backbone is cleavable specifically at the cleavage site by a site specific enzyme, for example TEV protease, caspase, Factor Xa or a restriction enzyme, cleavage light, for example UV light, or by a temperature change. Specifically, this causes cleaving of covalent bonds of the backbone with restriction enzyme or proteolytic enzyme, UV light; or melting/denaturing of hybridised oligonucleotide backbone. This enables efficient cleaving of the cleavage site.

Preferably, the first affinity interactor and the second affinity interactor are configured to form a bioconjugate. This means that the first and second affinity interactor may covalently bind to each other. Examples of pairs of interactors that form bioconjugates are spytag/spycatcher, snooptag/snoopcatcher and dogtag/dogcatcher. This enables robust and specific binding of the first and second affinity interactor to each other.

Preferably, the first affinity interactor is one of biotin or streptavidin and the second affinity interactor is the other one of biotin or streptavidin. This enables robust and specific binding of the first and second affinity interactor to each other.

More preferably, the connector comprises a plurality of first affinity interactors for binding one second affinity interactor each.

Preferably, the label comprises at least a first fluorophore. This enables detection of the label by an optical read-out device such as a microscope.

In another aspect a marker is provided for analysing biological samples comprising the connector according to one of the preceding claims and further comprising a second affinity interactor with a label. The second affinity interactor with the label is bound to first affinity interactor to connect the label to the connector. The label may in particular comprise at least one fluorophore. The fluorophore may, for example, be a fluorescent protein, a fluorescent molecule, or a fluorescent quantum dot.

In a further aspect a method is provided for analysing a biological sample comprising the following steps: providing, in particular, generating, at least a first set of markers, wherein the first set of markers comprises at least a first plurality of connectors configured to bind directly or indirectly to a first target molecule, and each first connector comprising a first label; introducing at least the first set of markers into the sample in order for the markers to bind to their respective target molecule in the sample; directing excitation light onto the biological sample, the excitation light being configured to visualise or excite at least the first label; and generating at least one optical readout from light emitted by at least the first label. The marker may comprise only the first affinity reagent or additionally the second affinity reagent, thus the first target molecule may be bound directly by the first affinity reagent or indirectly by the second affinity reagent. The label may be bound to the connector by the first and second affinity interactors.

In order to introduce the marker into the sample, the sample may be permeabilised. Further, the marker may be introduced into the sample as individual parts or as with the parts bound together prior. Preferably, when the marker comprises the connector with the first affinity reagent and the label, the individual parts may be introduced individually into the sample. However, when the marker comprises the connector with the first and the second affinity reagent and the label, the individual parts may be assembled or bound together prior to introduction into the sample.

The optical readout may be generated by means of a read-out device capable of fluorescence multicolour reading or imaging. A readout device typically includes at least one excitation light source, a detection system including at least one detection channel and may contain filters and/or dispersive optical elements to route excitation light to the sample and/or to route emission light from the sample onto a detector. In particular, the read-out device may be a fluorescent microscope.

Further, optional washing steps may be included, for example, after introduction of the marker into the sample, in order to remove unbound markers.

Preferably, the first set of markers further comprises at least a second plurality of connectors configured to bind directly or indirectly to a second target molecule, and each second connector comprising a second label. The second target molecule is bound by the first affinity reagent of the second connector. This enables analysing a larger number of target molecules.

It is particularly preferred, that the first label and the second label have different fluorescent properties. In particular, the label is a fluorophore. The fluorescent properties may include an excitation wavelength/spectrum, an emission wavelength/spectrum and an emission duration or fluorescence lifetime. This enables distinguishing between the first and the second label.

Preferably, in a step e) the cleavage site of the connectors of at least the first set of markers is cleaved. For example, cleaving may be achieved by addition of an enzyme, or by illuminating the markers with UV light. The cleaving results in separating the label from the marker. This enables blanking of the markers, meaning the fluorescent signal is removed from the markers.

Preferably, in a step f) the cleaved off second affinity interactor is removed with the label from the biological sample. This may be achieved by washing the sample with buffer, for example. This enables removing background fluorescence of the cleaved labels.

