Detection of target analytes using enzyme-mediated signal amplification

Abstract

The present invention provides a method of detecting a target analyte in a sample comprising generating a nucleic acid product by a proximity assay, as a signal for the analyte, performing an enzyme-mediated Tyramine Signal Amplification (TSA) amplification of the nucleic acid product to generate a TSA product localised to the nucleic acid product, and detecting the TSA product. The method may be used in the context of any proximity assay that generates a nucleic acid product, but finds particular utility in proximity ligation assays (PLAs) that generate a rolling circle amplification product (RCP). By combining the signal amplification power of proximity-based detection methods with that of TSA, the sensitivity of such methods is increased. The signal provided by the proximity-based nucleic acid product generation reaction is further amplified, with the TSA reaction product acting as a label or signal for the nucleic acid product.

Description

[0001] Detection of target analytes using enzyme-mediated signal amplification

[0002] Field

[0003] The present disclosure and invention lie in the field of nucleic acid detection, and in particular in the detection of nucleic acid or non-nucleic acid target analytes using proximity assays which generate a nucleic acid product, particularly a rolling circle amplification product (RCP), as the means by which the target analyte is detected and distinguished from other analytes, in other words as the signal, or reporter, for the target analyte. According to the methods herein, the nucleic acid product (e.g. RCP) signal is subjected to signal amplification using a Tyramide Signal Amplification (TSA)-based signal amplification reaction. Also provided are kits for use in the methods.

[0004] Background

[0005] Many analyte detection assays involve the detection of a target nucleic acid molecule which has been generated either from a target nucleic acid analyte or a copy or amplicon thereof, or from assay reagents, e.g. probes or oligonucleotides used therewith, as a detection assay reaction product which acts as a reporter (i.e. a signal, or proxy) for the target analyte, and which is detected in order to detect the target analyte. Typically, in order to increase sensitivity an amplification reaction is employed to increase the amount of nucleic acid available for detection, and thus the target nucleic acid molecule actually detected is typically an amplification product (amplicon), and especially an amplification product which comprises multiple copies of a monomer, or repeating unit. In many cases the target nucleic acid molecule, which is detected as the assay product, is an RCP.

[0006] Rolling circle amplification (RCA) is an isothermal amplification technigue reguiring a circular amplification template. Amplification of the circular template using a strand-displacing polymerase provides a concatenated RCA product (RCP), comprising multiple copies (i.e. monomer repeats) of a seguence complementary to that of the RCA template. Such a concatemer typically forms a ball or “blob”, which may readily be visualised and detected, and thus RCA-based assays have been adopted for the detection of nucleic acids, and indeed, more generally, as reporter systems for the detection of any target analyte. Both target nucleic acids, which may themselves be circularised directly, or probes, e.g. padlock probes, or circular or circularised nucleic acids more generally used or generated during the course of an assay reaction may provide template nucleic acid circles for RCA (for example as used in immunoRCA reactions, or as generated in proximity ligation assays (PLAs), including the well-known in situ PLA commercially available from Sigma-Aldrich under the Duolink brand name. In the case of the PLA, as explained in more detail below, the nucleic acid domains of proximity probes, when bound in proximity to the target analyte, template the ligation of added oligonucleotide(s) to form a circle and prime its amplification by RCA. Proximity assays are particularly advantageous as analyte detection assays as they employ dual recognition to detect the target - that is the binding of two proximity probes together to their respective target binding sites is required for a signal to be generated; only when the proximity probes are both bound in proximity, directly or indirectly, to the target analyte, can nucleic acid domains of the proximity probes interact to generate the nucleic acid signal (e.g. the RCP). Such dual recognition improves the specificity of the assay.

[0007] RCA may thus be employed as a signal amplification method, to increase the signal which is detected in a detection assay method. In this regard, the monomer repeats of the concatemeric RCP provide a large number of binding sites for a detection oligonucleotide which is detected, in order to detect the RCP. Similarly, other nucleic acid products can be generated in proximity assays, which can provide multiple binding sites for detection oligonucleotides or other labels. This may include the product of amplification methods such as hybridisation chain reaction (HCR), where large numbers of monomers, each comprising a label or a detectable sequence to which a detection oligonucleotide may be hybridised, are incorporated. A proximity assay based on HCR is described for example in WO 2015 / 118029. It is also known, for example, that signal amplification methods may involve the build-up of a sequence of probes which hybridise to each other, to provide multiple binding sites for labelled detection probes, or other labelling systems, as exemplified by the RNAscope™ technology, as described in WO 2011 / 094669 for example. Whilst RNAscope™ was developed for in situ hybridisation for detection of RNA, it exemplifies the principle of using sandwich-type, or intermediate, hybridisation probes each providing multiple binding sites for labelled detection probes, to generate a detectable nucleic acid product (a so-called hybridisation assembly) comprising multiple labels. Such nucleic acid products may be viewed as so-called “nucleic acid accumulations” (NAAs), which comprise multiple target sites for detection or, in other words, multiple detectable sites (i.e. binding sites for a detection oligonucleotide).

[0008] However, even RCA, or HCR or hybridisation assembly, assays may in some circumstances be limited, for example in samples which generate high background signal, or for detecting low abundance analytes or for single molecule detection or detection of analytes in single cells. Thus, it is desirable to increase the signal, and hence the sensitivity, of RCA-based and similar assays.

[0009] Tyramide Signal Amplification (TSA) is a well-known method of signal amplification which is used in analyte detection methods, typically immunohistochemical (IHC) or target nucleic acid sequence detection methods, to provide high density localised labelling of a target protein or nucleic acid sequence in situ. Traditional TSA is an enzyme-mediated detection method which employs a peroxidase enzyme to catalyse the conversion, in the presence of hydrogen peroxide, of a tyramide substrate to a highly reactive short-lived product (a tyramide radical, also referred to as activated tyramide) which reacts with molecules at or near the enzyme (principally tyrosine residues in proteins), covalently to link the tyramide to the molecules, resulting in deposition of the tyramide at or near the site of the enzyme. Each peroxidase molecule can cause the deposition of multiple tyramide molecules, which mediates the signal amplification; the tyramide substrate can be conjugated to a label, or to a moiety for binding to a subsequently added label, resulting in high density labelling at the site of deposition. The peroxidase can be localised to the target analyte by coupling it, or targeting it, to an analyte-binding probe (e.g. an antibody, or oligonucleotide probe etc.).

[0010] More recently, the TSA method has been developed to include the use of different enzymes, and different substrates, and further to employ enzyme- oligonucleotide conjugates, tyramide-oligonucleotide conjugates and / or label- oligonucleotide conjugates to target the enzyme, substrate, and / or label to the analyte, for example in a manner which allows the enzyme or label to be removed, and for the method to be performed serially in cycles to increase multiplexing capacity (see e.g. WO 2021 / 226516 (Leland Stanford Junior University) and US 2021 / 0222234 (Akoya Biosciences, Inc.)).

[0011] Whilst TSA has been used to provide amplified signals in the context of analyte detection, it has not previously been combined with a signal amplification method such as RCA, HCR, or hybridisation assembly methods.

[0012] Summary

[0013] It is proposed herein to use the TSA technology to detect the nucleic acid products such as RCPs, HCR products (HCRPs) or hybridisation products generated in proximity-based analyte detection assays, and thereby to increase, or amplify, the signal which is detected in the detection assay method.

[0014] The present inventors propose a new method to increase the sensitivity of proximity-based detection methods which rely on generating nucleic acid products as amplified signals, by combining the signal amplification power of such methods (e.g. RCA or HCR etc.) with that of TSA, further to amplify the signal provided by the proximity-based nucleic acid product generation reaction (e.g. the RCA, HCR, or nucleic acid hybridisation assembly reaction). The nucleic acid product (e.g. the RCA product (RCP)) provides an amplified signal to detect the analyte, which advantageously can be localised to the analyte, and this is further amplified by performing a TSA reaction which is targeted, or localised, to the nucleic acid product. The TSA reaction product in effect becomes a “label” or “signal” for the nucleic acid product, which is detected to detect the nucleic acid product, which in turn is a reporter or signal for the target analyte. Each of the multiple copies of the “detection sequence” in the nucleic acid product (e.g. the monomer repeats of the RCP concatemer, or HCRP etc.) may be targeted by (bound to) an enzyme molecule, leading to the generation of a TSA signal, itself an amplified signal, for each copy / monomer, resulting in multiple TSA signals per nucleic acid product. In this way, single molecule detection events may be amplified. Further, the method allows for signal capture in large tissue sections without high magnification.

[0015] Although of particular benefit for the localised detection of proteins or protein interactions or modifications in tissue samples, the method is of more general applicability, and provides an improved method of detecting, in any sample, any analyte molecule which may be detected by a proximity assay involving the generation of nucleic acid product as the assay signal, e.g. an RCP.

[0016] Accordingly, in a first aspect provided herein is a method of detecting a target analyte in a sample by a proximity assay, wherein a nucleic acid product has been generated as a signal for the analyte, said method comprising performing an enzyme-mediated Tyramine Signal Amplification (TSA) amplification of the nucleic acid product signal to generate a TSA product localised to the nucleic acid product, and detecting the TSA product, thereby to detect the target analyte.

[0017] In other words, the nucleic acid product of a proximity assay for detection of a target analyte is detected using a TSA signal amplification reaction, in order to detect the target analyte.

[0018] In particular, the TSA product is labelled with a detectable label, and the label is detected in order to detect the TSA product, and thereby the nucleic acid product and the target analyte. The label may be provided to the TSA product during or after the TSA reaction, e.g. by means of a labelled substrate for the TSA reaction, or by labelling the TSA product after it has been generated.

[0019] TSA in its typical format uses a tyramide substrate with the enzyme peroxidase, but, as will be described in more detail below, the term “TSA” as used herein includes also the use of analogous, or related, enzymes and substrates, which act in the same or similar way to the action of peroxidase on tyramide to generate a reactive product which reacts with molecules in the vicinity and is deposited.

[0020] Accordingly, the term “TSA” as used herein may alternatively be referred to as “TSA- like”.

[0021] Accordingly, defined another way, provided herein is a method of detecting a target analyte in a sample, wherein a nucleic acid product has been generated by a proximity assay as a signal for the analyte, said nucleic acid product comprising multiple copies of a detection sequence, and wherein the method comprises:

[0022] (i) hybridising to the multiple copies of the detection sequence an enzyme-capture oligonucleotide, wherein the enzyme-capture oligonucleotide comprises a binding site complementary to the detection sequence, and wherein the enzyme capture oligonucleotide is attached, particularly removably attached, to an enzyme;

[0023] (ii) contacting the reaction mixture from (i) (i.e. the hybridised nucleic acid product-capture oligonucleotide-enzyme) with a signal localisation agent comprising a substrate for the enzyme, wherein the enzyme catalyses a reaction which deposits a substrate reaction product in the vicinity of the enzyme, and hence of the nucleic acid product, wherein the localisation agent comprises either a label, (and the label is deposited along with the substrate reaction product) or a first binding moiety for binding to a labelling agent;

[0024] (iii) when the localisation agent does not comprise a label, contacting the reaction mixture of (ii) with the labelling agent, wherein the labelling agent comprises a label linked to a second binding moiety cognate to the first binding moiety, and allowing the second binding moiety to bind to the first binding moiety (thereby to attach the labelling agent to the substrate reaction product);

[0025] (iv) detecting the label.

[0026] The copies of the detection sequence may alternatively be referred to as repeats. They may be monomers which are comprised in a multimeric, e.g. concatemeric, nucleic acid product. The nucleic acid product may be generated in a nucleic acid extension, ligation and / or hybridisation reaction. It may accordingly be a ligation, extension or hybridisation product. In an embodiment it is an RCP, HCRP or a nucleic acid hybridisation assembly product. In a particular embodiment it is an RCP. In an embodiment, the enzyme capture oligonucleotide comprises an enzyme directly attached or conjugated to the oligonucleotide.

[0027] In another embodiment, the enzyme-capture oligonucleotide comprises a first capture moiety which is removably attached or attachable to a cognate second capture moiety comprised in an enzyme agent comprising said second capture moiety linked to the enzyme.

[0028] In such an embodiment, in step (i) the nucleic acid product, or the sample containing the nucleic acid product, is contacted with an enzyme-capture oligonucleotide comprising a binding site that hybridises to the detection sequence and a first capture moiety, and step (i) further comprises contacting the nucleic acid product with hybridised enzyme-capture oligonucleotide with the enzyme agent, and allowing the cognate first and second capture moieties to bind, to attach the enzyme to the nucleic acid product.

[0029] The first and second capture moieties and / or the first and second binding moieties may be an affinity binding pair, for example selected from (i) biotin and avidin or streptavidin; (ii) hapten and antibody; or (iii) first and second oligonucleotides comprising complementary binding sites.

[0030] In an embodiment, the affinity binding pair may be digoxigenin and an anti- digoxigenin antibody.

[0031] In certain embodiments of the methods above the TSA is performed using a peroxidase enzyme, for example horseradish peroxidase (HRP). The substrate for the enzyme may be a tyramide compound, a compound that contains a tyramine derivative, p-hydroxy-cinnamic acid, or a derivative of p-hydroxy cinnamic acid.

[0032] In an embodiment, the TSA amplification reaction / enzyme-mediated enzyme signal amplification reaction uses a tyramide compound as substrate with the enzyme peroxidase.

[0033] In an embodiment, the nucleic acid product has been generated in the sample and is localised to the target analyte in the sample. More particularly, the nucleic acid product may be generated in an in situ detection reaction for the analyte. In certain embodiments, the nucleic acid product is attached to the target analyte, e.g. by means of being attached to a probe itself bound directly or indirectly to the target analyte.

