WO2020193977A1

WO2020193977A1 - Method for detecting an analyte

Info

Publication number: WO2020193977A1
Application number: PCT/GB2020/050803
Authority: WO
Inventors: Christian Soeller; Tobias Lutz; Alexander CLOWSLEY; Lorenzo DI MICHELE; William KAUFOLD
Original assignee: University Of Exeter
Priority date: 2019-03-25
Filing date: 2020-03-25
Publication date: 2020-10-01

Abstract

The present invention relates to a method for detecting an analyte in a sample. The method comprises contacting the sample with a first probe and a second probe, wherein each probe comprises an analyte-binding domain and a nucleic acid domain. The nucleic acid domain of the first probe comprises a second probe-binding region and an imager nucleic acid- binding region comprised within a secondary structure, such that said imager nucleic-acid binding region is not available for binding by an imager nucleic acid. The nucleic acid domain of the second probe comprises a first probe-binding region capable of interacting with the second probe-binding region of the first probe when the first and second probes bind the analyte. Binding of the first probe-binding region to the second probe-binding region makes available the imager nucleic acid-binding region for binding by an imager nucleic acid. The method further comprises contacting the sample with an imager nucleic acid and detecting the imager nucleic acid using a super resolution imaging technique.

Description

Method for Detectinq an Analyte

Field of the Invention

The present invention relates to a method for detecting an analyte in a sample, for example proximity of two or more analytes. The present invention further relates to a kit for detecting an analyte in a sample.

Background to the Invention

Characterising protein interactions by detection of protein-protein complexes is the basis of understanding many processes in molecular biology. Often, these protein interactions are detected by in vitro methods such as co-immunoprecipitation, cross-linking or affinity blotting. However, it is increasingly evident that besides the presence of protein-protein interactions, it is important to determine where these occur within a cell or tissue. To this end, methods have been developed that rely on labelling the features of interest with synthetic DNA oligonucleotides, conjugated to antibodies or other molecular markers. The oligonucleotides act as proximity probes, and a subsequent amplification step is implemented to produce a fluorescent signal detectable by a conventional microscope. In proximity ligation assay (PLA), enzymatic amplification occurs via the rolling circle method, while in ProxHCR amplification is non-enzymatic and relies on a hybridisation chain reaction. However, the high fluorescent amplification in both methods also effectively limits them to diffraction-limited imaging. This means that the presence of protein pairs can be pinned down to a certain region but generally precludes accurate quantification and visualisation of their distribution at the nanometre scale, which is becoming increasingly important with the realisation that the nanoscale organisation of signalling directly controls cell function.

Imaging with spatial resolution beyond the diffraction-limit can now be achieved by a wide range of super-resolution techniques, such as structured illumination microscopy (SIM), stimulated emission depletion (STED) microscopy, (fluorescence) photoactivated localisation microscopy ((F)PALM) or (direct) stochastic optical reconstruction microscopy (dSTORM). A super-resolution imaging technique which relies on oligonucleotide interactions, is DNA-PAINT (Point Accumulation Imaging in Nanoscale Topography) (Schnitzbauer et al., 2017). In DNA- PAINT, the proteins of interest are labelled with a short DNA oligonucleotide, or“docking” strand. The transient binding of fluorescently labelled“imager” (DNA) strands to the docking strand is then detected and localised. The localisation data is used to reconstruct a super resolution image. Fluorescent super-resolution techniques can be used to acquire a multi-target image which, in principle, can be used to estimate the presence of protein-protein interaction sites by fluorescence co-localisation. However, unequivocal identification is complicated by the fact that co-localisation can be prone to false-positive signals, as the co-localisation precision directly depends on the local imaging resolution. Resolution can vary considerably, especially in optically complex samples such as thick fixed cells or tissue sections. An often very limited resolution along the optical axis (several 100 nm in 2D super-resolution techniques, >40 nm in 3D methods) can lead to additional false positives. In addition, registration errors between multiple channels, e.g. due to chromatic aberrations or sample drift, can lead to incorrect co localisation estimates.

It is an object of the present invention to obviate or mitigate one or more of the abovementioned problems.

Summary of the Invention

The present invention relates to a method for detecting an analyte, for example proximity or co-localisation of two or more analytes and is based, in part, on studies by the inventors in which they have shown that it is possible to detect and visualise proximity or co-localisation of analytes with high sensitivity and specificity by utilising super-resolution imaging.

In a first aspect of the present invention there is provided a method for detecting an analyte in a sample. The method of the first aspect of the invention comprises:

i) contacting the sample with a first probe and a second probe, wherein each probe comprises an analyte-binding domain and a nucleic acid domain, wherein a. the nucleic acid domain of the first probe comprises a second probe-binding region and an imager nucleic acid-binding region comprised within a secondary structure, such that said imager nucleic-acid binding region is not available for binding by an imager nucleic acid;

b. the nucleic acid domain of the second probe comprises a first probe-binding region capable of interacting with the second probe-binding region of the first probe when the first and second probes bind the analyte;

c. wherein binding of the first probe-binding region to the second probe-binding region makes available the imager nucleic acid-binding region for binding by an imager nucleic acid;

ii) contacting the sample with an imager nucleic acid; and

iii) detecting the imager nucleic acid using a super resolution imaging technique.

The inventors of the present invention have found that by developing the abovementioned method, which allows the study of nanoscale distribution of analytes, for example protein-protein complexes, proximity detection can be decoupled from the local imaging resolution. This results in a highly sensitive and specific method for detecting an analyte.

Analytes

As mentioned above, the method of the present invention allows an analyte of interest to be detected with high sensitivity and selectivity. The“target” analyte may be any molecule or entity of interest for which detection is required. For example, the analyte may include a peptide or protein, nucleic acid (RNA, DNA or any modification thereof), lipid or small molecule. Such an analyte may be present in or on a cell (i.e. the analyte may be present in its native form) or alternatively, may have been isolated. The analyte may itself be a cell, microorganism or fragment thereof. Provided that the analyte can be bound by analyte-binding domains of the first and second probes simultaneously or sequentially, the analyte is suitable for use in the method of the present invention.

The analyte may be a single analyte which requires detection, for example a peptide, protein, nucleic acid, lipid or small molecule of interest. Provided that the single analyte can be bound by analyte-binding domains of the first and second probes simultaneously, such a single analyte can be detected using the method of the invention.

In alternative embodiments, the analyte may comprise two or more analytes. For example, in embodiments in which the analyte comprises two analytes, the method may be used to detect proximity or co-localisation of said two analytes. In such an embodiment, the analyte-binding region of the first probe is capable of binding one of the analytes and the analyte-binding region of the second probe is capable of binding the second analyte.

The present invention therefore also provides a method for detecting proximity or co localisation of first and second analytes in a sample. The method comprises:

i) contacting the sample with a first probe and a second probe, wherein the first probe comprises an analyte-binding domain, which binds the first analyte, and a nucleic acid domain, and the second probe comprises an analyte-binding domain, which binds the second analyte, and a nucleic acid domain wherein a. the nucleic acid domain of the first probe comprises a second probe-binding region and an imager nucleic acid-binding region comprised within a secondary structure, such that said imager nucleic-acid binding region is not available for binding by an imager nucleic acid;

b. the nucleic acid domain of the second probe comprises a first probe-binding region capable of interacting with the second probe-binding region of the first probe when the first and second probes bind the first and second analytes; c. wherein binding of the first probe-binding region to the second probe-binding region makes available the imager nucleic acid-binding region for binding by an imager nucleic acid; ii) contacting the sample with an imager nucleic acid; and

As is discussed further below, in embodiments in which the method is used to detect proximity of first and second analytes, the design of the first and second probes determine the maximum distance between analytes that is required in order for proximity to be detected.