Preferably, the steps a) to d) are repeated with a second set of markers, wherein the second set of markers comprises at least a third plurality of connectors configured to bind directly or indirectly to a third target molecule, and each connector comprising a third label.

The method and the marker have the same advantages as the connector described above. In particular, the method and the marker may be supplemented using the features of the dependent claims directed at the connector.

Further features and advantages of the invention result from the claims and the following description of preferred embodiments, which are described with reference to the accompanying drawings.

<FIG> shows a schematic view of a connector <NUM> for analysing biological samples. The connector <NUM> comprises a first affinity reagent <NUM>, which is configured to specifically and directly bind to a target molecule <NUM>. The first affinity reagent <NUM> is a nanobody, for example.

In addition, the connector <NUM> comprises a backbone <NUM> connected to the first affinity reagent <NUM>. The backbone <NUM> comprises a cleavage site <NUM> for irreversibly separating the backbone <NUM> into two parts <NUM>', <NUM>", for example, by an enzyme <NUM>.

Moreover, the backbone comprises at least one first affinity interactor <NUM>. The first affinity interactor <NUM> is configured to specifically bind to a second affinity interactor <NUM> comprising a label <NUM>, in order to enable the binding of the label <NUM> to the backbone <NUM> of the connector <NUM>. The first affinity interactor <NUM> and the second affinity interactor <NUM> can thus bind to each other specifically. Upon binding, the second affinity interactor <NUM> with the label <NUM> is connected to the connector <NUM>. The binding of the first affinity interactor <NUM> to the second affinity interactor <NUM> may be substantially irreversible. Once the first and second affinity interactors <NUM>, <NUM> are bound to each other, the cleavage site <NUM> enables irreversibly separating the label <NUM> from the label from the connector <NUM>, or rather from the first affinity reagent <NUM>. The label <NUM> may be a dye, in particular, one or more fluorophores.

<FIG> and <FIG> show schematic views of exemplary embodiments of the connector <NUM>.

<FIG> shows a schematic view of nucleotide-based connectors <NUM>, <NUM>. The connectors <NUM>, <NUM> each comprise a backbone <NUM>, which may be an oligonucleotide, in particular a DNA or RNA oligonucleotide, and/or comprise nucleic acid analogues such as PNA, LNA, XNA, and morpholinos. At one end of the backbone <NUM>, the backbone <NUM> is connected to a first affinity reagent <NUM>, which is oligonucleotide-based, similar to the backbone <NUM>, and which comprise a specific sequence enabling the first affinity reagent <NUM> to directly or indirectly bind to an target molecule <NUM>, <NUM>.

In case of the connector <NUM>, the first affinity reagent <NUM> is indirectly bound to the target molecule <NUM>, for example, a protein, via a second affinity reagent <NUM>, such as an antibody. The second affinity reagent <NUM> comprises, for example, at its fragment crystallisable region (Fc region), an oligonucleotide sequence complementary to the specific sequence of the first affinity reagent <NUM>. The antigen-binding fragment (Fab region) of the second affinity reagent <NUM> may bind specifically to the target molecule <NUM>. Thus, the connector <NUM> specifically binds to the target molecule <NUM> indirectly via the second affinity reagent <NUM>.

Alternatively and in case of the connector <NUM>, the target molecule <NUM> is an RNA or DNA molecule, for example a mRNA molecule. The first affinity reagent <NUM> binds to a specific sequence of the target molecule <NUM> complementary to the sequence of the first affinity reagent <NUM>.

At the other end of the backbone <NUM>, the backbone <NUM> comprises a first affinity interactor <NUM> configured to specifically bind to a second affinity interactor <NUM>. The first affinity interactor <NUM> may, for example, be an oligonucleotide-based aptamer, similar to the backbone <NUM>. The second affinity interactor <NUM> may be a further aptamer or biotin, for example. The second affinity interactor <NUM>, in turn, is connected to a label <NUM>, such as a dye molecule.