[0034] The analyte may be any nucleic acid or non-nucleic acid analyte. In an embodiment the analyte is selected from a nucleic acid or a protein or any molecule, complex, interaction or aggregate comprising or involving a protein. In a particular embodiment the method is used to detect a modification on a protein analyte (i.e. a modified, e.g. phosphorylated, protein). In another embodiment the method is used to detect two epitopes on the same protein.

[0035] In an embodiment the sample is a biological or clinical sample, for example a tissue sample, or a biopsy sample. The sample may be fresh, fixed or frozen. In an embodiment, the sample is immobilised on a solid support.

[0036] The method may be used in the context of any proximity assay that generates a nucleic acid product (e.g. an RCP etc.) as an assay product which is detected. In a particular embodiment, the nucleic acid product has been generated in a proximity ligation assay (PLA) for the target analyte, and more particularly it is an RCP generated in a PLA. The probes used in the proximity assay, e.g. PLA, may be primary probes, capable of binding directly to the target analyte, or secondary probes capable of binding to one or more primary probes bound to the target analyte.

[0037] The method may include a step of providing the nucleic acid product. In an embodiment, the method may comprise a step of generating the nucleic acid product. In other words, the method may include performing a proximity detection assay to detect a target analyte, wherein said assay generates a nucleic acid product as a signal, or reporter, for the target analyte, and the nucleic acid product is detected by a TSA signal amplification reaction in order to detect the target analyte.

[0038] In any embodiment of the above methods, the label may directly or indirectly detectable. In an embodiment, it is an optical label, for example a fluorescent, coloured or chromogenic label, e.g. a dye or stain.

[0039] In an embodiment the label is detected microscopically, more particularly by imaging.

[0040] The method is particularly suited to multiplex detection, to detect two or more target analytes, different nucleic acid products being generated for the different analytes.

[0041] Various multiplex formats are possible, including generating multiple (i.e. two or more) nucleic acid products together in the same reaction, or sequentially, and / or performing the TSA reaction serially for each nucleic acid product, or in the same reaction mixture.

[0042] In an embodiment, the TSA reactions may be performed serially for the different nucleic acid products, and the step of labelling the deposited TSA reaction product with a labelling agent may be performed together in one step.

[0043] In an embodiment, two or more cycles of TSA amplification are performed, for example to detect a single target analyte in each cycle, or to detect a subset of different analytes together in each cycle, different subsets being detected in different cycles. In an embodiment, proximity assays are performed sequentially in cycles to generate one or more (e.g. two or more) nucleic acid products, and a TSA reaction is performed in each cycle to generate the TSA product. In an embodiment, the TSA product is labelled in each cycle (whether by means of a labelled localisation agent, or by means of a subsequent labelling agent). In an embodiment, after each cycle the enzyme is removed or inactivated before the next cycle takes place. The TSA product (label) may be detected in each cycle, or the step of detecting the labels may take place after all the cycles have been performed. Various other embodiments are described below.

[0044] Accordingly, in certain embodiments, the detection step may be performed individually to detect the label for each TSA product serially, or together in one step. In an embodiment, serial images are obtained, and are then consolidated, or merged, or superimposed. In another embodiment, all the labels corresponding to different analytes are detected together, e.g. in a single imaging step.

[0045] In certain embodiments, the methods may further comprise counterstaining the sample, before the step of detecting the TSA product, or detecting the label.

[0046] Further, the method may be combined with other detection assays performed on the same sample. In other words, it may be performed as part of an integrated workflow, or protocol, combining different detection or assay steps, including, for example, other immunoassay or detection steps, to detect other target analytes, for example in a sequential fashion.

[0047] A further aspect provides a kit for use in a method as described above, said kit comprising:

[0048] (i) reagents for a proximity assay which generates a nucleic acid product comprising multiple copies of a detection sequence corresponding to a target analyte;

[0049] (ii) an enzyme-capture oligonucleotide comprising an oligonucleotide comprising a binding site complementary to the detection sequence, linked to an enzyme, or to a first capture moiety;

[0050] (iii) when said enzyme-capture oligonucleotide comprises a first capture moiety, an enzyme agent comprising an enzyme linked to a second capture moiety which is cognate and capable of binding to the first capture moiety;

[0051] (iv) a signal localisation agent comprising a substrate for the enzyme linked to a label or to a first binding moiety, wherein the enzyme is capable of catalysing a reaction using said substrate which deposits a substrate reaction product; (v) when said signal localisation agent comprises a first binding moiety, a labelling agent comprising a label linked to a second binding moiety cognate and capable of bind to the first binding moiety.

[0052] In an embodiment, the reagents (i) comprise:

[0053] (i) one or more proximity probes pairs, each pair constituting a primary reagent specific for a different target analyte, and each member of a pair being capable of binding specifically and simultaneously to their target analyte; or

[0054] (ii) one or more proximity probe pairs, each pair constituting a secondary reagent capable of binding to a primary reagent bound to a target analyte, wherein the primary reagent comprises a single binding agent capable of binding specifically to the target analyte or a pair of binding agents each capable of binding specifically and simultaneously to the target analyte, and each member of the proximity probe pair is capable of binding specifically and simultaneously to the single binding agent, or to a separate single member of the primary binding agent pair (such that each member of a pair of primary binding agents is bound by a single member of a proximity probe pair); wherein each proximity probe of a proximity probe pair comprises a binding domain capable of binding specifically to its target, and a nucleic acid domain, and the nucleic acid domains are together capable of templating the ligation of one or more circularisable oligonucleotides to from a template circle for RCA and priming the RCA reaction, wherein said circularisable oligonucleotides are (a) separately provided, or (b) generated by cleavage of the nucleic acid domains; and

[0055] (iii) optionally, in the case of part (ii)(a), for each proximity probe pair, one or more circularisable oligonucleotides, each comprising at least one binding site capable of hybridising to a complementary binding site in each of the nucleic acid domains of the proximity probe pair; optionally wherein the reagents further comprise:

[0056] (iv) a polymerase for the RCA reaction; and / or

[0057] (v) dNTPs.

[0058] More particularly, in part (iii), the one or more circularisable oligonucleotides comprise a detection sequence (or more particularly a complement of a detection sequence) which is incorporated into the RCA template circle.

[0059] Analogously, the nucleic acid domains of the proximity probes may alternatively interact together to allow a different nucleic acid product to be formed, for example an HCR product, for example where the interaction of the nucleic acid domains with each other releases an initiator for an HCR reaction, or where the nucleic acid domains of the probes (or indeed the proximity probes themselves, where they are composed entirely of nucleic acids, i.e. where the analyte binding domain of the probe is also a nucleic acid) together allow the initiation of a hybridisation assembly reaction, for example they act as Z-probes, or provide binding sites for Z-probes.

[0060] Description of Figures

[0061] Figure 1 is a schematic showing an embodiment of the method, in which an RCP is generated by a PLA-RCA reaction, comprising a proximity ligation assay (PLA) in which the nucleic acid domains of a pair of proximity probes, upon binding of the probes in proximity to their target, template the ligation of two circularisable oligonucleotides into an RCA template circle which is amplified in an RCA reaction primed by one of the nucleic acid domains to form an RCP, which is then detected by a TSA reaction. The proximity probes are shown as antibodies, but may comprise any binding domain. They may be primary or secondary binding reagents for a target analyte (not shown). A capture oligonucleotide comprising a first capture moiety, or linking group is hybridised to the repeats of a detection seguence in the RCP, and the first capture moiety binds to a cognate second capture moiety or to a second linking group reactive with the first, which is comprised in an enzyme agent comprising an enzyme, here depicted as HRP, linked to the second capture moiety / second linking group, thereby to capture, or bind, the enzyme to the RCP. The addition of a labelled tyramide substrate, here depicted as labelled with a fluorophore (F), leads to the enzyme-catalysed deposition of the labelled tyramide substrate in the vicinity of the enzyme, and hence of the RCP and target analyte.

[0062] Figure 2 shows in the top panel the results of a Proximity Ligation Assay (PLA) to study the interaction between PDGFR and pan-tyrosine in BJ hTERT cells, comparing a first sample using standard PLA with a second sample using TSA- enhanced PLA. Non-TSA Sample Detection: For the non-TSA sample, after the RCA amplification step of the PLA, an oligonucleotide labeled with Alexa 594 was used for detection. TSA Sample Detection: For the TSA-enhanced sample, post-amplification, the rolling circle amplification (RCA) product was detected using an oligonucleotide labelled with biotin. Subseguently, the sample was incubated with streptavidin conjugated with horseradish peroxidase (HRP). After this incubation, TSA conjugated with Alexa 594 was added to visualize the amplified signals. In the bottom panel, BT474 cells were stained to detect the presence of HER2 using both standard PLA and TSA-enhanced PLA. For this set of cells, the fluorophore was changed to Alexa 647 for detection. Figure 3 shows combined PLA and immunofluorescence (IF) multiplex staining on formalin-fixed, paraffin-embedded (FFPE) non-small cell lung cancer (NSCLC) tissue samples. The NSCLC tissue samples were first subjected to Proximity Ligation Assay (PLA) to detect the interactions between PD1 and PDL1. Following the primary antibody binding specific for these interactions, tissues underwent PLA and TSA. Immediately after the PLA-TSA procedure, the sample was subjected to successive cycles of IF-TSA detection of primary antibodies to panCK, pSTAT3, pSMAD and CD8 respectively, with deactivation of HRP activity between cycles, after TSA visualisation. Images captured from fluorescence microscopy are shown, including a scale bar reference of 20 pm for size context.

[0063] Figure 4 shows the results of TSA-enhanced PLA, combined with IF-TSA detection of several markers, including CD31, DAPI, Active Yap, FAP and panCK in formalin-fixed, paraffin-embedded (FFPE) non-small cell lung cancer (NSCLC) tissue samples. A multiplex immunofluorescent (IF-TSA) panel was employed, using the Bond RXm Autostainer, with antigen retrieval between each cycle of staining for a individual marker. Subseguently, following another step of antigen retrieval, a PLA- TSA protocol was manually implemented to detect the PDGFRP-GRB2 interaction. The images obtained from fluorescence microscopy are shown.

[0064] Detailed Description

[0065] The present methods provide improved and highly sensitive methods for detecting target analytes. The methods are particularly well-suited to multiplex analyses. Further, they are particularly well-suited to in situ detection of analytes in cell or tissue samples, for example on slides or other solid supports. However, this does not detract from their wider use, including for example in homogenous assays with cells in suspension, based for example on flow cytometry detection.

[0066] As noted above, the present methods combine the signal amplification power of nucleic acid-based signal amplification methods used in proximity assays, such as RCA, HCR, or hybridisation assembly, with that of TSA. The use of the TSA signal amplification reaction in particular affords the advantage of allowing cycling of the method - as will be described in more detail below, the steps of generating the TSA product and / or labelling of the TSA reaction product may be performed seguentially. This allows multiplexing capacity to be extended. For example, the number of different proximity probe pairs that can be used together at one time in a proximity assay is limited. The method allows proximity assays for different target analytes to be performed seguentially, to generate TSA products, or labelled TSA products in different cycles. The labelled products may be detected together in one step, or individually in the separate cycles. This may further allow the same labels to be used in different cycles, to extend the multiplexing capacity of the method even further.

[0067] The very high signal amplification afforded by the method circumvents the problems with high background that can occur in some tissues, for example tissues with high autofluorescence, which limit even the use of RCA-based or similar detection using fluorescent labels in some cases. Further, the high signal amplification of the method allows for signal to be detected across a large region or area of the sample without the need for high magnification. Thus, a larger area of sample can be analysed, e.g. imaged. This is particularly advantageous in the case of heterogenous samples, where there is heterogeneity in the presence of the target analyte between different cells or regions of the tissue, for example in tumours, where it is becoming apparent that most cancer types harbour a high degree of intratumour heterogeneity and that the spatial distribution of cells expressing a biomarker might also represent a prognostic or predictive factor. The present methods are able not only quantitatively to determine the expression of a target analyte in a sample, and therefore provide a measure of its abundance, but can also reflect the spatial distribution and heterogeneity of the target analyte inside the sample.

[0068] As noted above, and described in more detail below, the present methods may be used with any proximity-based analyte detection assay that generates a nucleic acid product as the assay product which is detected. This includes in particular proximity assays, e.g. PLAs, in which an RCA template circle is generated or used, resulting in an RCA step generating an RCP. The present methods may be referred to generally as proximity-TSA assays, and PLA assays in particular are referred to as PLA-TSA assays.

[0069] The combination of the TSA signal amplification reaction with a proximity assay is particularly advantageous. Proximity assays are well known in the art, and have the advantage of dual target recognition, where 2 binders (i.e. a pair of proximity probes) are used to bind to a target in proximity, and generate the nucleic acid product signal only when both proximity probes have bound, thereby increasing specificity.

[0070] A proximity probe for use in the methods herein comprises a binding domain capable of binding, directly or indirectly, to its target analyte, and a nucleic acid domain. The binding domain may be any binding partner for the target analyte, or where the proximity probes are secondary reagents, it may be a binding partner for a primary binding partner for the target analyte. A binding partner may be any moiety capable of binding specifically to the analyte or to a primary binding partner therefor. In the case of non-nucleic acid analytes, the binding domain will typically be an antibody. The term “antibody” as used herein includes all types of antibody, whether natural or synthetic, and antibody derivatives and fragments etc. It may alternatively be any binding molecule or affinity binding partner, e.g. receptor / ligand, lectin, aptamer etc. Where the target analyte is a nucleic acid, the binding domain may be a nucleic acid comprising a sequence capable of hybridising to the target analyte. The nucleic acid domain of a proximity probe mediates or contributes to the generation of the nucleic acid product which is generated in the proximity assay. Thus, the nucleic acid domains of a pair of proximity probes interact with one another, for example by hybridising to one another, or to one or more common oligonucleotides, and said interaction gives rise to the generation of the nucleic acid product, for example by ligation and / or extension reactions, as is well known in the art, and as described in more detail below.