In yet further embodiments, the method may be used to detect modification, for example post-translational modification, of an analyte, for example a peptide or protein. For example, the method of the invention may be used to detect phosphorylation, glycosylation, acetylation, methylation, amidation, hydroxylation, sulfation, ubiquitination, biotinylation, pegylation, SUMOylation, Neddylation, Pupylation, glycation or carbonylation of an analyte of interest. In such examples, the analyte-binding region of the first probe is capable of binding the analyte in an un-modified region and the analyte-binding region of the second probe is capable of binding the analyte when it has been modified. The method of the present invention may also be used to detect proximity of more than one modification, for example first and second modifications of an analyte or multiple analytes.

The present invention therefore also provides a method for detecting modification of an analyte in a sample. The method comprises:

i) contacting the sample with a first probe and a second probe, wherein the first probe comprises an analyte-binding domain, which binds an un-modified region of the analyte, and a nucleic acid domain, and the second probe comprises an analyte-binding domain, which binds a modified region of the analyte, and a nucleic acid domain, wherein

a. the nucleic acid domain of the first probe comprises a second probe-binding region and an imager nucleic acid-binding region comprised within a secondary structure, such that said imager nucleic-acid binding region is not available for binding by an imager nucleic acid;

ii) contacting the sample with an imager nucleic acid; and

The skilled person will appreciate that the method of the present invention need not be limited to the detection of one or two analytes, but could be used to detect three, four or five analytes, for example proximity or co-localisation of three, four or five analytes in a multimer. As will be appreciated, in order to detect three analytes, the method may comprise contacting the sample with three probes. In such an embodiment, the method may comprise:

i) contacting the sample with a first probe, a second probe and a third probe, wherein the first probe comprises an analyte-binding domain, which binds the first analyte, and a nucleic acid domain, the second probe comprises an analyte binding domain, which binds the second analyte, and a nucleic acid domain and the third probe comprises an analyte-binding domain, which binds the third analyte, and a nucleic acid domain wherein

b. the nucleic acid domain of the second probe comprises a first probe-binding region comprised within a secondary structure, such that said first probe binding region is not available for binding to the first probe, and a third-probe binding region,

c. the nucleic acid domain of the third probe comprises a second probe-binding region capable of interacting with the third probe-binding region of the second probe when the second and third probes bind the second and third analytes; d. wherein binding of the second probe-binding region of the third probe to the second probe makes available the first probe-binding region of the second probe for binding to the second probe-binding region of the first probe, wherein binding of the first probe-binding region of the second probe to the second probe-binding region of the first probe makes available the imager nucleic acid-binding region for binding by an imager nucleic acid;

ii) contacting the sample with an imager nucleic acid; and

The skilled person will appreciate that the method can be adapted in order to detect the proximity or co-localisation of multiple analytes, for example in a multimer, by modifying the number of probes required. For example, in order to detect the proximity or co-localisation of four analytes, four probes may be required.

Analyte-binding domains

The probes of the present invention each comprise an analyte-binding domain. The analyte-binding domain may be any domain capable of binding the target analyte. The analyte binding domain may be capable of binding the target analyte directly or indirectly, for example via a further molecule which binds to the target analyte. However, in embodiments, the analyte binding domain binds directly to the target analyte and may be a specific binding partner for the target analyte. For example, the analyte-binding domain may bind to the target analyte with greater affinity and/or specificity than to other components in the sample.

The analyte-binding domain may be selected to have a high binding affinity for its target, for example a binding affinity with a dissociation constant lower than about 10⁹ M, 10⁶ M or 10 ⁴ M. By selecting analyte-binding domains which have a high binding affinity for the target analyte, the selectivity and sensitivity of the method of the present invention may be increased.

In embodiments of the invention, the analyte-binding domain may be selected from a protein, for example a monoclonal or polyclonal antibody or fragment thereof, lectin, soluble cell surface receptor, peptide, carbohydrate, nucleic acid, for example an aptamer or a nucleic acid molecule comprising the complementary sequence for a target nucleic acid, fragments thereof or any combination thereof.

In preferred embodiments of the invention, the analyte-binding domain comprises an antibody, for example a monoclonal or polyclonal antibody or fragment thereof. The present inventors have shown that by utilising an antibody as the analyte-binding domain, particularly high affinity binding to a target analyte can be achieved thereby increasing the selectivity of the method.

The term“antibody” as used herein may be used to encompass any antibody fragment, derivative or mimetic thereof, provided that such fragments, derivatives or mimetics possess high binding affinity for the target analyte. For example, the term“antibody” as used herein may include Fv, F(ab)₂ and Fab fragments, recombinantly or synthetically produced antibody fragments or derivatives such a single chain antibodies, scFvs, chimeric antibodies or CDR- grafted antibodies. As mentioned above, any such antibodies may be used provided they retain a high binding affinity for the target analyte.

In alternative embodiments of the invention, the analyte-binding domain is a nucleic acid molecule. The nucleic acid molecule may comprise, for example, ribonucleotides and/or deoxyribonucleotides and/or synthetic nucleotide residues. The nucleic acid molecule may comprise RNA and/or DNA or any suitable modification thereof.

The skilled person will appreciate that by designing analyte-binding domains which bind to particular regions of the analyte(s) of interest, information regarding the orientation of the analyte(s) can be obtained. By placing the probes at specific sites on the analytes, the distance between these sites provides information about the relative orientation of the two analytes. For example, if the sites are chosen so that the two sites are further than 10 nm apart unless protein 1 binds to protein 2 in a specific relative orientation, the presence of a signal shows that protein 1 and protein 2 have adopted this specific relative orientation. For example, posttranslational modification of proteins by ubiquitin is responsible for controlling protein localization, function, and lifetime. Ubiquitination occurs on lysine residues, so for single proteins there are multiple possible ubiquitination sites. The method outlined here presents a direct method of assaying the relative orientation of ubiquitin and the substrate protein, and for visualization of the nanoscale localization of proteins whose ubiquitination varies in orientation. Such a method could be extended to direct investigation of arbitrary orientations of proteins in protein-protein interactions and their nanoscale localization.

Nucleic acid domains

The method of the present invention comprises contacting a sample with at least first and second probes, wherein each probe comprises a nucleic acid domain. The nucleic acid domain, may comprise, for example, ribonucleotides and/or deoxyribonucleotides and/or synthetic nucleotide residues. The nucleic acid domain may comprise RNA, DNA, L-DNA (left handed DNA), PNA and/or XNA or any suitable modification thereof. The nucleic acid domain may be natural or synthetic. In preferred embodiments, the nucleic acid domain comprises DNA.