Moreover, the backbone <NUM> comprises a cleavage site <NUM>. The cleavage site <NUM> is preferably a restriction site for a restriction enzyme, in particular a rare cutter such as Notl. This enables separating the label <NUM> from the connector <NUM>, <NUM>.

Since the connector <NUM>, <NUM>, including the first affinity reagent <NUM>, the backbone <NUM> with the cleavage site <NUM> and the first affinity interactor <NUM> are all oligonucleotide-based, the connector <NUM>, <NUM> may each be synthesised in a single reaction or synthesised in a single piece.

<FIG> shows a schematic view of peptide-based connectors <NUM>, <NUM>. The connectors <NUM>, <NUM> each comprise a backbone <NUM>, which may be a peptide. At one end of the backbone <NUM>, the backbone <NUM> is connected to a first affinity reagent <NUM>, <NUM>, which are peptide-based, similar to the backbone <NUM>, and which directly or indirectly bind specifically to a target molecule <NUM>.

In case of the connector <NUM>, the first affinity reagent <NUM> is indirectly bound to the target molecule <NUM>, for example, a protein, via a second affinity reagent <NUM>, such as an antibody. The first affinity reagent <NUM> is, for example, a nanobody or a single domain antibody, which specifically binds to the second affinity reagent <NUM>, for example, at its fragment crystallisable region (Fc region). The antigen-binding fragment (Fab region) of the second affinity reagent <NUM> may bind specifically to the target molecule <NUM>. Thus, the connector <NUM> specifically binds to the target molecule <NUM> indirectly via the second affinity reagent <NUM>.

Alternatively and in case of the connector <NUM>, the first affinity reagent <NUM> is, for example, a nanobody or a single domain antibody, which specifically binds directly to the target molecule <NUM>.

At the other end of the backbone <NUM>, the backbone <NUM> comprises a first affinity interactor <NUM> configured to specifically bind to a second affinity interactor <NUM>. The first affinity interactor <NUM> may be peptide-based, similar to the backbone <NUM>, for example, it may be streptavidin. In this case, the second affinity interactor <NUM> may be biotin, for example. The second affinity interactor <NUM>, in turn, is connected to a label <NUM>, such as a dye molecule. Alternative pairs of first and second affinity interactors <NUM>, <NUM> that bind specifically to each other may be pairs of peptides that form bioconjugates, such as SpyTag and SpyCatcher, SnoopTag and SnoopCatcher, or DogTag and DogCatcher.

Moreover, the backbone <NUM> comprises a cleavage site <NUM>. The cleavage site <NUM> is preferably a specific peptide sequence at which a respective protease, for example, TEV protease from Tobacco etch virus or Factor Xa, or scUlp1 (from Saccharomyces cerevisiae with substrate scSUMO), or bdSENP1 (from Brachypodium distachyon with substrate bdSUMO), or bdNEDP1 (from Brachypodium distachyon with substrate bdNEDD8), or sNEDP1 (from Salmo salar with substrate ssNEDD8), or scAtg4 (from Saccharomyces cerevisiae with substrate Atg4p), or xlUsp2 (from Xenopus laevis with substrate xlUsp2), may cleave the peptide-based backbone <NUM>. This enables separating the label <NUM> from the connector <NUM>, <NUM>. Full length versions, truncated versions, or fragments of the aforementioned substrates and proteases may be used as cleavage site/protease pair.

In a particularly preferred embodiment of the present invention, bdSENP1 or a bdSENP1 derivative is used to perform cleavage. This provides numerous advantages including very efficient substrate cleavage at concentrations in the nanomolar (nM) regime, i.e. <NUM> at <NUM> or <NUM>-<NUM> at <NUM>° for near quantitative cleavage (conditions described in the literature: <NPL>), and good activity at temperatures between <NUM>-<NUM>.

Since the connector <NUM>, <NUM>, including the first affinity reagent <NUM>, the backbone <NUM> with the cleavage site <NUM> and the first affinity interactor <NUM> are all oligonucleotide-based, the connector <NUM>, <NUM> may each be synthesised by recombinant expression of a single DNA construct and/or synthesised as a continuous single piece.