[0071] The combined proximity assay-TSA methods, particularly PLA-TSA, provided herein are particularly advantageous in providing analyte detection methods of specificity and high sensitivity. This in particular allows detection at a single cell, or single molecule level. Single proteins can be detected allowing e.g. the detection of cell markers, protein interactions and protein modifications. This is exemplified, for example, in Example 2 below, which shows the detection of the PDL1-PD1 interaction (see Figure 3). The PLA-TSA assay allows a sensitive and robust detection of PDGFRp activation in diagnostic tissue samples. This may similarly be applied in the detection and analysis of other biomarkers.

[0072] The target analyte to be detected by the methods herein may be any analyte which it is desired to detect. It may thus be any substance, molecule or entity it is desired to detect. The method herein relies upon the generation of a nucleic acid product, e.g. an RCP, in order to detect the analyte. The RCP is generated by RCA of a circular RCA template molecule, that is a circular nucleic acid molecule. The RCA template may be used or generated in a proximity assay for the target analyte. Thus, it may be provided or generated as a proxy, or a marker, or an indicator for the analyte. As noted above, many assays are known for the detection of numerous different analytes, which use an RCA-based detection system, i.e. where the signal is provided by generating an RCP from a circular RCA template which is provided or generated in the assay, and the RCP is detected to detect the analyte. The RCP may thus be regarded as the ultimate reporter, or signal, which is detected to detect the target analyte. However, the RCA template may also be regarded as a reporter or signal for the target analyte; the RCP is generated based on the RCA template, and comprises complementary copies of the RCA template. The RCA template determines the signal which is detected, and is thus indicative of the target analyte. As will be described in more detail below, the RCA template may be a probe, or a part or component of a probe, or may be generated from a probe, or it may be a component of a detection assay (i.e. a reagent in a detection assay), which is used as a reporter for the assay, or a part of a reporter, or signal-generation system. The RCA template used to generate the RCP may thus be a circular (e.g. circularised) reporter nucleic acid molecule, namely from any RCA-based proximity detection assay which uses or generates a circular nucleic acid molecule as a reporter for the assay. Since the RCA template generates the RCP reporter, it may be viewed as part of the reporter system for the assay.

[0073] Analogously, for other nucleic acid products such as HCRPs or hybridisation assemblies, these may be generated as the proxy, or reporter, or signal etc. for the target analyte, in a proximity detection assay using precursors for such products, e.g. HCR monomers which interact with the one or both of the nucleic acid domains of the proximity probes, or hybridisation probes which are used to build a hybridisation assembly.

[0074] The term “reporter” is thus used synonymously with the term “signal” when used in the context of the nucleic acid product, or the RCA template or product (RCP). The terms thus denote a molecule which is used to report on the presence or absence of the analyte - it is a molecule which is detected in the assay method in order to detect the analyte, or which is used or generated as part of the signal generating system to detect the analyte. The nucleic acid product or the RCP / RCA template may also be referred to as a marker, or proxy or indicator for the analyte.

[0075] The analyte is the ultimate target of the detection method and may accordingly be any biomolecule or chemical compound, including a protein (which term includes peptides or polypeptides), a lipid, a carbohydrate (e.g. a glycosyl group which may occur on a protein) or a nucleic acid molecule, or a small molecule, including organic or inorganic molecules, or any complex or interaction between such molecules. The analyte may be a cell or a microorganism, including a virus, or a fragment or product thereof. An analyte can be any substance or entity for which a specific binding partner (e.g. an affinity binding partner, for example an antibody) can be developed. Such a specific binding partner may be a nucleic acid probe (for a nucleic acid analyte). Whilst such a probe may lead directly to the generation of an RCA template (e.g. a padlock or other circularisable probe), in the present methods, such probes are used in the context of proximity assays, where at least two target analyte-binding nucleic acid probes are used (for example they may together provide a binding site for a padlock probe, or for other assay reagents which lead to the generation of a nucleic acid product, e.g. Z-probes, or HCR monomers etc.). Alternatively, the specific binding partner may be coupled to a nucleic acid, which may be detected using an RCA strategy, e.g. in an assay which uses or generates a circular nucleic acid molecule which can be the RCA template, or a similar strategy which generates an alternative nucleic acid product.

[0076] Analytes of particular interest may thus include nucleic acid molecules, such as DNA (e.g. genomic DNA, mitochondrial DNA, plastid DNA, viral DNA, etc.) and RNA (e.g. mRNA, microRNA, rRNA, snRNA, viral RNA, etc.), and synthetic and / or modified nucleic acid molecules, (e.g. including nucleic acid domains comprising or consisting of synthetic or modified nucleotides such as LNA, PNA, morpholino, etc.), proteinaceous molecules such as peptides, polypeptides, proteins or prions or any molecule which includes a protein or polypeptide component, etc., or fragments thereof, or a lipid or carbohydrate molecule, or any molecule which comprise a lipid or carbohydrate component. The analyte may be a single molecule or a complex that contains two or more molecular subunits, e.g. including but not limited to protein- nucleic acid, e.g. protein-DNA, complexes, which may or may not be covalently bound to one another, and which may be the same or different. Thus, in addition to cells or microorganisms, such a complex analyte may also be a protein complex or protein interaction, or more generally a complex of interaction comprising or involving a protein. Such a complex or interaction may thus be a homo- or hetero-multimer. Aggregates of molecules, e.g. proteins, may also be target analytes, for example aggregates of the same protein or different proteins. The analyte may also be a complex between proteins or peptides and nucleic acid molecules such as DNA or RNA, e.g. interactions between proteins and nucleic acids, e.g. regulatory factors, such as transcription factors, and DNA or RNA. The individual members of an interaction may be detected using proximity probes, each specific for a member of the interaction. An exemplary interaction is for example the PD-1 / PD-L1 interaction. Analogously interactions with other immune checkpoints or regulatory proteins may be detected.

[0077] Since a proximity assay detects proximity between two target molecules, the analyte may simply be the proximal localisation of two or more target molecules or entities together, without necessarily requiring binding, or a physical association between them. Such a proximity may indicate that the molecules / entities are interacting, but this is not a requirement. Proximity in this context means that the two target molecules / entities are sufficiently close to one another to enable them to be detected by a proximity assay. For example, this may in practice meant that they lie, or are located, within a distance of no more than 100, 90, 80 or more particularly 70 nm of each other, e.g. 10-80, 20-80, 20-70, 30-70, 40-70nm of each other etc. A single molecule target such as a protein may be detected by detecting two separate epitopes on the molecule.

[0078] The target analyte may be a modified protein, for example with a post- translational modification (PTM) which is detected in the detection method. Thus, for example, a proximity assay may be performed using proximity probes which on the one hand detect the protein and on the other the modifying group (e.g. a phosphorylation).

[0079] In another embodiment, the target analyte may be a protein or component of a proteinaceous molecule which is detected on the surface of a cell, or vesicle or other cellular or sub-cellular compartment.

[0080] The target analyte may be a variant of a target sequence. Target sequences may commonly occur in variant forms, for example allelic variants, or mutant and wild-type sequences and it may be desirable which variant is present. Thus, the target nucleotide sequence may be one of a number of different variants of the nucleic acid sequence which may occur in a target nucleic acid molecule. The term “nucleotide sequence” is used herein synonymously and interchangeably with “nucleic acid sequence”.

[0081] The term "detecting" is used broadly herein to include any means of determining the presence of the analyte (i.e. if it is present or not) or any form of measurement of the analyte. Thus "detecting" may include determining, measuring, assessing or assaying the presence or absence or amount or location of analyte in any way. Quantitative and qualitative determinations, measurements or assessments are included, including semi-quantitative. Such determinations, measurements or assessments may be relative, for example when two or more different analytes in a sample are being detected, or absolute. As such, the term "quantifying" when used in the context of quantifying a target analyte(s) in a sample can refer to absolute or to relative quantification. Absolute quantification may be accomplished by inclusion of known concentration(s) of one or more control analytes and / or referencing the detected level of the target analyte with known control analytes (e.g. through generation of a standard curve). Alternatively, relative quantification can be accomplished by comparison of detected levels or amounts between two or more different target analytes to provide a relative quantification of each of the two or more different analytes, i.e., relative to each other.

[0082] In one embodiment the method may be for the localised detection of target analyte. "Localised" detection means that the signal giving rise to the detection of the analyte is localised to the analyte, in this case the nucleic acid product and the TSA product are localised to the target analyte. The analyte may therefore be detected in or at its location in the sample. In other words, the spatial position (or localization) of the analyte within the sample may be determined (or "detected"). This means that the analyte may be localised to, or within, the cell in which it is expressed, or to a position within a cell or tissue sample. Thus "localised detection" may include determining, measuring, assessing or assaying the presence or amount and location, or absence, of the analyte in any way.

[0083] More particularly, the method may be used for the in situ detection of an analyte. In a particular embodiment, the method may be used for the localised, particularly in situ, detection of nucleic acids, e.g. mRNA, or proteins or protein interactions or modifications, including more particularly the localised, particularly in situ, detection of proteins or protein interactions or modifications in a sample of cells.

[0084] As used herein, the term "in situ" refers to the detection of a target analyte in its native context, i.e. in the cell or tissue in which it normally occurs. Thus, this may refer to the natural or native localization of a target analyte. In other words, the analyte may be detected where, or as, it occurs in its native environment or situation. Thus, the analyte is not moved from its normal location, i.e. it is not isolated or purified in any way, or transferred to another location or medium etc. Typically, this term refers to the analyte as it occurs within a cell or within a cell or tissue sample, e.g. its native localization within the cell or tissue and / or within its normal or native cellular environment. In particular, in situ detection includes detecting the target analyte within a tissue sample, and particularly a tissue section. In other embodiments the method can be carried out on a sample of isolated cells, such that the cells themselves are not in situ.

[0085] In other embodiments, the detection is not localized, or not in situ. In other words, the method includes embodiments in which the target analyte is not present (e.g. is not fixed) in its native context. This may include embodiments in which a target analyte is immobilized, e.g. on a solid support, for example in or on an array. In still other embodiments, the method can be carried out in solution or in suspension. In particular the analyte can be in solution. Thus, for example, the method can be performed on a sample comprising an isolated analyte. In another embodiment the method can be performed where the analyte is suspended in a sample, for example where the analyte is a cell, or an aggregate etc. In still another embodiment, the analyte may be present in or on a cell which is in suspension in the sample, or which is immobilized in the sample etc.

[0086] The analyte is present within a sample. The sample may be any sample which contains any amount of target analyte which is to be detected, from any source or of any origin. A sample may thus be any clinical or non-clinical sample, and may be any biological, clinical or environmental sample in which the target analyte may occur. All biological and clinical samples are included, e.g. any cell or tissue sample of an organism, or any body fluid or preparation derived therefrom, as well as samples such as cell cultures, cell preparations, cell lysates etc. Environmental samples, e.g. soil and water samples or food samples, are also included. The samples may be freshly prepared for use in the method of the present invention, or they may be prior-treated in any convenient way e.g. for storage.

[0087] As noted above, in one embodiment, the target analyte may be detected in situ, as it naturally occurs in the sample. In such an embodiment the target analyte may be present in a sample at a fixed, detectable or visualisable position in the sample. The sample will thus be any sample which reflects the normal or native (" / n situ") localisation of the target analyte, i.e. any sample in which it normally or natively occurs. Such a sample will advantageously be a cell or tissue sample. Particularly preferred are samples such as cultured or harvested or biopsied cell or tissue samples in which the target analyte may be detected to reveal the localisation of the target analyte relative to other features of the sample. In some embodiments, the sample may be a cell or tissue sample possessing a high autofluorescence, in particular a human tissue sample. In some embodiments, the sample may be a cancer tissue sample.

[0088] As well as cell or tissue preparations, such samples may also include, for example, dehydrated or fixed biological fluids, and nuclear material such as chromosome / chromatin preparations, e.g. on microscope slides. The samples may be freshly prepared or they may be prior-treated in any convenient way such as by fixation or freezing. Accordingly, fresh, frozen or fixed cells or tissues may be used, e.g. FFPE tissue (Formalin Fixed Paraffin Embedded). Analytes, including cells, or cells which carry or contain an analyte, may be immobilised on a solid support or surface, e.g. a slide, well or beads or other particles etc., using techniques and reagents well known the art, e.g. capture probes and such like, or by chemical bonding or cross-linking etc.

[0089] Thus, representative samples may include any material which may contain a target analyte, including for example foods and allied products, clinical and environmental samples, etc. The sample may be a biological sample, which may contain any viral or cellular material, including all prokaryotic or eukaryotic cells, viruses, bacteriophages, mycoplasmas, protoplasts and organelles. Such biological material may thus comprise all types of mammalian and non-mammalian animal cells, plant cells, algae including blue-green algae, fungi, bacteria, protozoa etc. Representative samples thus include clinical samples, e.g. whole blood and blood- derived products such as plasma, serum and buffy coat, blood cells, other circulating cells (e.g. circulating tumour cells), urine, faeces, cerebrospinal fluid or any other body fluids (e.g. respiratory secretions, saliva, milk, etc.), tissues, biopsies, as well as other samples such as cell cultures, cell suspensions, conditioned media or other samples of cell culture constituents, etc. The sample may be pre-treated in any convenient or desired way to prepare for use in the present methods, for example by cell lysis or purification, fixing of cells, isolation of the analyte, immobilisation etc.