The nucleic acid domain of the second probe comprises a first probe-binding region capable of interacting with the second probe-binding region of the first probe when the first and second probes bind the analyte. Preferably the interaction between the first probe-binding region and the second probe-binding region involves, or is achieved by, hybridisation between the two regions. In such embodiments, the first and second probe-binding regions may contain regions which are capable of hybridising to one another i.e. are complementary. As set out above, the first and second probe-binding regions can only interact or hybridise when the first and second probes bind the analyte. When the first and second probes are bound to the analyte they are in molecular proximity which enables binding of the complementary regions in the first and second probes. The first and second probes may be designed so that interaction of the complementary regions in the first and second probes only occurs when the proximity is smaller than a maximum distance, for example 50 nm, 30 nm or 10 nm. The maximum distance can be controlled by the length, and sequence of the nucleic acid domains, as well as control over their secondary structure.

The nucleic acid domain of the first probe comprises an imager nucleic acid-binding region comprised within a secondary structure. In embodiments of the invention, binding of the first probe-binding region to the second probe-binding region makes available the imager nucleic-acid binding region for binding by an imager nucleic acid.

The presence of the imager in the secondary structure results in the imager nucleic-acid binding region not being available for binding by an imager nucleic acid. The skilled person will appreciate how this may be achieved. For example, the imager nucleic-acid binding region may be hybridised to another portion of the nucleic acid domain of the first probe, such that the imager nucleic-acid binding region cannot be accessed/bound by the imager nucleic acid.

In embodiments of the invention, binding of the first probe-binding region to the second probe-binding region unfolds the secondary structure in which the imager nucleic acid-binding region is comprised. Such unfolding of the secondary structure may make available the imager nucleic acid-binding region for binding by an imager nucleic acid.

As the skilled person will appreciate, only when the first and second probes are in proximity can the first probe-binding region bind to the second probe-binding region and unfold the secondary structure. If the distance between the first and second probes is not sufficiently close, the first probe-binding region cannot bind to the second probe-binding region and therefore the imager nucleic acid-binding region is not made available for binding by an imager nucleic acid. When the first and second probes are in proximity, the first probe-binding region of the second probe competes with the secondary structure forming mechanism of the first probe, thereby unfolding the secondary structure and making available the imager nucleic acid-binding region for binding by an imager nucleic acid.

The skilled person will appreciate that the first and second probes are designed so that when in free solution at moderate concentration, for example, during labelling reactions and similar procedures, the probability of in-solution interactions between first and second probes are negligible both due to the large average distance between these probes when in solution and the brief nature of any chance encounters between first and second probes when in solution. The effect of proximity is to increase the effective mutual concentration (activity) of two constructs, such that while their interaction in solution is improbable (for example at concentrations of 500 nM), interaction when bound is probable due to their higher activity (for example an effective activity of 500 mM).

The skilled person will have an understanding of nucleic acid secondary structures and will be able to design such nucleic acid secondary structures according to their knowledge in the art. The secondary structure of the first probe may comprise at least one loop, for example a single stranded nucleic acid loop. The secondary structure may further comprise a double- stranded region, for example a self-complementary region where two portions of the nucleic acid domain hybridise together. In embodiments of the invention, the secondary structure may comprise or consist of a stem-loop structure, for example a single stem-loop.

In embodiments of the present invention, the second-probe binding region of the first probe may be within, or partially within, the secondary structure. In embodiments in which the secondary structure is a stem-loop structure, the second probe-binding region may be within, or partially within, the double-stranded stem region of the secondary structure. In such embodiments, the first-probe binding region of the second probe can displace the stem of the hairpin, by binding to the second-probe binding region, thereby unfolding the loop and enabling the imager to bind to the imager nucleic acid-binding region. The present inventors have found that by locating the second-probe binding region within the double-stranded stem region of the secondary structure, efficiency of unfolding of the secondary structure on binding of the first probe-binding region to the second probe-binding region may be improved. ln embodiments of the present invention, a portion of the second probe-binding region may be external to the secondary structure of the first probe. In such embodiments, the second probe-binding region may be protected from interaction with the second probe. For example, interaction of the second probe-binding region with the second probe may be prevented by hybridisation of the second probe-binding region or the first probe-binding region to a blocking strand (e.g. a blocking nucleotide sequence). The blocking nucleotide sequence may have a preferred length of 15-30 base complementarity to the first probe or the second probe, or interaction may be prevented by formation of the second probe-binding region or the first probe binding region into a secondary structure, which may be metastable or can be altered by addition of further nucleic acid structures. Such a protection from interaction of second probe and second probe-binding region may be utilised during sample-labelling steps in order to prevent unwanted interactions between the first and the second probe. The interaction may then be initiated at a specific time point, for example once labelling is complete. For example, the interaction between the first and the second probe may be initiated by addition of an nucleic acid structure which may remove a blocking strand or alter secondary structures of the first probe or second probe.

Having part of the second probe-binding region external to the secondary structure can speed up the kinetic of loop opening in the first probe and increase the overall probabilities that the loop opens.

In the method of the present invention, an imager nucleic-acid binding region is comprised within the secondary structure of the first probe. In embodiments in which the secondary structure comprises a stem-loop structure, the imager nucleic-acid binding region may be within, or partially within, the stem-loop structure, optionally within the double-stranded stem region. For example, in embodiments, the imager nucleic-acid binding region may be at least partially complementary to the second probe-binding region. By the imager nucleic-acid binding region being at least partially complementary to the second probe-binding region, the imager nucleic-acid binding region remains inaccessible to the imager nucleic acid unless the first probe-binding region is bound to the second-probe binding region. The imager nucleic-acid region would, under conditions where the second probe is not in molecular proximity to the first probe, hybridise, at least partially to the second-probe binding region, therefore rendering the imager nucleic-acid region unavailable for binding by the imager nucleic acid.

Other suitable nucleic acid secondary structures include for example G quadruplexes or I motifs. Any secondary structure will be suitable provided that the secondary structure is stable enough so that the imager nucleic acid-binding region is inaccessible to an imager nucleic acid until such time that the first probe-binding region of the second probe interacts with the second- probe binding region of the first probe. The skilled person will appreciate how sufficient stability of secondary structure can be imparted, for example by altering the length of the nucleic acid or the G/C content of the nucleic acid. For example, in embodiments in which the secondary structure is a single stem-loop structure, the stability of the single stem-loop can be altered by changing the length of the stem or the G/C content of the stems, for example. The skilled person will also appreciate how the condition dependent stability of G-quadruplexes (in the presence of ions), or l-motifs (at specific pH values) may provide further conditions required for the imager binding domain to be exposed.

In embodiments in which the secondary structure comprises a stem-loop structure, the double-stranded stem may comprise between 10 and 50 nucleotides, preferably between 15 and 30 nucleotides, more preferably about 20 nucleotides. The loop may comprise between 3 and 10 nucleotides, more preferably about 6 nucleotides. A length of loop between 3 and 10 nucleotides, particularly 6 nucleotides, is advantageous in the method of the present invention. Surprisingly the inventors found that further increasing the length of the loop increased the likelihood of the imager nucleic acid binding to the stem in its“folded” form.

The skilled person will appreciate that by altering the length, sequence, and secondary structure of nucleic acid domains of the first and second probes, the maximum and minimum distance over which interaction of the first and second probes takes place can be altered.

Probes

The probes of the invention each comprise an analyte-binding domain and a nucleic acid domain. The analyte-binding domain and nucleic acid domain of each probe are coupled to each other. This coupling may be achieved by any means suitable, as will be understood by the skilled person. The coupling may be direct or indirect.