Alternatively to the first affinity reagents <NUM>,<NUM> discussed in connection with <FIG> and <FIG>, the first affinity reagents <NUM>, <NUM>, <NUM> may be a multimer of a nanobody or single-domain antibody, a conventional antibody, an aptamer, or a drug, small molecule or toxin.

Alternatively, the cleavage sites <NUM>, <NUM>, <NUM> may be cleaved by a temperature shift, for example in case the backbone is oligonucleotide-based, in particular a DNA-origami, and the cleavage site is a hybridised part of two oligonucleotide strands, or by activation light, such as UV light, in case the cleavage site is a photocleavable linker.

<FIG> shows connector <NUM>, <NUM>, <NUM> that each have a label attached. Elements with the same structure and the same function have the same reference signs. The connector <NUM> comprises the first affinity reagent <NUM>, as previously described, the label attached to a backbone <NUM> of the connector <NUM> comprises four fluorophores <NUM>. The backbone <NUM> may, for example, be peptide-based. The backbone <NUM> comprises four first affinity interactors <NUM> that are each configured to specifically bind to four second affinity interactors <NUM> in order to individually attach the fluorophores <NUM> connected to the second affinity interactors <NUM>. The fluorophores <NUM> may be attached to the connector <NUM> by mixing both, the fluorophores <NUM> with second affinity interactors <NUM> and the connector <NUM>. As previously described, the first and second affinity interactors <NUM>, <NUM> may be streptavidin and biotin, respectively, for example.

Connector <NUM> has a label <NUM> attached, specifically the label <NUM> is attached to a first affinity interactor <NUM> of the connector <NUM> via a second affinity interactor <NUM> that specifically binds to the first affinity interactor <NUM>. The label <NUM> comprises a DNA-origami backbone <NUM> that is connected to the second affinity interactor <NUM> and that comprises four first auxiliary affinity interactors <NUM>. The DNA-origami backbone <NUM> may, for example, be a nanoruler. Four fluorophores <NUM> are individually attached to backbone <NUM>, by a second auxiliary affinity interactor <NUM> that specifically binds to the first auxiliary affinity interactor <NUM> and that is connected to each of the fluorophores <NUM>.

In a particularly preferred embodiment of the present invention, the first and second auxiliary affinity interactor are one of the biotin-streptavidin pair. In this case biotinylated staple strands are allowed to bind to streptavidin-conjugated dyes or fluorophores before the staple strands are allowed to interact with the DNA-origami backbone <NUM> (preincubation). As the interaction between biotin-streptavidin is practically irreversible this can be leveraged to attach multiple copies of the same dye as in example <NUM> or multiple dyes (single or multiple copy) as in example <NUM> to the DNA-origami backbone <NUM>. In this case it may be desirable to use a version of streptavidin with <NUM>, <NUM>, <NUM> or only one active biotin-binding site.

Connector <NUM> has a label <NUM> attached to the first affinity interactor <NUM> via a second affinity interactor <NUM>, as described for connector <NUM>. Similarly, the label <NUM> comprises the DNA-origami backbone <NUM> with four first auxiliary affinity interactors <NUM>. In contrast to the connector <NUM> with the label <NUM>, the label <NUM> comprises four fluorophores <NUM>, <NUM>, <NUM>, <NUM> that each differ in their fluorescent properties. These properties may include excitation wavelength, fluorescent wavelength, and fluorescent duration. Each fluorophore <NUM>, <NUM>, <NUM>, <NUM> comprises one of the second auxiliary affinity interactors <NUM> that enable the fluorophores <NUM>, <NUM>, <NUM>, <NUM> to be attached to one of the first auxiliary affinity interactors <NUM> of the label <NUM>. By using differing fluorophores <NUM>, <NUM>, <NUM>, <NUM> in the label <NUM>, connectors with a larger diversity of fluorescent properties may be generated. The diversity of fluorescent properties enables being able to distinguish between a large variety of connectors.