[0090] Although the present methods may be used to select a target analyte in an in situ (i.e. a native) setting, it is also contemplated that the method may be employed to select a target analyte in any detection system, including where a target analyte has been isolated or purified from its native setting. The sample may thus be a direct product of a target analyte isolation procedure, or of a cell lysis procedure, or it may further be fractionated or purified in some way. Thus, the analyte may be a synthetic molecule such as a cDNA or an amplicon etc., and the sample may be any material or medium containing such a molecule, e.g. a reaction mixture. In an embodiment, the sample can be a preparation of cells, e.g. a cell suspension.

[0091] According to the present methods, a nucleic acid product is provided which has been generated as a signal / reporter for said analyte, and which comprises multiple repeats or copies of a detection sequence indicative of said analyte. The detection sequence is in turn involved in the TSA reaction, the product of which is ultimately detected in order to indicate the presence of the target analyte. In certain embodiments, e.g. where multiple analytes are detected in multiplex in the same reaction (e.g. where multiple RCPs are generated in multiplex), the detection sequence for a given target analyte is therefore specific to that analyte, or unique, such that multiple target analytes can be distinguished from each other. In methods which are cycled, or carried out sequentially, the same detection sequence may be used in different cycles.

[0092] A “detection sequence” is thus a sequence which marks or identifies a given analyte, or a given analyte in a particular assay. It is a sequence by which a given analyte may be detected and distinguished from other analytes in the assay, or the assay cycle. Where an “analyte” comprises a group of related molecules e.g. isoforms or variants or mutants etc., or molecules in a particular class or group, it is not required that a detection sequence is unique or specific to only one particular analyte molecule, and it may be used to denote or identify the analyte as a group. However, where desired, a detection sequence may be unique or specific to a particular specific analyte molecule, e.g. a particular variant. In this way different variants, or isoforms, or mutants may be identified or distinguished from one another. The detection sequence may be incorporated into the nucleic acid product as a tag or identifier (ID) sequence (e.g. a barcode) for the analyte (including for a nucleic acid analyte). It may thus be a synthetic or artificial sequence.

[0093] In the case of RCA-based detection methods, the detection sequence is a complementary copy of a sequence present in the RCA template which is used to generate the RCP. Analogously, this would be the case for other nucleic acid generation reactions based on a polymerase-catalysed extension reaction. The detection sequence complement may be provided in the RCA template as a tag or identifier sequence for the analyte, for example where the RCA template is or is generated from a probe (e.g. a circularisable probe such as a padlock probe, or a circularisable oligonucleotide as used in a PLA-RCA reaction). It will be understood in this regard that the sequence in the RCA template which is complementary to the detection sequence present in the RCP may itself be regarded as a detection sequence. The RCA template may be provided or generated from a probe or reporter molecule which is designed to detect a particular analyte, and thus such a probe or reporter molecule may be viewed as comprising a detection sequence for that analyte - the detection sequence is then copied, as a complementary sequence, into the RCP. The term “detection sequence” can therefore encompass both a detection sequence present in the RCP and its complement (more particularly reverse complement) present in the RCA template. Accordingly, a “detection sequence” can include the complementary sequence.

[0094] As is clear from the above, the target analyte may be any nucleic acid molecule, including DNA, RNA, or a mixture thereof. Moreover, the target analyte may be any form of nucleic acid, such as mRNA, cDNA, etc. The sample may undergo any necessary treatments to prepare the target analyte for detection. In some embodiments, the RNA present in the sample may be reverse transcribed into cDNA, for example by contacting the sample with a reverse transcriptase enzyme and appropriate primers. Such enzymes and primers are well known in the art, and any suitable enzymes and primers may be employed. This reverse transcription reaction may be carried out in situ, following fixing of cells in the sample. In such an embodiment, the cDNA produced by the reverse transcription reaction can then be considered as the target analyte to be detected.

[0095] As mentioned above, RCA is widely known as an amplification technique, and many detection assays have been proposed and described using RCA to generate a detectable product.

[0096] As noted above, the template circle for an RCA reaction may be produced by the circularisation of a probe for a target nucleic acid sequence, notably a padlock probe, according to principles well-known in the art. Padlock probes may take many forms, and may be provided in 1-part form, or multi-part (e.g. 2-part) form. They include gap-fill padlock probes. The RCA template may alternatively be a pre-formed circle, which forms part of a target-specific proximity probe (e.g. is hybridised to a target-specific proximity probe, or to a nucleic acid part or domain thereof). Analogously, it may be a circularisable oligonucleotide which is ligated to form a circle during the course of the assay reactions. Thus, an RCP may be a product of an any type of proximity-based detection reaction which comprises an RCA step, for example a proximity probe assay in which a circular nucleic acid molecule is generated - see the Duolink™ PLA of SigmaAldrich for example, and the modified PLA which uses so-called Unfold proximity probes, which comprise hairpins which are opened, or unfolded, by cleavage to release nucleic acid domains which may be circularised to form an RCA template (see Klaesson et al, 2018, Scientific Reports 8, 5400). A typical PLA generates a template circle upon interaction of the nucleic acid domains of proximity probes, when bound in proximity to their target. Such an assay method is referred to herein as a PLA-RCA method and is depicted schematically in Figure 1. More specifically, the nucleic acid domains of a pair of proximity probes may, upon binding of the proximity probes in proximity to their respective targets, hybridise to one or more circularisable oligonucleotides (which may be viewed as a padlock probe specific for one or both of the nucleic acid domains of the proximity probe pair), allowing a ligation reaction to be templated to generate a nucleic acid circle which may be subjected to RCA. The proximity probes may be secondary reagents which bind to specific binding partners which are themselves bound to the target analyte. However, the proximity probes may alternatively be primary reagents which bind directly to the target analyte. A single circularisable oligonucleotide (padlock probe) may be used, which hybridises to both nucleic acid domains of the two proximity probes. However, various configurations are possible, including the use of a 2-part padlock probe (2 circularisable oligonucleotides) which are hybridised to the nucleic acid domains, such that the hybridisation brings the respective 5’ and 3’ ends of the oligonucleotides together in juxtaposition for ligation. The ligation may be templated by one or both of the nucleic acid domains. In an embodiment, RCA of the resulting circle may be primed by a nucleic acid domain. In such an embodiment, one nucleic acid domain may template ligation, and the other may prime RCA. In another embodiment both nucleic acid domains may template a ligation, and one nucleic acid domain may also prime RCA. In another embodiment, a separate RCA primer is used. The advantage of a nucleic acid domain priming the RCA reaction is that the resulting product (RCP) is localised to the proximity probe, which is bound to the target analyte, and hence to the target analyte, thereby allowing localised detection of the analyte in the sample. This is particularly useful in in situ applications, e.g. in a cell or tissue sample, where information about the location of different target analytes within the cell or tissue can be obtained.

[0097] Accordingly, in more detail, a padlock probe may alternatively be defined as a circularisable probe. The use of padlock or circularisable probes is well known in the art, including in the context of RCA reactions. A circularisable probe comprises one or more linear oligonucleotides which may be ligated together to form a circle. Padlock probes are well known and widely used and are well-reported and described in the literature. Thus, the principles of padlock probing are well understood and the design and use of padlock probes is known and described in the art. A padlock probe is typically a linear circularisable oligonucleotide which hybridizes to its target nucleic acid sequence or molecule in a manner which brings 5’ and 3’ ligatable ends of the probe into juxtaposition for ligation together, either directly or indirectly, with a gap in between. By ligating the hybridized 5' and 3' ends of the probe, the probe is circularized. It is understood that for circularization (ligation) to occur, the ligatable 5’ end of the padlock probe has a free 5' phosphate group.

[0098] To allow the juxtaposition of the ends of the padlock probe for ligation, the padlock probe is designed to have the target-binding sites at or near its 5' and 3' ends. That is, the regions of complementarity which allow binding of the padlock probe to its target lie at or near the ends of the padlock probe.

[0099] To allow ligation, the 3’ and 5’ ends which are to be ligated (the “ligatable” 3’ and 5’ ends) are hybridized to their target sequence (i.e. to a complementary binding site), which acts as the ligation template. The ligatable ends of a padlock probe may be brought into juxtaposition for ligation in various ways, depending on the probe design. Where the target-binding sites are located at the ends of the padlock probe, the binding of the padlock probe may bring the ends into said juxtaposition. Where the complementary binding sites in the target molecule or sequence lie directly adjacent (or contiguous) to one another, the ends of the padlock probe will hybridise directly adjacent to each other (i.e. with no gap) and may be ligated to each other directly. Thus, in this case the ligatable ends of the probe are provided by the actual ends of the probe. However, in an alternative configuration the padlock probe is a gap-fill padlock probe, and hence the binding sites at the ends of the padlock probe do not hybridise to adjacent binding sites, but rather to non-adjacent (noncontiguous) binding sites in the target sequence. In such an arrangement, the 5’ ligatable end of the probe is provided by the actual 5’ end of the probe. However, the ligatable 3’ end of the probe is generated by extension of the hybridized 3’ end of the probe, using the target sequence as extension template to fill the gap between the hybridized ends of the probe. The extension reaction brings the extended 3’ end of the probe into juxtaposition for ligation. In this case, the ligatable 3’ end of the probe is thus the extended 3’ end of the probe.

[0100] Padlock probes may be provided in 2 or more parts that are ligated together. In the context of a proximity probe assay, the nucleic acid domains of a pair of proximity probes may each act as a ligation template. In another embodiment, a 2- part padlock may take the form of a “connector” oligonucleotide with two targetbinding regions at or near the 5’ and 3’ ends respectively, which hybridise to the target with a gap in between them, and a gap oligonucleotide which hybridizes in the gap between the ends. The gap oligonucleotide may partially or fully fill the gap.

[0101] In the case of padlock probes, in one embodiment the ends of the padlock probe may be brought into proximity to each other by hybridisation to adjacent sequences on a target nucleic acid molecule (such as a nucleic acid domain of a probe), which acts as a ligation template, thus allowing the ends to be ligated together to form a circular nucleic acid molecule, allowing the circularised padlock probe to act as a template for an RCA reaction. In such an example the terminal sequences of the padlock probe which hybridise to the target nucleic acid molecule will be specific to their target sequence in question, and will be replicated repeatedly in the RCP. They may therefore act as a detection sequence indicative of that target, and hence of the target analyte. Accordingly, it can be seen that the detection sequence in the RCP may be equivalent to a sequence present in the target nucleic acid sequence itself (e.g. the nucleic acid domain of a proximity probe). Alternatively, a detection sequence (e.g. tag or barcode sequence) may be provided in the nontarget complementary parts of the padlock probe. In still a further embodiment, the detection sequence may be present in the gap oligonucleotide which is hybridised between the respective hybridised ends of the padlock probe, where they are hybridised to non-adjacent sequences in the target molecule.

[0102] As noted above, in embodiments of the present method where an RCP has been generated as a reporter for the target analyte, the RCA reaction may amplify an RCA template which is itself a reporter for the presence of the target analyte. Such an RCA template may contain a detection sequence (or more particularly a complement thereof), in order to produce an RCP comprising multiple repeat copies of a detection sequence indicative of said target analyte. In such embodiments, the detection sequence may be present in a probe molecule (such as padlock probe, or any other probe described or mentioned above), or in a circular or circularisable nucleic acid reporter molecule which is used in conjunction with a probe to detect the target analyte.

[0103] Accordingly, a new nucleic acid molecule may be generated in a sample (i.e. a nucleic acid molecule that was not present in the original sample and was not one of the components added to the sample) by one or more molecules that interact with, e.g. bind to, the target analyte. The detection of the generated nucleic acid molecule is indicative of the target analyte in a sample. The generated molecule may be a circular molecule, or it may template the circularisation of another molecule, such as a padlock probe for the generated molecule.

[0104] Analogously to proximity assays using antibody-based proximity probes to bind to proteins, hybridisation probes may be used to detect nucleic acid analytes, which are provided with, or designed to hybridise to, circular RCA templates or circularisable RCA template molecules, e.g. proximity variants of FISH and smFISH assays in which the hybridisation probes comprise, in addition to a target-specific binding domain for hybridisation to a target molecule, a second domain, which does not hybridise to the target nucleic acid but which functions analogously to the nucleic acid domains described above, e.g. which contains a binding site for a circular or circularisable probe, which may, upon circularisation if necessary, be subjected to RCA. Such hybridisation proximity probes may be provided as hairpins which open upon binding of the probe to its target, releasing the second domains for interaction.

[0105] Similarly, in such a proximity assay, the nucleic acid domain of a proximity hybridisation probe may prime the RCA reaction, resulting in the RCP being attached, and localised to the target, via the binding domain of the bound proximity probe.

[0106] PLAs are described above. Proximity extension assays (PEAs) may generate an extended nucleic acid molecule wherein the nucleic acid domain of one proximity probe is extended using the nucleic acid domain of another proximity probe as extension template. The extended molecule may be detected by hybridisation of a circular or circularisable oligonucleotide which acts as an RCA template for an RCA reaction.

[0107] Alternatively, rather than generating an RCP as the nucleic acid product which is detected by the TSA reaction, other types of nucleic acid product may be generated. Such products may be the result of nucleic acid amplification, nucleic acid polymerisation or nucleic acid hybridisation reactions.