In embodiments in which the analyte-binding domain is a nucleic acid, the coupling may be by way of a nucleotide bond. Alternatively, in embodiments in which the analyte-binding domain is a protein, for example an antibody, the coupling may be by way of a covalent or non- covalent linkage (for example by chemical crosslinking of the two domains or by way of biotin- streptavidin based connection, for example). The analyte-binding and nucleic acid domains may be coupled by linking groups. The skilled person working in this field will be aware of a variety of linking groups which could be utilised, but these could include, for example via maleimide- PEG2-succinimidyl ester, via DBCO-sulfo-NHS ester and copper-free Click Chemistry using azide-modified nucleotides, via thiolated nucleic acids or via commercially available conjugation kits.

Imager nucleic acid

In the method of the present invention, an imager nucleic acid is used as a‘detector’ to detect whether an analyte is present in a sample of interest. The imager nucleic acid of the present invention binds an imager nucleic-acid binding region of the first probe, when made available for binding, and the imager nucleic acid is subsequently detected using a super resolution imaging technique. The imager nucleic acid may comprise RNA and/or DNA or any suitable modification thereof, provided that it can bind to the imager nucleic acid binding region of the first probe and can be detected by a super resolution imaging technique.

The imager nucleic acid comprises a moiety which can be detected by way of a super resolution imaging technique. For example, the moiety may be a fluorescent marker, tag or dye, for example green fluorescent protein (GFP), yellow fluorescent protein (YFP), blue fluorescent protein (BFP), cyan fluorescent protein (CFP), red fluorescent protein (RFP), Cyanine dyes (e.g. Cy2, Cy3, Cy3B, Cy3,5, Cy5, Cy5.5, Cy7), Atto dyes (Atto 488, Atto 550, Atto 647N, Atto 655, Atto 700, Atto 750), Alexa Fluor dyes. In preferred embodiments, the moiety comprises an Atto dye, more preferably Atto 655 or Atto 700. In other embodiments the moiety may be a plasmonic particle or a marker with specific scattering properties that can be detected by super resolution.

The imager nucleic acid may be any suitable length, provided that it is at least partially complementary to the imager nucleic-acid binding region of the first probe. Preferably, the imager nucleic acid is between 7 and 20 nucleotides in length, yet more preferably 8-9 nucleotides in length.

As will be appreciated, the imager nucleic acid must show highly specific binding to the imager nucleic acid binding region such that the specificity of the method of the present invention remains high.

In embodiments, the mean binding time of the imager nucleic acid to the imager nucleic acid binding region is at least around 20 ms, preferably at least around 100 ms. In embodiments of the invention, the mean binding time of the imager nucleic acid to the imager nucleic acid binding region is between around 20 and 1500 ms.

In embodiments of the present invention where the super resolution technique comprises STORM or STED, for example, the imager nucleic acid may bind effectively permanently to the imager nucleic acid binding region or a larger concentration of imager will be used so that many or most accessible imager binding sites are occupied.

In embodiments of the present invention, the imager nucleic acid which binds to the imager nucleic-acid binding region may comprise a nucleotide nanostructure, for example, with several secondary imager nucleic-acid binding sites. The imager nucleic acid may comprise several secondary imager nucleic-acid binding sites which may, for example, have an alternative sequence compared to the imager nucleic-acid binding site on the first probe. In such embodiments, the method of the present invention may further comprise contacting the sample with a secondary imager nucleic acid. In such embodiments, detecting the imager nucleic acid using a super resolution imaging technique may comprise detecting the secondary imager nucleic acid using a super resolution imaging technique.

Detection In the method of the present invention, the imager nucleic acid is detected using a super resolution imaging technique. In the method of the present invention, the imager nucleic acid can only be detected (i.e. localised) when bound to the imager nucleic-acid binding region of the first probe. If the nucleic-acid binding region of the first probe is not available for binding, the imager will be free in the sample (for example in solution), or removed by washing in some embodiments, and cannot be detected (i.e. localised) using the imaging technique.

Therefore, in embodiments of the present invention step (iii) of the method comprises detecting imager nucleic acid bound to the imager nucleic-acid binding region using a super resolution imaging technique.

The method of the present invention makes use of super resolution imaging to detect the imager nucleic acid. Super-resolution imaging is a class of techniques that enhance the resolution of an imaging system. Imaging with spatial resolution beyond the diffraction-limit can be achieved by a wide range of super-resolution techniques, as will be appreciated by the skilled person. Super-resolution techniques suitable for use in the present invention include, but are not limited to, structured illumination microscopy, stimulated emission depletion microscopy, fluorescence photoactivated localisation microscopy, direct stochastic optical reconstruction microscopy, and DNA-PAINT (DNA-points accumulation for imaging in nanoscale topography). Preferably, the super-resolution technique used in step (iii) of the method is DNA-PAINT. The use of DNA-PAINT in the method of the present invention allows detection of the analyte of interest with extremely high resolution. Compared to methods currently used to detect analytes, for example co-localisation or proximity of analytes, the use of super resolution imaging techniques, particularly DNA-PAINT, allows the spatial distribution of analytes to be determined on the nanometer scale and allows quantitative analysis of the results, for example the number of analytes can be determined. By integrating a super-resolution imaging technique in the method of the invention, the present inventors have surprisingly been able to develop a highly specific and sensitive method for detecting analytes in which quantitative information regarding the analytes can be obtained.

Amplification

In embodiments of the present invention, the detecting step does not require amplification. Many of the methods used at present, for example proximity ligation assays (PLAs) and ProxHCR, require amplification of oligonucleotides bound to a fluorescent marker to allow detection of a fluorescent signal in a conventional microscope. However, the fluorescent amplification utilised in both of these methods limits the techniques to diffraction limited imaging of the protein-protein interactions. In the present invention, amplification techniques are not required in order to detect the analyte which results in a highly sensitive method in which quantitative information can be obtained from the image data. For example, if amplification is not utilised it is possible to count the interaction sites and determine the spatial resolution of the analytes of interest with high sensitivity. By not utilising amplification no additional enzymes are required and therefore the method is both simpler and quicker to implement. The term “amplification” as used herein, includes, for example PCR, HCR and rolling circle amplification.

Therefore, in a preferred embodiment of the invention, detecting the imager nucleic acid does not include amplification of the imager nucleic acid. As described above, such amplification, for example uncontrolled, strong amplification of the imager nucleic acid, leads to substantial loss of spatial resolution and/or stochastic local variation in the amplification factor.

As mentioned above, the present invention does not require that the imager nucleic acid is amplified in order for detection to occur. Imaging of single imager nucleic acids can therefore be performed which allows the resultant images to be used to obtain information regarding the number of interaction sites and spatial distribution with extremely high resolution. In embodiments, step (iii) of the present invention comprises detecting the imager nucleic acid using super resolution imaging of single imager nucleic acids. As will be appreciated by the skilled person the phrase“single imager nucleic acids” does not necessarily require that a single imager nucleic acid is detected in total, but rather, a single imager nucleic acid is detected for each analyte detected at any one time (for example a single analyte, a modified analyte, co localisation or proximity of two or more analytes). However, if the interaction of the imager nucleic acid and analyte is transient, over time, multiple imager nucleic acids can be detected for each analyte subsequently.