<FIG> shows a schematic view of a biological sample <NUM> with a plurality of connectors. The biological sample <NUM> may, for example, be a cell, in particular, a mammalian cell comprising a nucleus <NUM>. Several connectors <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> with labels are used to specifically bind to a particular target molecule in the sample <NUM>, illustrating the use of the labelled connectors. The labels may differ in their fluorescent properties.

For example, connector <NUM> is indirectly bound to a cytoplasmic protein target molecule <NUM> via a second affinity reagent <NUM>, here an antibody, and a nanobody first affinity reagent <NUM>. Connector <NUM> is directly bound to a cytoplasmic protein target molecule <NUM> via first affinity reagent <NUM>, here a nanobody of the connector <NUM>. Connector <NUM> is directly bound to a mRNA target molecule <NUM> via an oligonucleotide-based first affinity reagent <NUM>. The connector <NUM> is directly bound to a cytoplasmic protein target molecule <NUM> via a small molecule-based first affinity reagent <NUM>. Similar to connector <NUM>, connector <NUM> is indirectly bound to a cytoplasmic protein target molecule <NUM>, however, connector <NUM> is attached to a label <NUM> comprising a plurality of fluorophores. Similarly to connector <NUM>, connector <NUM> is indirectly bound to a nuclear protein target molecule <NUM>. Similarly to connector <NUM>, connector <NUM> is directly bound to a nuclear mRNA target molecule <NUM>.

By means of the connector <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> the individual components <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> of the biological sample <NUM> may be visualised by fluorescent imaging, for example, of the biological sample <NUM> with the connector <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> bound to their target molecules. Further, when using distinct labels, with unique fluorescent properties for a particular connector, the components <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> can be localised within the image of the biological sample <NUM> depending on where a respective fluorescent signal was detected.

As explained above, the combination of one of the connectors with one of the labels specifically bound to the connector via the first and second affinity interactors, may also be termed a marker. This is also the case, when the connector with the label is optionally specifically bound to one of the second affinity reagents. In order to generate the marker, the connector, the label and optionally the second affinity reagents may be mixed together. This allows binding of the label to the connector, more precisely, the binding of the second affinity interactor of the label to the first affinity interactor of the connector and thereby attachment of the label to the connector.

In case the second affinity reagent is to be attached to the connector, the second affinity reagent may be added at the same time, prior or after adding the label. Since the first affinity interactor and the second affinity interactor bind to each other specifically, as do the first and second affinity reagents, the first affinity interactor will only bind to the second affinity interactor, and the first affinity reagent will only bind to the second affinity reagent, even when mixing all components together.

In order to stabilise the binding between the individual parts, the connector and the label and/or the connector and the second affinity reagent may optionally be crosslinked, for example, with glutaraldehyde.

<FIG> shows a flowchart for a method for analysing the biological sample <NUM>, for example, which may be used in the context of multiplex imaging. The process starts in step S600. In step S602 a set of markers is provided. The first set of markers may comprise a first plurality of connectors with a first label and a first affinity reagent configured to specifically bind to a first target molecule. Alternatively, the first plurality of connectors may further comprise a second affinity reagent configured to specifically bind to the first target molecule and the first affinity reagent being configured to bind to the second affinity reagent. Preferably, the first set of markers includes at least a second plurality of connectors with a second label and a first (or second) affinity reagent configured to specifically bind to a second target molecule. The first and second labels have different fluorescent properties such as excitation wavelength, emission wavelength or emission duration.

The step of providing the first set of markers may include generating the markers from the respective connector, the label and optionally the second affinity reagent. This may include mixing the respective connector, the label, and optionally the second affinity reagent, in step S602, as explained above.

In step S604 the sample is stained with the markers. This means that the markers are added to the sample, which causes binding of the affinity reagents to their respective target molecules. Alternatively to mixing the respective connector, the label and optionally the second affinity reagent in step S602, the respective connector, the label and optionally the second affinity reagent may be added individually to the sample in step S604. This is particularly the case, when the marker only comprises a first affinity reagent and not a second affinity reagent. When the connector and the label are introduced into the sample individually, the marker is generated in-situ. The marker then stains the sample by binding to the respective target molecule with the first or second affinity reagent.