[0108] Typically, as discussed above, nucleic acid products are generated in detection assays as a means of signal amplification. Accordingly, a nucleic acid product generated in the methods herein may be characterized by comprising a detection sequence, at high concentration or high copy number, including in certain embodiments, at a spatially defined site or position. As indicated above, such a product may originate from localized amplification or polymerization reactions (such as RCA or HCR), or from the hybridization of multiple hybridization probes at proximal locations on the same nucleic acid molecule. Such a hybridization system may allow a branched nucleic acid structure to be built up, or assembled, for example akin to a RNAscope™ product as mentioned above. A template, or scaffold, molecule may be provided which comprises multiple binding sites for further hybridization probes. These may comprise detection sequences, or they may comprise binding sites for further hybridization probes etc. Thus, a “layered” or “branched” structure, or assembly, may be made up, or composed of multiple hybridization probes. The hybridization probes, or a subset thereof (e.g. the last or final hybridization probes added to the structure), may comprise a detection sequence. The nucleic acid product may in such an embodiment be referred to as a hybridization assembly.

[0109] It is known in the art that such hybridization assembly methods may employ proximity probes in the form of so-called Z-probes. A Z-probe is a hybridization probe comprising a first domain which provides a binding site for the target nucleic acid molecule (i.e. hybridizes to the target) and a second domain, which is not complementary to, and does not hybridise to the target, but comprises a binding site for a further molecule in the assembly process. For example, in the context of RNAscope™, Z-probes hybridise to the target nucleic acid molecule, and together provide a binding site for a pre-amplifier oligonucleotide. The pre-amplifier oligonucleotide comprises multiple binding sites for amplifier oligonucleotides, and the amplifier oligonucleotides comprises multiple binding sites for labelled detection oligonucleotides (i.e. multiple detection sequences). Thus, in an embodiment, the proximity probe may be, or may comprise a Z-probe. Thus, the nucleic acid domain of a proximity probe may be, or may comprise a Z-probe, or an analogous component, or they may each comprise a binding site for a Z-probe. More generally, the nucleic acid domains of a pair of proximity probes may together provide binding sites for components of a hybridization assembly reaction, such that the hybridization assembly may only be generated when both proximity probes have bound to their target analyte in proximity (directly or indirectly, i.e. as primary or secondary binding reagents for the target analyte).

[0110] As noted above, proximity assays using proximity probes which, when bound, in proximity to their target analyte, are able to initiate an HCR reaction, are known in the art, for example as described in WO 2015 / 118029. Accordingly, whilst in certain embodiments the nucleic acid products may be RCA or HCR products (RCPs or HCRPs) or hybridisation assemblies, they may be produced by any amplification reaction which can be employed or adapted to produce such a product, including for example, PCR, recombinase polymerase amplification (RPA), helicase-dependent amplification (HAD), LAMP, multiple strand displacement amplification (MDA) etc. Amplification methods such as MDA may be used to generate branched amplification products.

[0111] Whether produced by an amplification, polymerisation, or hybridisation reaction etc., the nucleic acid products can be seen as comprising multiple monomers, joined together, whether covalently or by hybridisation, wherein the monomers may each comprise a detection sequence which can be labelled by hybridisation of a labelled detection oligonucleotide. The monomer may be a repeat in an RCP or other amplification product, an HCR monomer in an HCRP, or a hybridisation probe in a hybridisation product (e.g. akin to an amplifier probe in an RNAscope reaction).

[0112] The nucleic acid product may have a linear or a branched structure. In the case of a linear product such as an RCP, as noted above, this may be coiled into a ball, or blob, a structure which is a discrete visualisable entity.

[0113] The nucleic acid product is typically a DNA molecule. However, it may also be composed of or may comprise other nucleic acids, natural or synthetic. Thus, it may for example be a chimeric construct comprising both RNA and DNA. The nucleic acid product may be made up of ribonucleotides and / or deoxyribonucleotides as well as synthetic nucleotides that are capable of participating in Watson-Crick type or analogous base pair interactions. Thus, the nucleic acid product may be or may comprise, e.g. bisulphite-converted DNA, LNA, PNA or any other derivative containing a non-nucleotide backbone.

[0114] It will be evident from the fore-going that the nucleic acid product generated in the method may be used to detect any target analyte in a sample. Alternatively put, the nucleic acid product is any nucleic acid product which can be generated in the course of a detection assay.

[0115] Since the nucleic acid product may be generated according to known assay methods, the performance of steps of the method leading to the generation or provision of the nucleic acid product will thus generally be according to methods and principles well known and understood in the art. Thus, a sample containing the analyte may be incubated with proximity probes, to allow the proximity probes to bind or interact with the analyte, e.g. to hybridise to a nucleic acid analyte, or for antibodybased or analogous probes to bind to the analyte. Conditions for such an incubation step are known in the art, and may be varied according to the sample, or analyte, or probes used, etc. This may include washing steps to remove unbound probes etc.

[0116] In the case of RCA-based methods where an RCP is generated, where necessary, the binding of proximity probes may be followed by a reaction to circularise a circularisable probe or added circularisable oligonucleotide(s), again according to well-known procedures. Ligation reactions for circularisation of such probes or oligonucleotides are also well known and described in the art, and a variety of different template-directed ligases may be used, including temperature sensitive and thermostable ligases, such as bacteriophage T4 DNA ligase, bacteriophage T7 ligase, E. coli ligase, Taq ligase, Tth ligase, Ampligase® and Pfu ligase. A suitable ligase and any reagents that are necessary and / or desirable may be combined with the sample / reaction mixture and maintained under conditions sufficient for ligation to occur. Ligation reaction conditions are well known in the art and may depend on the ligase enzyme used.

[0117] The next step following a ligation step (if required) is to generate the RCP. RCA is well known in the art, and procedures are widely described in the literature. The primer for the RCA will depend on the assay format, and may be provided by a probe or a part thereof, e.g. by the nucleic acid domain of a proximity probe, or it may be separately provided. An RCA primer will be of sufficient length, to provide for hybridization to the RCA template under annealing conditions.

[0118] In addition to the above components, the RCA reaction mixture includes a polymerase, e.g. phi29 polymerase, and other components required for a DNA polymerase reaction. The desired polymerase activity may be provided by one or more distinct polymerase enzymes. In some embodiments the polymerase has exonuclease activity, e.g. 5' and / or 3' exonuclease activity. 3’ exonuclease activity may be desirable, in order to digest the 3’ end of a probe or target molecule to generate a hybridised 3’ end (to the RCA template) which can act as a primer for the RCA reaction.

[0119] Procedures and conditions for HCR reactions are well-known in the art, as are procedures for hybridisation assembly reactions.

[0120] In performing the methods herein, including the steps of producing the RCP or other nucleic acid product, and performing the TSA reactions, various reagents are brought together or combined, and reaction mixtures are prepared. In all these steps, the various reagents and constituent components of a reaction mixture may be combined in any convenient order. For example, all of the various constituent components may be combined at the same time to produce the reaction mixture, and / or they may be added sequentially, according to the needs or steps of the method. Thus, the method may comprise various steps of contacting reagents with the sample, or with a reaction mixture.

[0121] The term “contacting” is used broadly herein to include bringing the reagents in question into contact. Thus, one may be added to the other and vice versa, or they may each be introduced to each other etc. This time or order of addition, or contact with the sample etc., may depend on the precise nature of the method, or method step, which is performed.

[0122] Regardless of the mechanism by which the nucleic acid product is generated, or more particularly by which an RCA template is provided or generated, the RCP or other nucleic acid product that is provided or generated in the method herein comprises multiple copies of a detection sequence which is indicative of the target analyte. In the case of an RCP, it comprises multiple repeat tandem copies of the detection sequence. The detection sequence is used in the initiation of the TSA reaction. In particular, the detection sequence provides a binding site for localisation or capture of the enzyme that performs the TSA reaction. Since the RCP is a concatemer of monomer repeats, multiple binding sites for the enzyme are provided. Analogously, other nucleic acid products also comprise multiple binding sites for the enzyme.

[0123] Thus, the method comprises hybridising a multiplicity of oligonucleotides to the nucleic acid product, or more particularly to the detection sequences present in the repeats / copies (monomer units) thereof, which allow an enzyme to be bound to the nucleic acid product. As used herein the term "multiple" or "multiplicity" means two or more, e.g. at least 2, 3, 4, 5, 6, 10, 20, 30, 50, 70 or 100 or more. It will be understood that whilst each of the repeat units / copies or monomers of the nucleic acid product comprise the binding site for the enzyme, in practice not all of these binding sites may (or will) be occupied by an enzyme. It suffices that a number, or multiplicity, of such binding sites are bound by an enzyme. Thus, in the method the enzyme is bound to a detection sequence in at least one monomer of the nucleic acid product, but preferably to multiple detection sequences.

[0124] To localise the TSA enzyme to the detection sequences in the nucleic acid product, an enzyme capture oligonucleotide may be used, which targets the enzyme to the detection sequences in the nucleic acid product. The enzyme-capture oligonucleotide thus comprises a sequence, or binding site, which is complementary to the detection sequence, or a part thereof, i.e. it is capable of hybridising to the detection sequence. The capture oligonucleotide may be attached, directly or indirectly, to the enzyme. This attachment may be reversible, or in other words, the enzyme may be removably attached to the capture oligonucleotide.

[0125] In an embodiment, the capture oligonucleotide may be directly conjugated to the enzyme, as is done in some TSA methods. Methods and reagents for coupling oligonucleotides to enzymes or other moieties are well known in the art and any convenient method may be used. However, in other embodiments the enzyme is attached in a manner that allows its ready removal, or for it to be attached in sequential steps. Advantageously, this may allow the method to be cycled. Thus, in such an embodiment the enzyme may be attached to the capture oligonucleotide by means of a binding pair, that is a pair of moieties that bind to each other, wherein one of the pair is attached (i.e. linked or conjugated) to the capture oligonucleotide, and the other is attached to the enzyme.

[0126] Accordingly, in an embodiment, the enzyme-capture oligonucleotide comprises a first capture moiety which is cognate to, i.e. capable of binding specifically to, a second capture moiety comprised within an enzyme agent. The enzyme agent comprises the second capture moiety linked to the enzyme. This allows the enzyme to be bound, or captured to, the nucleic acid product in two steps. Thus, in a first step, the nucleic acid product, or more particularly the sample or reaction mixture comprising the nucleic acid product, is contacted with an enzymecapture oligonucleotide comprising a binding site that hybridises to the detection sequence and a first capture moiety, and in a second step the nucleic acid product with the hybridised enzyme-capture oligonucleotide is contacted with an enzyme agent comprising the enzyme and the second capture moiety. The cognate first and second capture moieties are allowed to bind, to attach the enzyme to the RCP. Such a scheme is depicted in Figure 1. These two steps may be performed sequentially, or simultaneously. Thus, in the case of the latter, the enzyme-capture oligonucleotide and the enzyme agent may both be added together to the sample / reaction mixture containing the nucleic acid product.

[0127] The first and second capture moieties may be members of any affinity binding pair it is desired or convenient to use. Conveniently, they may be biotin and a biotinbinding protein such as avidin or streptavidin (which terms include biotin-binding derivatives or mutants of native or wild-type avidin or streptavidin). Conveniently, biotin may be the first capture moiety and avidin / streptavidin the second capture moiety. Alternatively, they may be hapten / hapten-binder, e.g. antibody. Haptens are well known and available in the art and include for example DNP (2,4-dinitrophenol) and digoxigenin. The term antibody as used herein includes antibody fragments, and any format of antibody, including single chain antibodies and such like (in other words it may be any binding protein comprising an antigen-binding site obtained or derived from an antibody). In another embodiment, the first and second capture moieties may be first and second oligonucleotides comprising complementary binding sites. As indicated above, it is well known in the art that there are a variety of ways and means by which a capture moiety may be attached, or coupled, to an oligonucleotide, and any known coupling chemistry may be used.

[0128] In a particular embodiment, the enzyme-capture oligonucleotide comprises biotin. The enzyme-capture oligonucleotide is allowed to hybridise to its complementary binding sites in the nucleic acid product (i.e. to the detection sequences). Subsequently, the nucleic acid product with the hybridised enzymecapture oligonucleotide is contacted with an enzyme agent comprising the enzyme linked to streptavidin, thereby attaching the enzyme to the nucleic acid product.

[0129] In the subsequent steps of the TSA reaction, the captured enzyme (i.e. the nucleic acid product with attached enzyme) is brought into contact with a substrate for the enzyme. The enzyme catalyses a reaction which deposits a substrate reaction product in the vicinity of the enzyme. Since the enzyme is attached to the nucleic acid product, the substrate reaction product is deposited in the vicinity of the nucleic acid product, allowing the localised detection of the nucleic acid product. Conveniently, the substrate is labelled to allow its detection. The labelling may be direct or indirect. Thus, the substrate may be directly conjugated with, or attached to, a label (as is depicted in Figure 1 for example), or it may be attached to a moiety which allows it to be attached to a label, e.g. subsequently attached to the label. Thus, the labelling may take place directly during the substrate deposition reaction, or subsequently, after the substrate has been deposited. In other words, the substrate or the substrate reaction product may be labelled. Thus, the captured enzyme is contacted with a TSA reagent which comprises the substrate.

[0130] Accordingly, in the TSA reaction, the hybridised nucleic acid product-capture oligonucleotide-enzyme is contacted with a signal localisation agent comprising (i) a substrate for the enzyme and (ii) either a label or a first binding moiety for binding to a labelling agent. Where the signal localisation agent comprises a label, the label is deposited along with the substrate reaction product. As noted above, the signal localisation agent may alternatively be referred to as a TSA reagent. Where the signal localisation agent comprises a first binding moiety, the reaction mixture comprising the deposited substrate reaction product is contacted with a labelling agent comprising a label linked to a second binding moiety cognate to the first binding moiety, and the first and second binding moieties are allowed to bind. In this manner, the labelling agent, and hence the label, is attached to the substrate reaction product. The label is then detected, to detect the nucleic acid product, and hence the target analyte. The use of signal localisation and labelling agents comprising cognate binding moieties allows for the label to be removed, for example in the context of a sequential method, performed in cycles, as discussed below.