In preferred embodiments of the present invention, step (iii) of the method comprises detecting single imager nucleic acids bound to the imager nucleic-acid binding region using a super resolution imaging technique.

In alternative embodiments, step (iii) of the method comprises detecting a small group of imager nucleic acids bound to the imager nucleic-acid binding region using a super resolution imaging technique. In such embodiments, the imager nucleic acid which binds to the imager nucleic-acid binding region may comprise a nucleotide nanostructure, for example, with several secondary imager nucleic-acid binding sites. The imager nucleic acid may comprise several secondary imager nucleic-acid binding sites which may, for example, have an alternative sequence compared to the imager nucleic-acid binding site on the first probe. In such embodiments, the method of the present invention may further comprise contacting the sample with one or more secondary imager nucleic acids. In such embodiments, detecting the imager nucleic acid using a super resolution imaging technique may comprise detecting the secondary imager nucleic acid using a super resolution imaging technique. In such embodiments, the imager nucleic acid may comprise between 2 and 10 secondary imager nucleic-acid binding sites, for example. The features described above in relation to the imager nucleic acid, for example the presence of a moiety which can be detected by super-imaging resolution, are equally applicable to the secondary imager nucleic acid.

In another preferred embodiment a small or moderate amount of amplification of the imager signal, e.g. 4x, 10x, 100x, may be used to increase the resultant signal but without loss of spatial resolution beyond the spatial resolution of the super-resolution imaging technique used. The amplification process may be linear. By utilising linear amplification non-linearities and saturation are avoided. This can be achieved, for example, by utilising an imager nucleic acid with several secondary imager nucleic-acid binding sites and secondary imager nucleic acids, as described above. The greater number of new imaging binding sites on the nanostructure, compared to the single binding site on the first probe, will result in an amplified signal. Preferably, if amplification is utilised the variation in amplification factor is small so that quantitation is not adversely affected.

Sample

In the present invention, a sample which may comprise an analyte is contacted with first and second probes. The sample may include any sample which may contain an analyte of interest, for example an environmental sample (e.g. soil, water or food samples), synthetic sample (e.g. DNA origami, labelled colloidal particles or other synthetic nanostructures), clinical sample or biological sample (e.g. cell or tissue sample, e.g. a sample comprising prokaryotic or eukaryotic cells, viruses, bacteriophages, mammalian or non-mammalian cells, plant cells, algae, fungi, bacteria or protozoa), bodily fluid (for example plasma, serum, blood, urine, faeces, cerebrospinal fluid, saliva, respiratory secretions or any other suitable bodily fluid), cell culture sample, cell lysate sample).

In embodiments of the invention, the sample is present as a suspension in an imaging buffer. Imaging buffers will be known to the skilled person. In embodiments, the pH of the imaging buffer may be between pH 7 and 9, preferably around pH 8.

In embodiments, additional components may be added to the sample prior to use in the method of the present invention. For example, additional components may be added to the sample which improve marker specificity and enhance the signal obtained, such as antibody blocking solutions, refractive index matching media etc.

In embodiments of the invention, the method may include a step of chemically fixing the sample to preserve the biological sample. The skilled person will appreciate how such fixation might be achieved, but this could include, for example using aldehyde crosslinking fixatives such as formaldehyde, glutaraldehyde, glyoxal, paraformaldehyde, using precipitating fixatives for example acetone, ethanol, methanol, using oxidising crosslinking fixatives for example chromic acid, osmium tetroxide, potassium dichromate, potassium permanganate, and/or by using HOPE fixative, mercurials, picrates, or other fixatives. In embodiments of the invention, the method may include attaching the sample to a suitable substrate, for example a microscopic slide. The skilled person will appreciate how such immobilisation might be achieved, but this could include, for example via a laminin coating, a poly-L-lysine coating, a BSA-biotin coating in combination with a neutravidin or streptavidin coating.

Other

In the method of the present invention, the method may further comprise obtaining quantitative information regarding the number of analytes present, the number of analytes in proximity/co-localising or the number of analytes which are modified, for example by quantification of the association or dissociation rates of the imager nucleic acid to the proximity probe complex, for example by utilising the linear relationship between the number of available binding sites with the mean duration separating observed binding events in equilibrium.

In the method of the present invention, the method may further comprise multiplexed or parallelised detection of more than one population of analytes, analyte pairs or assemblies in proximity/co-localisation or modified analytes in the same sample, for example by distinguishing different populations by using separate imager nucleic acids with different spectral properties which can be separated in different detection channels by optical means, or by using the ratio of the intensity of the signal between two channels or binding kinetics of the imager nucleic acids, or instead by distinguishing populations by temporal multiplexing by subsequent removal and addition of imager nucleic acids targeting different probe populations.

Kit

The present invention also provides a kit for use in detecting an analyte. The kit may comprise:

i) first and second probes wherein each probe comprises an analyte-binding domain and a nucleic acid domain, wherein

c. wherein binding of the first probe-binding region to the second probe-binding region makes available the imager nucleic acid-binding region for binding by an imager nucleic acid; and

ii) an imager nucleic acid. The kit may be for use in the method of the present invention described above.

The described and illustrated embodiments are to be considered as illustrative and not restrictive in character, it being understood that only the preferred embodiments have been shown and described and that all changes and modifications that come within the scope of the inventions as defined in the claims are desired to be protected.

The optional features set out herein may be used either individually or in combination with each other where appropriate and particularly in the combinations as set out in the accompanying claims. The optional features for each aspect or exemplary embodiment of the invention as set out herein are also to be read as applicable to any other aspect or exemplary embodiments of the invention, where appropriate. In other words, the skilled person reading this specification should consider the optional features for each exemplary embodiment of the invention as interchangeable and combinable between different exemplary embodiments.

It should be understood that while the use of words such as“preferable”,“preferably”, “preferred” or“more preferred” in the description suggest that a feature so described may be desirable, it may nevertheless not be necessary and embodiments lacking such a feature may be contemplated as within the scope of the invention as defined in the appended claims. In relation to the claims, it is intended that when words such as“a,”“an,” or“at least one,” are used to preface a feature there is no intention to limit the claim to only one such feature unless specifically stated to the contrary in the claim.