In step S606 markers that are not bound to their target molecules are washed out, for example, by washing the sample with a buffer solution. This is to reduce background staining by the label of unbound markers.

In step S608 an image readout is performed, for example, an image of the sample is acquired by illuminating the sample with fluorescent light and capturing the emission light. This may be done by means of a fluorescent microscope. The image reveals locations within the sample of the target molecules, which are stained by a marker with an affinity reagent configured to bind to the particular target molecule.

In step S610 the label is separated from the connector by cleaving of the cleavage site of the connector. This removes the fluorescent signal from the target molecule that the marker is bound to. This may also be called blanking.

In step S612 the cleaved off label is washed out of the sample, for example, by washing the sample with the buffer solution. The method ends in step S614.

Alternatively to ending the method in step S614, the method may proceed by repeating steps S602 to S612 with a different set of markers. This enables using a new set of connectors with affinities to further target molecules whilst at least partially using labels with the same fluorescent properties as the first set of markers. By iterating through steps S602 to S612 repeatedly, a number of target molecules larger than the number of markers with unique fluorescent properties may be stained, imaged and localised.

The connector, or the markers as described herein, may further be used in conjunction with methods and workflows for analysing biological samples disclosed in applications <CIT> and <CIT>, the contents of which are incorporated herein.

<FIG> shows a schematic view of several markers <NUM>, <NUM>, <NUM>. The markers <NUM>, <NUM>, <NUM>, comprise antibodies <NUM>, <NUM>, <NUM> of the same isotype as second affinity reagents. This means, their Fc region <NUM> is the same. The first affinity reagents <NUM> bind to the Fc region <NUM> of the antibodies <NUM>, <NUM>, <NUM>. This means that based on one type of connector comprising only the same first affinity reagent <NUM>, markers may be generated that nevertheless bind specifically to different target molecules <NUM>, <NUM>, <NUM>. Further, this allows using antibodies of the same isotype in a single set of markers. The markers <NUM>, <NUM>, <NUM> have to be prepared separately to avoid mixing the different antibodies <NUM>, <NUM>, <NUM> and labels.

Claim 1:
A set of markers for analysing biological samples, each marker (<NUM>, <NUM>, <NUM>) comprising a connector (<NUM>) and a label (<NUM>) with a second affinity interactor (<NUM>), the connector comprising :
at least one first affinity reagent (<NUM>, <NUM>) configured to bind indirectly to a target molecule (<NUM>, <NUM>, <NUM>, <NUM>),
a second affinity reagent (<NUM>, <NUM>) bound to the first affinity reagent (<NUM>, <NUM>) and the second affinity reagent (<NUM>, <NUM>) is configured to bind to the target molecule (<NUM>, <NUM>, <NUM>, <NUM>), and
a backbone (<NUM>) connected to the first affinity reagent (<NUM>, <NUM>) and comprising at least one first affinity interactor (<NUM>),
wherein the second affinity reagent (<NUM>, <NUM>) is an antibody (<NUM>, <NUM>, <NUM>) and the first affinity reagent (<NUM>, <NUM>) is configured to bind to a fragment crystallisable region (<NUM>) of the antibody (<NUM>, <NUM>, <NUM>),
wherein the first affinity interactor (<NUM>) is configured to specifically bind to the second affinity interactor (<NUM>) comprising the label (<NUM>) in order to bind the label (<NUM>) to the backbone (<NUM>),
wherein the backbone (<NUM>) comprises a cleavage site (<NUM>) for irreversibly separating the first affinity reagent (<NUM>, <NUM>) and the first affinity interactor (<NUM>), and
wherein the antibodies (<NUM>, <NUM>, <NUM>) of the markers (<NUM>, <NUM>, <NUM>) of the set of markers are of the same isotype and the antibodies (<NUM>, <NUM>, <NUM>) are specific to different target molecules (<NUM>, <NUM>, <NUM>).