[0131] The first and second binding moieties may be members of an affinity binding pair as described above for the first and second capture moieties.

[0132] As noted above, the TSA reaction and various detection methods using TSA are known in the art and, generally speaking, known methods and reagents may be used to perform the various steps of the TSA reaction as set out above. TSA protocols and reagents have been commercialised. Reference may be made in this regard to the ThermoFisher website pages relating to TSA. Further, Akoya Biosciences, Inc. have developed the TSA method for use in connection with spatial biology, and the localised detection of analytes in cells. Reference may be made in this regard, for example, to US 2021 / 0222234 of Akoya Biosciences, Inc., which describes multiplexed imaging assays based on TSA, in particular using enzyme- oligonucleotide and substrate-oligonucleotide conjugates. The TSA reagents are used in conjunction with an analyte targeting binding agent-oligonucleotide conjugate. US 2021 / 0222234 of Akoya Biosciences, Inc. describes barcoded antibodies for use with their TSA method. WO 2020 / 163397 of Akoya Biosciences, Inc. describes the detection of biological samples using antibodies conjugated to barcode oligonucleotides. A readout molecule (such as an enzyme, more specifically horseradish peroxidase) is conjugated to an oligonucleotide which has a complementary sequence to a barcode oligonucleotide. As such, the readout moiety and detection moiety hybridise, the readout of which can be detected (for example, by utilising TSA). The barcode oligonucleotide may thus be seen as a capture oligonucleotide, which operates in a manner akin to the capture oligonucleotides, enzyme agents, and capture moieties etc, as described herein. Reference may also be made to WO 2021 / 226516 of Leland Stanford Junior University. The disclosures of all references mentioned herein are incorporated by reference.

[0133] Thus, the TSA reaction may be performed using reagents, known and available in the art, notably known TSA enzymes and substrates, which can be used or adapted for use in accordance with the methods herein, e.g. used to prepare enzyme-capture oligonucleotides, enzyme agents, TSA reagents or signal localisation agents etc. This incudes for example reagents available commercially from, amongst others, Akoya Biosciences, Inc. or ThermoFisher. For example, the enzyme is typically a peroxidase, which term refers broadly to any enzyme having peroxidase activity, e.g. soybean peroxidase or, more commonly, horseradish peroxidase (HRP). The peroxidase acts in the presence of hydrogen peroxide to catalyse the deposition of the substrate, i.e. to convert the substrate into a reactive species, or reactive reaction product, which is able to react with molecules in its vicinity, and thereby bind to, or to become localised to, those molecules, and hence to the vicinity of enzyme and / or proteins in the vicinity of the enzyme. These may include the analyte, or an antibody or other binding molecule bound directly or indirectly to the analytes.

[0134] As noted above, other enzymes can be used in place of peroxidase to catalyse an analogous TSA reaction. For example, the enzyme can be a hemincontaining complex which can mimic HRP, for example haematin.

[0135] The substrate can be any substrate that may be converted by a peroxidase or analogous enzyme to a reactive product capable of being deposited. In other words, the reactive product is capable of reacting with molecules in its vicinity, most notably with proteins, and in particular with tyrosine residues in proteins. In an embodiment, the substrate is a tyramide compound, that is a derivative of tyramine, a compound that contains a tyramine or tyramide derivative, p-hydroxy-cinnamic acid, or a derivative of p-hydroxy cinnamic acid.

[0136] The label may be any label known or used in the art in detection assays. In an embodiment it is an optical label. This includes any optical label that may be detected directly or indirectly. In other words, the label may directly or indirectly signal-giving. By indirectly signal-giving it is typically meant that the label undergoes a process or conversion before it can be detected, for example it is enzymatically converted, or converted by a chemical reaction to a product that may be detected.

[0137] A wide variety of labels are known in the art, any of which may be used. For example, the label may be a fluorescent label, a coloured label, a chromogenic label, a quantum dot, a mass-tag label, or a particle. Conveniently, the label is dye or stain. It is convenient to use a label that may be visualised, and particularly which may be detected microscopically, and more particularly by imaging.

[0138] Very many different fluorescent or coloured labels are known in the art and are commercially available. This includes traditional well-known labels such as C3, Cy5, Cy7, FITC, FAM and TAMRA etc. Further, the Opal dyes available from Akoya Biosciences may be used. This includes Opal fluorophores developed or commercialised for in TSA detection protocols. Suitable labels are also available from ThermoFisher, e.g. Alexa Fluor dyes. The detection step will depend on the label which is used and also on the sample, and the format off the detection method, but generally any convenient or desired detection modality may be used (and the label may be selected in accordance with that).

[0139] The method may be carried out in heterogenous or homogenous formats. That is, it may be performed on a solid phase (or support), or in solution or suspension (i.e. without a solid phase or support), or indeed both, since a solid phase may be introduced at a later stage.

[0140] The format of the method may be selected based on the nature of the sample, or the target nucleic acid molecule, or the desired readout or detection technology used.

[0141] As indicated above, conveniently, the label is detected microscopically, and particularly by imaging. Techniques and instruments for such detection, e.g. fluorescent microscopes and image analysis software etc are well known in the art. Similarly, techniques for detecting coloured labels, e.g. by bright field microscopy are also well known in the art.

[0142] Other detection methods may also be used, for example flow cytometry. The use of flow cytometry to detect fluorescently labelled products in detection assays is well known, for example.

[0143] More generally, the labelled TSA products may be detected using any of the well-established methods for analysis of target analytes known from the literature which use detection of fluorescence, including for example microscopy, and imaging techniques.

[0144] Depending on the level of multiplexing, combinatorial labelling methods may be used, according to techniques well known in the art. For example, ratio labelling may be performed with different fluorescently labelled detection oligonucleotides.

[0145] Although various detection modalities may be employed, conveniently the labelled nucleic acid molecules may be detected by microscopy or flow cytometry. In particular, in a microscopy-based method, the labelled products may be detected by imaging.

[0146] The use of such detection techniques advantageously allows the nucleic acid molecules to be digitally recorded. Indeed, since the degree of signal amplification afforded by the methods herein allows them to be visualised, and they may be detected by a camera or any device including a camera, such as a mobile phone.

[0147] To detect products generated in a homogenous format, they may be captured or brought down to a solid support, or surface, to facilitate imaging, or microscopic detection more generally. The methods herein are particularly suited to multiplex analyses. Whilst this includes detecting multiple analytes in the sample simultaneously, this is limited both by the number of proximity probe pairs that can be employed at any one time, and by the number of different labels that may be detected and distinguished from one another. This means that often multiplexing is achieved by performing the method sequentially to detect different analytes in different reactions. Thus, the methods may be serially, in cycles, on the same sample, to detect a different analyte, or a different sub-set of analytes in different cycles.

[0148] Protocols for performing multiplexed TSA detection assay in such a manner are known and described in the art, for example in US 2021 / 0222234 (Akoya Biosciences, Inc.) or WO 2021 / 226516 (Leland Stanford Junior University), and such protocols may be followed, or adapted for use in the methods herein. Such methods typically involve removing enzymes, and / or probes, between cycles. Labelling of the TSA product may take place in individual cycles, or the labelling steps may be performed together. Detection may take place in each cycle, or it may take place in one step, after all the cycles have been performed, and all TSA products have been labelled. Different permutations are available, and will depend on the label, detection modalities, and how the label is attached to the TSA substrate or TSA reaction product etc., as well as the degree of multiplexing. For example, in a microscopic method, images may be taken in each cycle, and may then be combined or overlaid, or otherwise merged or consolidated. Alternatively, a single imaging step may be performed at the end of all the cycles.

[0149] Typically, in a multiplexing method, at least the enzyme is removed between cycles. In an embodiment the proximity probes are also removed. The term “removed” as used herein includes any means of physically removing, or inactivating the enzyme so that it cannot participate in any subsequent reaction, and new enzyme needs to be added for the next cycle (to be localised to a different nucleic acid product, representative of a different target analyte). Where the proximity probes are concerned, particularly proximity probes comprising an antibody-based binding domain, it is advantageous to remove the proximity probes between cycles.

[0150] Antigen retrieval processes which are known in the art may be used for this purpose, which may remove both the proximity probes, and the enzyme. These may use heat and / or enzymatic or chemical processes for the removal, for example HIER (heat-induced antigen (epitope) retrieval). HIER methods involve heating to high temperatures, e.g. to at least 60, 70, 80, 85, 90 or 95 °C for a period of time, e.g. for at least 10, 15 or 20 minutes, or longer. Generally, lower temperatures require longer incubations, for example, 60 °C overnight. HIER may include the use of steamers, microwave ovens, pressure cookers and hot water baths etc. Suitable solutions, e.g. buffers for use in antigen retrieval, are known in the art. Enzymatic methods include protease-induced epitope retrieval (PIER), using for example, pepsin, trypsin, or proteinase K.

[0151] In such processes, removal of the enzyme, and optionally also the proximity probes, does not necessarily remove the label from the TSA product. For example, where the enzyme substrate is directly conjugate to a label, i.e. where the signal localisation agent comprises a label, the label remains following removal of the enzyme, or enzyme and proximity probes. However, in other protocols and variants of the method, for example such as those described by Akoya Biosciences, the labels can be removed. This can readily be achieved for example in embodiments where signal localisation agents and labelling agents are used, which hybridise to one another via complementary oligonucleotides (which are used as the cognate first and second binding moieties). Further, in other embodiments, labels can be removed by bleaching protocols, e.g. by light bleaching as is known in the art.

[0152] In a representative example of a multiplexed method, in the first cycle the steps are performed as described above, with a first pair, or a first set of two or more pairs, of proximity probes to detect a selected (first) target analyte or first sub-set of target analytes, and the TSA product(s) is / are labelled with a first label or first set of labels. Following the labelling step, the enzyme, whether attached directly to the capture oligonucleotide or via a separate enzyme agent which binds to the capture oligonucleotide, is removed, for example by HIER. If HIER is used, the proximity probes are also removed from the sample. The next cycle is then performed, using a second pair, or a second set of proximity probes to detect a second target analyte or second sub-set of target analytes. In this second cycle, different labels are used to label the TSA product(s), which may be distinguished from the first label / set of labels. Further cycles may be performed in an analogous manner, with a removal step in between cycles. The detection of the labelled TSA products may take place in a single step at the end. In such a situation, the number of cycles will be limited by the number of available labels that can be distinguished. In other embodiments, the labelled products may be detected at the end of each cycle, as described above.

[0153] The term "hybridisation" or "hybridises" as used herein refers to the formation of a duplex between nucleotide sequences which are sufficiently complementary to form duplexes via Watson-Crick base pairing, or any analogous base-pair interactions. Two nucleotide sequences are "complementary" to one another when those molecules share base pair organization homology. Hence, a region of complementarity in a molecule or probe or sequence refers to a portion of that molecule or probe or sequence that is capable of forming a duplex. Hybridisation does not require 100% complementarity between the sequences, and hence regions of complementarity to one another do not require the sequences to be fully complementary, although this is not excluded. Thus, the regions of complementarity may contain one or more mismatches. Accordingly, "complementary", as used herein, means "functionally complementary", i.e. a level of complementarity sufficient to mediate a productive hybridisation, which encompasses degrees of complementarity less than 100%. The degree of mismatch tolerated can be controlled by suitable adjustment of the hybridisation conditions. Those skilled in the art of nucleic acid technology can determine duplex stability empirically considering a number of variables including, for example, the length and base pair composition of the respective molecules or probe oligonucleotides, ionic strength, and incidence of mismatched base pairs, following the guidance provided by the art. Thus, the design of appropriate probes and oligonucleotides etc., for example for ligation or hybridization, or primers etc. for any of the reaction steps describe herein, and binding regions thereof, and the conditions under which they hybridise to their respective targets is well within the routine skill of the person skilled in the art.

[0154] A region of complementarity, such as for example between a nucleic acid domain of a proximity probe and the binding region of a padlock probe, or between a detection sequence and an enzyme capture oligonucleotide etc., may be at least 6 nucleotides long, to ensure specificity of binding, or more particularly at least 7, 8, 9 or 10 nucleotides long. The upper limit of length of the region is not critical, but may for example be up to 50, 40, 35, 30, 25, 20 or 15 nucleotides. A complementary region may thus have a length in a range between any one of the lower length limits and upper length limits set out above. In the case of a padlock probe, the length of an individual target-binding region may be in the lower ranges, so that the total length of the two binding regions when hybridised to their target is within the upper ranges. For example, an individual target binding region may be 8-15, e.g. 10-12 nucleotides, so that the total hybridised length is 16-30 nucleotides long, e.g. 20-24. It may be desirable, within the constraints of conformation of the probes, and spacing of the domains, and desired or favoured hybridisations, to minimise the total length of a padlock probe to minimise the size of the circle which is subjected to RCA, and hence to minimise the lengths of the complementary regions where possible.