Detailed Description of the Invention

The present invention will now be further described with reference to the following figures which show:

Figure 1 : Principle of Proximity-Dependent PAINT (PD-PAINT). a: Strand S1 forms a stem loop structure which prevents imagers (P1) binding to the docking domain (5* -6), due to a high curvature of domain S1-6 and hybridization of domain S1-3-5* to S1-3-5. Strand S2 contains domains 1-5* which are complementary to 3-5 of the S1 stem and the unbound toe hold region 1-2 on S1. The corresponding toe-hold domain 1*-2* on S2, along with neighboring domains 7* and 3*, are initially protected by shield strand B in order to prevent unwanted S1-S2 dimerization during the sample functionalization stages b: Addition of the shield remover R strips the shield B, and, should S1 and S2 be in close proximity, triggers their dimerization initiated by the 1-2 toe-hold, which results in the opening of the S1 loop c: The now exposed docking domain 5*-6 allows the transient binding of an imager to the S1-S2 complex so that a super-resolution image can be obtained by DNA-PAINT. d: Alternative tethering geometry (geometry 2) in which strands S1 and S2 are attached to the substrate via attachment strands D1 and D2. In this configuration the double stranded domain formed by D1 and S1 can rigidly rotate around the attachment point of D1 whereas the flexible single-stranded section of S2 is free to move around the attachment point of D2. Figure 2: Thermodynamic properties of PD-PAINT from coarse-grained simulation a: The free energy change of S1-S2 hybridization is sensitive to the separation distance between tethers and to the structure connecting the tether to the loop or its complement. At close inter tether distances (< 5 nm), hybridization is essentially irreversible for both tested tethering geometries. At higher distances, hybridization free energy increases monotonically for the direct tethering geometry (geometry 1 , blue) due to stretching of the entropic spring formed by the two 15 polythymine linkers. For the indirect-tethering geometry (geometry 2, red), the 32 base pair dsDNA spacer reduces the dependency of free energy on inter-tether distance b: Dimerization probabilities are ~1 at inter-tether distances shorter than 10 nm (geometry 1), or 20 nm (geometry 2). At greater distances (~16 nm for geometry 1 and ~25 nm for geometry 2) dimerization probability sharply transitions to ~0. c: Hybridization of the imager to S1 as a closed loop is inhibited in the absence of S2 and restored by the presence of S2. Free energy profiles for formation of Watson-Crick bonds between the imager and S1 are shown, relative to the unhybridized state. We compare the closed loop state of S1 (blue line), the open state after hybridization of S2 (orange dashed) and conventional DNA-PAINT binding (green dotted). Note that closed loop states with >5 bonds resulted in large free energies whose contribution to the overall hybridization free energy is negligible d: simulation snapshots for the three configurations tested in panel c. All DNA sequences used are shown in Table S2 Snapshots from the simulation are visualized with UCSF Chimera²⁵. All error bars are given as one standard error.

Figure 3: PD-PAINT to detect multivalent binding of biotin by streptavidin. A) Biotinylated strands S1 and S2 were allowed to attach in equal amounts to the surface of streptavidin- coated 500 nm polystyrene spheres, which allows a considerable fraction of S2 to open up the S1 stem loop structure, whenever S1 and S2 attach to adjacent binding sites on the same protein, sequentially or simultaneously. B) Rendered image of the resulting DNA-PAINT signal, box indicates area of magnified inset in C. D) Widefield fluorescence image at high imager concentrations (5 nM), with most of the accessible docking domains occupied by imagers. E) If only S1 is used, the loop remains closed and does not allow imagers to bind. F, G) Rendered images, analogous to B and C, but displaying little signal. H) Widefield fluorescence image showing only a diffuse background fluorescence signal from comparatively high concentration of imagers (10 nM) in solution, outlining shadows of unlabelled nanospheres against the fluorescent background and very rare binding of single imagers (arrow). Scale bars: 2 pm.

Figure 4: PD-PAINT with multiple antibodies binding to ryanodine receptors (RyR) in cardiac tissue sections. A) Two populations of secondary antibodies, labelled with extended docking sequences P1 D and P5d containing P1 and P5 motifs, respectively, bind to single primary antibodies i, ii: Exchange-PAINT imaging steps with imagers P1i and P5i. iii: A strand S1 binds to the extended docking sequence P1d, preventing imagers from binding iv: Strand S2 binds to docking sequence P5d. It opens up the S1 loop, if in close proximity. This allows imager binding to the S1 docking domain. Bottom: rendered DNA-PAINT images of the respective steps. B) Overlay of the DNA-PAINT images described in a (P5 yellow, P1 magenta, PD-PAINT cyan). PD-PAINT signal typically only appears where P1 and P5 signals show co localisation (inset). The intensity profiles shows that the magnitude of PD-PAINT detected co localisation can differ from image-based co-localisation. C) PD-PAINT with two primary antibodies against RyR. Top: PD-PAINT signal, bottom: overlay of steps, colours equivalent to B. Scale bars: 2 pm, inset (b): 500 nm, inset (c): 1 pm.

Figure 5: Detail view of data shown in Fig. 3 c. A) sketch, showing PD-PAINT strands S1 and S2 binding to two different oligo-labelled secondary antibodies which in turn bind two populations of primary anti-RyR antibodies. B) P1 image, C) Conventional Exchange-PAINT image of the two populations of secondary antibodies with P1 image B and P5 image C D) only S1 added, E) both S1 & S2 added - PD-PAINT image, F) overlay, yellow - P5, magenta - P1 , cyan - PD-PAINT. Scale bars: 2 pm, inset 1 pm.

Figure 6: Negative control experiment. A) Schematic of locations of RyR and COLVI, B) RyR image (with P1) C) collagen-VI image (with P5), D) RyR - COLVI overlay. Some regions suggest apparent co-localisation (see arrows pointing at“yellow” regions). E) S1 attached to RyR ABs, S2 to COLVI ABs. Distance too large to give any PD-PAINT signal, suggesting apparent co-localisation of RyR and COLVI in d does not reflect molecular proximity. Scale bars: 2 pm, inset 1 pm.

Figure 7: Quantitative imaging with PD-PAINT. A) Widefield image of a streptavidin- coated nanoparticle (grey), with rendered DNA-PAINT image of a single biotinylated docking strand (yellow), used for qPAINT calibration. B) Mean dark time T_D,mean calculated by fitting an exponential to the cumulative dark time distribution of the calibration site shown in A. C) Nanoparticles coated with conventional P1 docking strands (left) and PD-PAINT strands S1 and S2. D) Using the calibration obtained in B, the number of accessible docking sites for PD- PAINT (black) and conventional DNA-PAINT (blue) per nanoparticle is calculated. Values shown in grey take into account that 50% of the biotinylated strands in PD-PAINT not containing docking sites (strand S2). A remaining reduction of available sites compared to conventional DNA-PAINT is expected, e.g. due to a stochastic distribution of S1/S2 pairs. Scale bars: A 250 nm, C 2 pm.

Figure 8: Multichannel PD-PAINT imaging of two populations of streptavidin-coated nanoparticles. Bead population 1 was labelled with biotinylated PD-PAINT strands S1 and S2 containing a P1 docking sequence (green), population 2 with S1 B and S2B containing a P5 docking sequence (red). A) Simultaneous imaging by spectral multiplexing, i.e. different imager sequences are labelled with dyes of different emission spectra. They are simultaneously excited by a single laser source but the detection path is chromatically split. For details on the optical setup see Baddeley et al (2011). Atto 655 is conjugated to imager P1 , Atto 700 to imager P5. B) Temporal multiplexing (“Exchange-PAINT”), both imagers are labelled with Atto 655 and imaged sequentially, details see Jungmann et al. 2014. C) Rendered image of PD-PAINT by spectral multiplexing. D) Rendered image of PD-PAINT by Exchange-PAINT. E) Proposed use of multiplexed PD-PAINT for imaging of different populations of protein pairs. Scale bars: 2 pm, insets: 500 nm.