[0155] As indicated above, the methods herein allow detection assays to be performed on all types of samples for all types of analyte, robustly and with enhanced sensitivity and high specificity. The methods facilitate multiplexing of proximity assays, and in particular the PLA-RCA assays described above. As noted above, the very high signal amplification afforded by combining nucleic amplification methods such as RCA with TSA circumvents background issues and allows for signal capture in large tissue sections without high magnification. Both low and high abundance signals may be detected accurately and robustly, including in the same sample (i.e. in a multiplex assay). Thus, the present methods allow the utility of various existing assays to be extended to any and all types of sample and / or target analytes. Particularly, the performance of in situ assays, and in particular isPLA may be improved.

[0156] In certain embodiments, the methods may further comprise counterstaining the sample to aid visualisation, before the step of detecting the TSA product, or detecting the label. This includes nuclear or cytoplasmic counterstaining. For example, the nuclear stain DAPI may be used. Other suitable stains are known in the art, and may be used according to choice.

[0157] As noted above, the methods herein may be used in combination with other detection assays, including commercially available assays, and may be integrated in a workflow with one or more other detection assays. Such other assays may be assays to detect other analytes in the sample. This may allow a particular target analyte, for example an interaction (e.g. a protein-protein interaction (PPI), such as PD-1 / PD-L1), to be assessed in the context of its local environment, for example the tissue environment, or tumour microenvironment (TME). Both cellular and functional data can be integrated in this way, for example, the proximity-TSA method revealing functional information, e.g. an interaction, for example between members of a signalling pathway or receptor-ligand interaction etc., or protein modification etc., with other assays providing other information, for example the presence of certain markers (e.g. cellular or tumour markers, immune markers etc.), expression of proteins and RNA etc. Accordingly, in combination with other detection assays, the present method can provide a powerful tool for characterising a particular tissue or environment in relation to a particular interaction etc. By way of example, the methods herein may be used for an in situ analysis of the PD-1 / PD-L1 interaction, or other checkpoint interactions, in the TME. In this way the understanding of immune responses may be deepened, and identification of spatial signatures improved, e.g. for improved patient stratification.

[0158] Such other detection assays thus include any assays for detecting an analyte in a sample, for example immunoassays, including immunohistochemical (IHC) procedures, or any other detection assay, e.g. immunofluorescence staining assays to detect one or more protein analytes present in the sample. The one or more other detection assays may be performed before or after the present method. Conveniently, the same sample preparation steps may prepare the sample for both the present method and the other assay(s), e.g. antigen retrieval, dewaxing, fixing etc. For example, once the present method has been completed up to generating the TSA product, or up to detecting the TSA product, further steps may be performed. For example, directly or indirectly labelled antibodies or other binding partners for one or more target analytes may be added to the sample, together in combination or sequentially.

[0159] The subsequent detection assay may be a multiplex assay, which may be performed simultaneously or sequentially.

[0160] The other detection assay may employ TSA-labelling technology to detect the antibody. Different labels, e.g. different Opal dyes, may be used to detect different analytes.

[0161] Accordingly, in an embodiment, the method provided herein may further comprise the following steps:

[0162] (v) contacting the reaction mixture with a detection antibody directed against a target analyte, wherein the detection antibody is conjugated to an oligonucleotide comprising a capture sequence (or “barcode”);

[0163] (vi) detecting said detection antibody of step (v) by:

[0164] (i) hybridising to the capture sequence (or barcode) an enzymecapture oligonucleotide, wherein the enzyme-capture oligonucleotide comprises a binding site complementary to the capture sequence (or barcode), and wherein the enzyme-capture oligonucleotide is attached to an enzyme;

[0165] (ii) contacting the reaction mixture from (i) (i.e. the hybridised detection antibody-capture oligonucleotide-enzyme) with a signal localisation agent comprising a substrate for the enzyme, wherein the enzyme catalyses a reaction which deposits a substrate reaction product in the vicinity of the enzyme, and hence of the unique oligonucleotide barcode, wherein the localisation agent comprises either a label, (and the label is deposited along with the substrate reaction product) or a first binding moiety for binding to a labelling agent;

[0166] (iii) when the localisation agent does not comprise a label, contacting the reaction mixture of (ii) with the labelling agent, wherein the labelling agent comprises a label linked to a second binding moiety cognate to the first binding moiety, and allowing the second binding moiety to bind to the first binding moiety (thereby to attach the labelling agent to the substrate reaction product);

[0167] (iv) detecting the label; and optionally

[0168] (vii) repeating steps (v) and (vi) with a further detection antibody for another target analyte, wherein step (vii) may be repeated one or more times.

[0169] The method will now be described in more detail with reference to the Figures and non-limiting examples.

[0170] Examples

[0171] Example 1 Demonstration of use of PLA-TSA to detect phosphorylation of PDGFR and HER2

[0172] In our comparative analysis, we sought to evaluate the efficiency of the traditional Proximity Ligation Assay (PLA) against its enhanced counterpart, PLA-TSA. To undertake this assessment, two cellular models were chosen: the BJ hTERT cells, highlighting PDGFR phosphorylation, and BT474 cells, emphasizing the HER2 expression. The rationale behind studying PDGFR phosphorylation in BJ hTERT cells is its notorious difficulty for detection, attributed to the lack of reliable antibodies and typically weak signal intensity. The BT474 cells, renowned for their HER2 expression, present a valuable model, especially when considering the crucial role of HER2 in oncological research, particularly in breast cancer pathophysiology. Accurate and sensitive detection of these molecular targets, especially in difficult studies involving human tissues like cancer, is vital. Unreliable antibodies can compromise data integrity, skewing interpretations and potentially leading to errant conclusions. Hence, our choice of these models was strategic, aiming to underscore the common challenges in molecular detection and the necessity for precise methodologies.

[0173] Two primary antibodies targeting PDGFR and pan-tyrosine were applied to the prepared cells. Following this, proximity probes specific to these antibodies were added. After the proximity probes were bound, the ligation and RCA amplification steps of the PLA were conducted.

[0174] Non-TSA Sample Detection: For the non-TSA sample, after the amplification step, an oligonucleotide labeled with Alexa 594 was used for detection. TSA Sample Detection: For the TSA-enhanced sample, post-amplification, the rolling circle amplification (RCA) product was detected using an oligonucleotide labelled with biotin. Subsequently, the sample was incubated with streptavidin conjugated with horseradish peroxidase (HRP). After this incubation, TSA conjugated with Alexa 594 was added to visualize the amplified signals.

[0175] BT474 cells were stained to detect the presence of HER2 using both standard PLA and TSA-enhanced PLA (as described above). For this set of cells, the fluorophore was changed to Alexa 647 for detection.

[0176] The results are shown in Figure 2.

[0177] The amplification process for the Alexa 647 fluorophore was observed to have comparable efficiency to the previously used Alexa 594 fluorophore in the PLA-TSA experiment. The detected signals showcased a marked difference between the conventional PLA and the enhanced PLA-TSA techniques. Notably, the intensity of the PLA-TSA method was augmented several-fold for both the PDGFR and pantyrosine antibody pairs, irrespective of the Alexa dyes used. This enhancement in the PLA-TSA approach highlights its heightened sensitivity and precision, making it especially valuable for probing interactions that have historically been elusive due to limitations in antibody efficacy and inherently weak signals.

[0178] Our findings highlighted the superior efficiency of PLA-TSA over conventional PLA. Notably, the clarity and robustness of signal detection were markedly enhanced with PLA-TSA. An ancillary observation was the consistency in signal amplification between two different Alexa dyes - Alexa 594 and Alexa 647, indicating the versatility and reproducibility of the PLA-TSA method. Collectively, our data suggest that the PLA-TSA methodology, with its demonstrable superiority in molecular detection, holds significant promise for translational applications, particularly in intricate histopathological assessments central to oncological research.

[0179] Example 2

[0180] A multiplex TSA-based immunofluorescent staining of a non-small cell lung cancer (NSCLC) tissue core The primary objective of this staining was to simultaneously visualize multiple markers - DAPI, PD1-PDL1 (PLA), panCK, pSTAT3, pSMAD2, and CD8. Among these, panCK, pSTAT3, pSMAD2, and CD8 were detected using a combination of immunofluorescence and Tyramide Signal Amplification (IF-TSA), with each marker differentiated using unique dyes. Furthermore, for a more distinct visualization of PD1-PDL1 interactions, the Proximity Ligation Assay (PLA) was integrated with TSA.

[0181] Formalin-fixed, paraffin-embedded (FFPE) non-small cell lung cancer (NSCLC) tissue cores were sectioned and placed on slides. These were then deparaffinized in xylene and rehydrated through a graded alcohol series. For antigen unmasking, sections were subjected to heat-induced epitope retrieval (HI ER) using a citric buffer. Tissue sections were blocked in 5% BSA in PBS to minimize non-specific binding.

[0182] The NSCLC tissue samples were first subjected to Proximity Ligation Assay (PLA) to detect the interactions between PD1 and PDL1. Following the primary antibody binding specific for these interactions, tissues underwent PLA and TSA. Post washing, secondary antibodies conjugated to unique oligonucleotides were introduced. After the probes were bound, the ligation and amplification steps were conducted. This RCA product was detected using complementary oligonucleotides labeled with biotin. Streptavidin conjugated with horseradish peroxidase (HRP) was added next, binding the HRP enzyme to the amplified DNA. The tissue was treated with a tyramide substrate conjugated to a fluorophore. Immediately after the PLA- TSA procedure, the tissue underwent treatment to deactivate any residual horseradish peroxidase (HRP) activity. This ensured that no cross-reactivity or overlap occurred in the subsequent IF-TSA steps. Tissue samples were next incubated with primary antibodies against panCK. Upon binding, they were subjected to TSA, enhancing the visual detection of panCK. Following the panCK staining, another round of HRP deactivation was performed to prepare the tissue for subsequent markers. The same cycle of primary antibody incubation, TSA visualization, and HRP deactivation was then repeated sequentially for the other markers: pSTAT3, pSMAD2, and CD8. After staining, tissue samples were examined under a fluorescence microscope. The results are shown in Figure 3. Images captured included a scale bar reference of 20 pm for size context.

[0183] In our examination of non-small cell lung cancer (NSCLC) tissues, the importance of the PD1-PDL1 interaction became strikingly evident. Utilizing the advanced sensitivity of the PLA-TSA method, we successfully detected the PD1-PDL1 interaction. When observed in the context of other significant proteins, namely panCK, pSTAT3, pSMAD2, and CD8, this interaction delineated clear spatial patterns integral to cellular dynamics. The spatial profiling emphasized the prominence of PD1-PDL1 within the cellular matrix, highlighting its role as a primary immune checkpoint, a mechanism often commandeered by malignancies for immune evasion. Moreover, the spatial relationships between PD1-PDL1 and other markers shed light on the intricate interplay of these proteins within the NSCLC tissue matrix. This profiling not only validated the relevance of the PD1-PDL1 interaction in the NSCLC context but also underscored the significance of understanding its spatial distribution in relation to other key signalling proteins, thereby offering a comprehensive perspective on cellular behaviour in NSCLC.

[0184] The pursuit of understanding cellular markers and their co-localizations, as depicted here with panCK, pSTAT3, pSMAD2, and CD8, is pivotal for unravelling the complexities of cellular mechanisms. This co-localization elucidates the spatial proximity and potential functional interactions between these molecular entities, thereby shedding light on their roles within the cellular milieu. To ensure clarity in these visualizations, signal amplification techniques, such as TSA, are indispensable. By enhancing inherently weak fluorescent signals or emphasizing markers in low abundance, TSA ensures a comprehensive representation of cellular interactions, leaving no subtle interplays unnoticed. Of the markers visualized, the interaction between PDL1 and PD1 stands out for its critical role in immune modulation, especially its exploitation by malignancies to subvert host immune surveillance.

[0185] When juxtaposed with other markers visualized through the TSA system, understanding the PD1-PDL1 dynamics becomes even more crucial. By doing so, researchers can postulate how signaling pathways, typified by markers like panCK, pSTAT3, pSMAD2, and CD8, potentially modulate or interact with immune processes. Unveiling these intricate interactions not only augments our cellular and molecular comprehension but also has significant implications for devising innovative therapeutic interventions.

[0186] Example 3 PDGFRB-GRB2 PLA combined with several markers including CD31, DAPI, Active YAP, FAP and panCK

[0187] To investigate associations between the PDGFRP-GRB2 interaction and panCK, YAP, FAP and CD31, a multiplex immunofluorescent (IF-TSA) panel was employed. Using the Bond RXm Autostainer for the multiplex process, we initiated deparaffinization and antigen retrieval using a pH9 buffer. Subsequent cycles only required antigen retrieval with a pH6 buffer. Post-retrieval, slides were blocked and washed. Primary antibodies, differing in diluents, were applied and incubated at RT. After washing, secondary antibodies were added based on the primary antibody's origin and incubated. Fluorophores were then added after another wash. Following staining cycles, another antigen retrieval was done, succeeded by multiple washes. The process then shifted from the autostainer to manual execution for the PLA protocol. For multiplex-stained tissue PLA detection of PDGFRP-GRB2, we began with a 10-minute endogenous enzyme block at room temperature and washed with TBS-T. Next, blocking solution was prepared and applied to tissues for 60 minutes at 37°C. Primary antibodies (PDGFRp and GRB2) were diluted in an antibody diluent and incubated on tissue slides overnight at 4°C. Probes anti-M (anti-mouse) and anti- R (anti-rabbit) were prepared, applied, and incubated for 60 minutes at 37°C. Ligation and amplification were sequentially prepared and applied for 30 and 60 minutes respectively at 37°C. HRP solution was prepared and applied for 30 minutes at RT. Slides were washed and treated with a Fluorescent Opal solution. Nuclear staining utilized DAPI, with a brief incubation in the dark. After washing, the slides were mounted.

[0188] The results are shown in Figure 4.