Figure 9. Evaluation of PD-PAINT properties with DNA origami a: Origami tiles assembled with fixed separation d (depending on design, d = 5, 10 or 15 nm) between sites D1 and, D2 and immobilized on a glass coverslip, were initially probed with imager P1 against a complementary domain in D1 to ascertain tile completeness (D1 control phase) b: Addition of both the S1 , binding to D1 , and S2+B strands, binding to D2 sites, prevents further signal from P1 imagers c: Introduction of the shield removal sequence R displaces B from S2, enabling SI- 82 hybridization if they are sufficiently close (P1 imaging phase) d: Complete origami tiles (all 6 sites visible during initial probing period, as in a), with a spacing of 5 nm between D1 and D2 sites yielded -52% of sites appearing in the S1-S2 interaction period (c) of the experiment, expressed as a fraction of sites sampled in the control period (a), n=153. As the distance between sites increased, (e) 10 nm and (f) 15 nm, the fraction of recovered sites decreased to -28% and -18%, n=272 & 369 complete tiles, respectively. All data acquired from two separate experiments for each origami tile version i-vi exhibit rendered example images of origami tiles of varying PD-signal. Top row, green, D1 site sampling to ascertain complete tile. Middle, red, PD signal reporting 1-6 sites during the S1-S2 interaction period (c). Bottom, overlay of the two channels. Frame numbers used to generate super-resolution images: D1 Control phase ~40k and S1 opening period ~100k. Scale bar: 30 nm.

Figure 10: Kinetics of PD-PAINT investigated on DNA origami a: Sequences of binding events at single origami binding sites were extracted during the D1 control phase with P1 imager binding directly to D1. b: Similar binding event traces were recorded in the S1 imaging phase, after labelling D2 binding sites with S2-B and removing B with the addition of R. For each site the first passage time f_fpt was determined, as the time distance between the addition of R and the first recorded bright event c: Visual comparison of a number of observed binding event time traces during the D1 control phase and the S1 imaging phase d: histogram of the recorded first passage times and the corresponding cumulative distribution function (CDF). The latter was fitted with Eq. (1) to extract the S1-S2 dimerization, rate k_open. The fit to the CDF and the one to the first passage time distribution histogram (derived by differentiation) are shown as dashed lines.

Figure 11 : PD-PAINT assay of the proximity of cardiac proteins JPH and RyR in isolated cardiomyocytes. Shown in a-c from left to right: DNA process schematic (not to scale), single molecule time trace of events, and final rendered image a: Initial imaging of P1 docking sites. SI introduced at -500 nM binding to D1 and preventing further P1 imager docking events. Single molecule events are reduced to background levels within an approximate 10 min incubation period b: Following an exchange-PAINT protocol P5 imagers are then used to probe RyR on D2. Pre-annealed S2-B then bind to D2 preventing further RyR sampling, taking -10 minutes to reduce events c: The addition of the displacement sequence R removes B and enables S1-S2 dimerization when in close enough proximity. This process is stochastic and the single molecule events gradually increase over time as more S1 loops are opened d: Overlay of super-resolution images obtained at each stage (a-c, rotated 90°) showing JPH (yellow), RyR (magenta), and PD-PAINT signal (Cyan). Magnification of boxed regions left (i) where JPH and RyR signal is clearly present within the cluster but are seemingly not in close enough proximity for S1-S2 dimerization and (ii) showing all 3 signals present (arrows highlight strongly overlapping signals which appear white). Frame numbers used to generate super-resolution images: D1 & D2 ~30k & open S1 sites ~60k. Scale bars: (a-d) 1 pm, (i-ii) 100 nm.

As discussed above, the method of the present invention allows proximity detection to be decoupled from local imaging resolution. The present inventors undertook significant investigation to develop the method of the present invention which has high specificity and selectivity and does not suffer from the disadvantages of presently used methods of analyte detection.

Materials and Methods

PD-PAINT materials and sample preparation

Oligonucleotide sequences (see Table 1) were designed and checked with the Nupack web application (www.nupack.org) and purchased from Eurofins Genomics (Eurofins Scientific, Luxemburg) and IDT (Integrated DNA Technologies, Coralville) with high-performance liquid chromatography (HPLC)-purification. The extended docking strand P5d was modified with a fluorescein dye to aid conjugation to secondary antibodies and to help with identifying region of interests within tissue sections using fluorescent widefield mode. Fluorescein was not detected in the DNA-PAINT imaging channel and was photobleached before DNA-PAINT imaging and thus did not interfere with super-resolution results. Lyophilized DNA for antibody conjugation was resuspended in phosphate buffered saline (PBS, pH 7.4, Sigma-Aldrich), all other DNA was resuspended and stored in Tris-EDTA (TE, pH 8.0, Sigma-Aldrich). Concentrations of oligonucleotides were checked at the DNA absorbance peak (260 nm) and the respective dye absorbance peaks on a NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham). Antibody conjugation of extended docking strands followed a click-chemistry protocol described by Schnitzbauer et al. (Schnitzbauer et ai, 2017). For super-resolution imaging, DNA-PAINT buffer (PBS, 500 mM NaCI, pH 8.0) was used, see“buffer C” in (Jungmann et a!., 2010). Table 1 : Oligonucleotide sequences

Name Sequence

Biotin-Sl GGA AGG AGG AGA GGA GAA TAC ATC TAT ATT CTC CTC TCC TCC TTC CTT TTT

TTT TTT TTT T [BIO] (SEQ ID No: l)

Biotin-S2 [BIO] TTT TTT TTT TTT TTT GGA AGG AGG AGA GGA GAA TA (SEQ I D No:2)

Biotin-SIB GGA AGG AGG AGA GGA GAT TAG GTA AAT AAT CTC CTC TCC TCC TTC CTT TTT CTT

CAT TAT T [BIO] (SEQ ID No:3)

Biotin-S2B [BIO] TTT TTA ATT GAG TAT GGA AGG AGG AGA GGA GAT TA (SEQ I D No:4)

51-Pld GGA AGG AGG AGA GGA GAA TAC ATC TAT ATT CTC CTC TCC TCC TTC CTT TTT

TTT TTT TTT TTA GTA AGT GAA TAA TGA AGA AAT AGA TGT ATA A (SEQ ID No:5)

52-P5d CTT CCT CAC AAT CAA AAT TTA CCT AAA ATT TTT TTT TTT TTT TGG AAG GAG

GAG AGG AGA ATA (SEQ I D No:6)

Pld [AzideN] TTA TAC ATC TAT TTC TTC ATT ATT CAC TTA CTA (SEQ I D No:7)

P5d [AmC6] TTT TAG GTA AAT T [FLUdT] TTG ATT GTG AGG AAG (SEQ I D No:8)

PI imager CTA GAT GTA T [At655] (SEQ I D No:9)

P5 imager CTT TAC CTA A [At655] (SEQ I D No: 10)

PI docking [BIO] TTA TAC ATC TA (SEQ I D No:ll)

Streptavidin-coated polystyrene particles (diameter: 500 nm, Microparticles GmbH, Berlin) were labelled with biotinylated DNA as described elsewhere (Lutz et al., 2018). For PD-PAINT with both S1 and S2 strands present, S1 and S2 were mixed prior to their addition of the nanoparticles to ensure a stochastic distribution of biotinylated strands S1 and S2 on biotin binding sites.