[0189] Elevated stromal platelet-derived growth factor receptor beta (PDGFRP) expression is a harbinger of poor prognosis in various solid tumors. Its role in NSCLC, however, remains ambiguous. Recognizing that mere PDGFRp expression does not equate to its active signaling, understanding its activation state might offer deeper insights. In this study, we employed the advanced PLA-TSA in situ technique to pinpoint activated PDGFRp, deciphering its clinical relevance and prognostic nuances in NSCLC.

[0190] We leveraged the unfold proximity ligation assay (PLA) to target the PDGFRP-GRB2 interaction, a hallmark of active receptor signaling. Notably, our adapted method integrates the precision of tyramide signal amplification, enabling simultaneous staining of pan-cytokeratin (panCK), YAP, FAP, and CD31 - each key to understanding the nuanced tumor microenvironment. Analysis of NSCLC patient- derived tissue microarrays revealed our PLA technique's prowess in discerning PDGFRp activation in diagnostic FFPE samples. Intriguingly, adenocarcinomas exhibited pronounced stromal PDGFRp activation, an ominous sign indicative of decreased survival rates. Our discoveries underscore the superior prognostic utility of assessing PDGFRp activation over mere protein expression levels in NSCLC. Furthermore, they spotlight the potential therapeutic avenues targeting activated PDGFRp signaling in NSCLC, underscoring the urgency for in-depth exploration. These results again underline the utility, and the advantages, of the PLA-TSA technique.

[0191] Example 4

[0192] Exemplary protocol for in situ proximity ligation assay (PLA)-TSA assay using Naveni® PLA reagents, including Naveni® PLA PD-1 / PD-L1

[0193] The Naveni® PLA protocols use primary antibodies to bind to the target analyte and secondary antibodies as the proximity probe pair. The secondary antibodies, which are coupled to the nucleic acid domains, bind to their respective primary antibodies and are termed Navenibodies.

[0194] 1. Sample Preparation

[0195] 1.1. Perform antigen retrieval if required, followed by incubation in horseradish peroxidase blocking solution to quench endogenous enzymes. Use a volume that covers each sample, and follow the manufacturer's recommended incubation times.

[0196] 1.2. Wash slides twice for 5 minutes each in 1x TBS-T.

[0197] 2. Blocking

[0198] 2.1. Prepare the blocking solution by adding 5 pL of Supplement 1 to every 40 pL of Blocking Buffer.

[0199] 2.2. Apply this solution to cover the sample area, using approximately 40 pL for each 1 cm2.

[0200] 2.3. Incubate for 60 minutes at 37°C in a preheated humidity chamber.

[0201] 3. Primary Antibody Incubation

[0202] 3.1. Prepare the primary antibody solution by adding 5 pL of Supplement 2 to every 40 pL of Primary Antibody Diluent.

[0203] 3.2. Apply the antibody solution to cover the sample area.

[0204] 3.3. Incubate for 60 minutes at 37°C or overnight at 4°C in a humidity chamber.

[0205] 3.4. Wash the slides three times for 5 minutes each in 1x TBS-T. 4. Navenibody Incubation

[0206] 4.1. Dilute Navenibody M1 and Navenibody R2 1 :40 in Navenibody Diluent.

[0207] 4.2. Apply sufficient volume of the Navenibody working solution to cover the sample area.

[0208] 4.3. Incubate for 60 minutes at 37°C in a preheated humidity chamber.

[0209] 4.4. Wash the slides in pre-warmed 1x TBS-T for 10 seconds twice and 15 minutes once.

[0210] 5. Enzyme Reaction (to generate the PLA nucleic acid product, namely the RCP)

[0211] 5.1. For Reaction 1, dilute Buffer 1 1 :5 in distilled water and add Enzyme 1 at a 1:40 ratio. Mix gently.

[0212] 5.2. Apply enough Reaction 1 to cover the sample area.

[0213] 5.3. Incubate for 30 minutes at 37°C in a preheated humidity chamber.

[0214] 5.4. Wash slides twice for 3 minutes each in 1x TBS-T.

[0215] 5.5. For Reaction 2, repeat the process with Buffer 2 and Enzyme 2, following the same dilution and application guidelines. Incubate for 90 minutes.

[0216] 6. HRP Incubation

[0217] 6.1. Dilute the HRP reagent 1 :800 in HRP Diluent.

[0218] 6.2. Apply the HRP solution, covering the sample area.

[0219] 6.3. Incubate for 30 minutes at room temperature with gentle agitation.

[0220] 7. Substrate Development (TSA)

[0221] 7.1. Prepare the TSA substrate solution immediately before use.

[0222] 7.2. Apply the TSA substrate solution to the sample area.

[0223] 7.3. Incubate at room temperature for 10 minutes, adjusting the time based on target abundance and desired signal intensity.

[0224] 7.4. Wash the slides twice in TBS and once in deionized water to stop the reaction.

[0225] 8. Nuclear Staining

[0226] 8.1. Apply a nuclear counterstain if desired, such as DAPI for fluorescence microscopy.

[0227] 8.2. Rinse the slides under running tap water.

[0228] 9. Dehydration and Mounting

[0229] 9.1. Dehydrate the slides through a graded alcohol series and clear in xylene. 9.2. Mount with VectaMount® Express Mounting Medium.

[0230] 9.3. Apply coverslips, ensuring no air bubbles. Allow slides to dry flat at room temperature for 10 to 20 minutes. 10. Imaging and Storage

[0231] 10.1. Analyze using appropriate microscopy settings.

[0232] 10.2. Store the slides at room temperature. The signal should remain stable for years.

Claims

Claims1. A method of detecting a target analyte in a sample, wherein a nucleic acid product has been generated by a proximity assay as a signal for the analyte, said method comprising performing an enzyme-mediated Tyramine Signal Amplification (TSA) amplification of the nucleic acid product signal to generate a TSA product localised to the nucleic acid product, and detecting the TSA product, thereby to detect the target analyte.

2. The method of claim 1 , wherein the nucleic acid product is a ligation product, extension product or hybridisation product.

3. The method of claim 1 or claim 2, wherein said nucleic acid product is a rolling circle amplification product (RCP).

4. The method of any one of claims 1 to 3, wherein the nucleic acid product has been generated in the sample and is localised to the target analyte in the sample.

5. The method of any one of claims 1 to 4, wherein the nucleic acid product has been generated in an in situ detection reaction for the analyte.

6. The method of any one of claims 1 to 5, wherein the analyte is:(i) a nucleic acid;(ii) a protein or proteinaceous molecule, or a protein complex or aggregate;(iii) a protein-protein interaction;(iv) a protein-nucleic acid complex or interaction; or(v) a post-translational modification of the protein, including a phosphorylated protein.

7. The method of any one of claims 1 to 6, wherein the sample:(i) is a clinical sample;(ii) comprises cells;(iii) is a tissue sample;(iv) is immobilised on a solid support; and / or(v) is fresh, frozen or fixed.

8. The method of any one of claims 1 to 7, wherein the analyte is detected in single cells.

9. The method of any one of claims 1 to 8, wherein the nucleic acid product is an RCP generated in a proximity ligation assay (PLA) assay for the target analyte.

10. The method of any one of claims 1 to 9, wherein the nucleic acid product comprises multiple copies of a detection sequence and the method comprises:(i) hybridising to the multiple copies of the detection sequence an enzyme-capture oligonucleotide, wherein the enzyme-capture oligonucleotide comprises a binding site complementary to the detection sequence, and wherein the enzyme-capture oligonucleotide is attached to an enzyme;(ii) contacting the reaction mixture from (i) (i.e. the hybridised nucleic acid product-capture oligonucleotide-enzyme) with a signal localisation agent comprising a substrate for the enzyme, wherein the enzyme catalyses a reaction which deposits a substrate reaction product in the vicinity of the enzyme, and hence of the nucleic acid product, wherein the localisation agent comprises either a label, (and the label is deposited along with the substrate reaction product) or a first binding moiety for binding to a labelling agent;(iii) when the localisation agent does not comprise a label, contacting the reaction mixture of (ii) with the labelling agent, wherein the labelling agent comprises a label linked to a second binding moiety cognate to the first binding moiety, and allowing the second binding moiety to bind to the first binding moiety (thereby to attach the labelling agent to the substrate reaction product);(iv) detecting the label.

11. The method of claim 10, wherein following step (iv) the method further comprises performing one or more other detection assays to detect one or more other analytes in the sample.

12. The method of any one of claims 10 or 11, wherein the enzyme-capture oligonucleotide comprises a first capture moiety which is removably attachedor attachable to a cognate second capture moiety comprised in an enzyme agent comprising said second capture moiety linked to the enzyme.

13. The method of claim 12, wherein in step (i) the nucleic acid product (or the sample containing the nucleic acid product) is contacted with an enzymecapture oligonucleotide comprising a binding site that hybridises to the detection sequence and a first capture moiety, and step (i) further comprises contacting the nucleic acid product with hybridised enzyme-capture oligonucleotide with the enzyme agent, and allowing the cognate first and second capture moieties to bind, to attach the enzyme to the nucleic acid product.

14. The method of any one of claims 12 or 13, wherein the first and second capture moieties and / or the first and second binding moieties are an affinity binding pair selected from:(i) biotin and avidin or streptavidin;(ii) hapten and antibody;(iii) first and second oligonucleotides comprising complementary binding sites.

15. The method of claim 14, wherein the first and second capture moieties and / or the first and second binding moieties are digoxigenin and an anti-digoxigenin antibody.

16. The method of any one of claims 1 to 15, wherein the enzyme is a peroxidase, optionally horseradish peroxidase (HRP).

17. The method of claim 16, wherein the substrate for the enzyme is a tyramine compound, a compound that contains a tyramine derivative, p-hydroxy- cinnamic acid, or a derivative of p-hydroxy cinnamic acid.

18. The method of any one of claims 1 to 17, wherein the label is a directly or indirectly detectable optical label.

19. The method of any one of claims 1 to 18, wherein the label is a fluorescent label, a coloured label, a chromogenic label, a quantum dot, a mass-tag label, or a particle.

20. The method of claim 18, wherein the label is a dye or stain.

21. The method of any one of claims 1 to 20, wherein the label is detected microscopically and / or by imaging.

22. The method of any one of claims 1 to 21, wherein the method is a multiplex method to detect two or more target analytes, and two or more different nucleic acid products are generated, each nucleic acid product corresponding to one of said two or more target analytes, and the nucleic acid products are individually detected, each by the TSA-like amplification reaction.

23. The method of claim 22, wherein:(i) the two or more nucleic acid products are generated together in the sample, or in a single reaction mixture; or(ii) the two or more nucleic acid products are generated individually, in serial reactions in the sample.

24. The method of claim 22 or claim 23, wherein the two or TSA-like amplification reactions are performed simultaneously in the sample or in a single reaction mixture.

25. The method of any one of claims 20 to 24, wherein TSA-like amplification reactions are performed serially to detect one or more different nucleic acid products in each cycle, optionally wherein in each cycle two or more different nucleic acids are detected.

26. The method of claim 25, wherein the enzyme is removed between cycles.

27. The method of any one of claims 1 to 26, wherein the probes used in the proximity assay are secondary reagents which bind to a primary reagent capable of binding specifically to a target analyte.

28. The method of any one of claims 1 to 26, wherein the probes used in the proximity assay are primary reagents which bind directly to the target analyte.

29. A kit for use in the method of any one of claims 1 to 28, said kit comprising:(i) proximity assay reagents for generating a nucleic acid product comprising multiple copies of a detection sequence corresponding to a target analyte;(ii) an enzyme-capture oligonucleotide comprising an oligonucleotide comprising a binding site complementary to the detection sequence, linked to an enzyme, or to a first capture moiety;(iii) optionally an enzyme agent comprising an enzyme linked to a second capture moiety which is cognate and capable of binding to the first capture moiety;(iv) a signal localisation agent comprising a substrate for the enzyme linked to a label or to a first binding moiety, wherein the enzyme is capable of catalysing a reaction using said substrate which deposits a substrate reaction product;(v) optionally a labelling agent comprising a label linked to a second binding moiety cognate and capable of binding to the first binding moiety.

30. The kit of claim 29, wherein the proximity assay reagents (i) comprise:(i) one or more proximity probes pairs, each pair constituting a primary reagent specific for a different target analyte, and each member of a pair being capable of binding specifically and simultaneously to their target analyte; or(ii) one or more proximity probe pairs, each pair constituting a secondary reagent capable of binding to a primary reagent bound to a target analyte, wherein the primary reagent comprises a single binding agent capable of binding specifically to the target analyte or a pair of binding agents each capable of binding specifically and simultaneously to the target analyte, and each member of the proximity probe pair is capable of binding specifically and simultaneously to the single binding agent, or to a separate single member of the primary binding agent pair (such that each member of a pair of primary binding agents is bound by a single member of a proximity probe pair); wherein each proximity probe of a proximity probe pair comprises a binding domain capable of binding specifically to its target, and a nucleic acid domain, and the nucleic acid domains are together capable of templating the ligation of one or more circularisable oligonucleotides to form a template circle for RCA and priming the RCA reaction, wherein said circularisableoligonucleotides are (a) separately provided, or (b) generated by cleavage of the nucleic acid domains; and(iii) optionally in the case of part (ii)(a), for each proximity probe pair, one or more circularisable oligonucleotides, each comprising at least one binding site capable of hybridising to a complementary binding site in each of the nucleic acid domains of the proximity probe pair; optionally wherein the reagents further comprise:(iv) a polymerase for the RCA reaction; and / or(v) dNTPs; and / or(vi) a ligase for ligation of the circularisable oligonucleotide(s).

Images