Porcine cardiac tissue was fixed in 2% paraformaldehyde (PFA, pH 7.4, Sigma-Aldrich) for 1 hour at 4°C. Tissue samples were cryo-protected using solutions with increasing concentrations of sucrose (up to 30%) and frozen in 2-Methylbutane (Sigma-Aldrich) cooled by liquid nitrogen. Cryo-sections were taken at -5-15 pm thickness onto pre-cleaned No. 1.5 coverslips coated with 0.05% poly-L-lysine (Sigma-Aldrich). Coverslips were attached to open-top PMMA imaging chambers (compare Crossman et al. (Crossman et al., 2015)). The tissue was permeabilised with 0.1 % Triton X-100 in PBS for 10 minutes and then incubated with a droplet of Image-iT™ FX signal enhancer for 1 hour at room temperature. Primary antibodies, (Ryanodine receptor (RyR) (MA3-916, mouse & HPA016697, rabbit) and collagen VI (ab6588, rabbit) were incubated overnight at 4°C in an incubation solution containing 1 % BSA, 0.05% Triton X-100 and 0.05% NaN₃ in PBS. Samples were washed at least three times for 10-20 minutes each in PBS The respective anti-mouse and anti-rabbit secondary antibody (Jackson ImmunoResearch, West Grove) conjugations were added in incubation solution for 2 hours at RT. Tissue sections were then finally washed three times with DNA-PAINT buffer before imaging. PD-PAINT strands Tissue-S1 and -S2 were added at a concentration of 100 nM in DNA-PAINT buffer.

Imaging setup

Images were acquired on a modified Nikon Eclipse Ti-E inverted microscope (Nikon, Tokyo) containing a 60* 1.49NA APO oil immersion TIRF objective (Nikon, Tokyo) with an Andor Zyla 4.2 sCMOS (scientific complementary metal-oxide-semiconductor) camera (Andor, Belfast). A piezo objective scanner (P-725, Physik Instrumente, Karlsruhe) was used for focus control. For imager excitation in super-resolution imaging, a 642 nm CW diode laser (Omikron LuxX, Rodgau) was used, at a power of approximately 15 mW on an illumination spot of approximately 30 pm diameter. Widefield fluorescence images were excited with a tunable LED-light source (CoolLED, Andover). While thermal drift was reduced with a custom objective holder, any residual focal drift was compensated by continuous feedback from an auxiliary camera in transmission mode at a non-interfering wavelength (Jayasinghe et al., 2018; Lutz et al., 2018), similar to a method described by McGorty et al. (McGorty et al., 2013). Tracking data from the auxiliary camera was used in post-analysis for lateral drift correction, deviating aberrations between the tracking and fluorescence emission imaging paths were corrected for.

Image acquisition and analysis

Streptavidin-coated nanoparticles were imaged in total internal reflection fluorescence (TIRF) mode with a focus just above the coverslip, tissue sections were imaged in highly inclined and laminated optical sheet (HILO) modes, focussing approximately 1 pm into the tissue. The entire imaging process, including hardware control, localisation and fitting and post-processing was carried out in the Python Microscopy Environment (PyME), available freely via: https://bitbucket.org/christian_soeller/python-microscopy-exeter/. Integration time for super resolution image acquisition was set to 100 ms. For localisation, individual binding events were detected and fitted to a 2D Gaussian photon distribution. Out-of-focus binding events were suppressed in a post-processing step by filtering with respect to parameters of the fitting, e.g. localisation errors and photon number per event. Lateral drift was corrected for using tracking data from an auxiliary camera as described above. Binding events which were detected over multiple subsequent frames were merged and the images rendered by jittered triangulation (Baddeley et ai, 2010).

Example 1

Fig. 1 shows the principle of PD-PAINT, where fluorescence signals are only detected if two epitopes of interest are within close distance to each other. To implement PD-PAINT, the two target epitopes are labelled with nanostructures S1 and S2 via suitable markers. Labelling can be done by linking the nanostructures to antibodies (or nanobodies) or by hybridizing them to other DNA strands, previously connected to the epitopes. Strand S1 contains the 9 nt docking sequence consisting of domains 5* (3 nt) and 6 (6 nt). The docking sequence is complementary to the imager strands P1 (6*-5). However, when S1 is spatially isolated, the docking site is protected by a closed stem-loop motif in which 5* is hybridized to the complementary domain 5, and 6 is wrapped to form a short loop. The loop is further stabilized by the complementary domains 3-4 and 3*-4* (11 bp). In the closed-loop configuration of S1 the docking site has negligible affinity for the P1 imager (Fig. 1a), as previously determined in the context of catalytic DNA reactions⁹ and further demonstrated here by means of coarse-grained computer simulations discussed below. The second target is labelled with nanostructure S2, complementary to the stem region of S1 (3-4) as well as the un-protected toe-holding domain 1- 2. The latter would promote rapid S1-S2 dimerization, should the two nanostructures be present in sufficient concentration and have unrestricted access to each other, e.g. during labelling steps. To prevent unwanted S1-S2 dimerization, S2 is therefore initially protected by a shield strand B, which occupies the toe-holding region 1*-2* and is further stabilized by adjacent complementary domains 3 and 7. Once both S1 and S2 have fully bound to their appropriate targets, and any excess washed away, a removal strand R is used to displace B from S2, freeing 1*-2* (Fig. 1 b). At this stage, S1-and S2 can dimerize, but only if their tethering locations are within a given maximal distance from each other. When S1 and S2 are fully hybridized, the open loop exposes the docking sequence 5*-6 of S1 , allowing transient binding of the complementary imager P1 , which results in frequent detection of these binding events as fluorescent blinks, i.e. , a DNA-PAINT signal (Fig. 1c). Whereas Fig. 1a-c show a direct tethering geometry (geometry 1), in the experiments described below an indirect tethering geometry (geometry 2) was employed, based on two attachment strands D1 and D2 which are anchored on the substrate. S1 and S2-B, when added in solution, attach to D1 and D2, respectively, as shown in Fig. 1d.

To explore the functionality of the proposed PD-PAINT scheme its thermodynamic properties were investigated by means of Monte Carlo (MC) simulations using the oxDNA coarse-grained model of nucleic acids. For PD-PAINT to elucidate the co-localization of epitopes, it must obey two thermodynamic design criteria. First, the S1 loop must be opened if S2 is nearby. Second, the closed loop should not interact with its imager in a way that leads to detectable blinks, i.e. any transient binding event of P1 to the closed S1 loop must be much briefer than those detected for the open S1 loop.

Free-energy calculations were implemented to determine the likelihood of the formation of S1- S2 dimers, resulting in the opening of the S1 loop, as a function of the distance d between their anchoring points. Simulations were performed for both direct anchoring (geometry 1 , Fig. 1 a-c) and indirect anchoring (geometry 2, Fig. 1d). In geometry 1 , the reactive domains of S1 and S2 are separated from their anchoring points by a 15-nt polyThymine sequence, while in geometry 2, directly relevant to experiments, the reactive domains of S1 are further spaced by a 32 base- pair rigid double-stranded (ds) DNA domain.

Figure 2a reports the computed S1-S2 dimerization free energy as a function of the tethering distance. In both tested geometries the free energy gain of dimerization is substantial at short separations, with minima between -12 and -10 k_BT. At large separation distances the dimerization free energy sharply rises, with the increase occurring at larger separations for geometry 2 (right hand side symbols), due to the additional 32bp dsDNA spacer connected to S1. The S1-S2 dimerization probability is shown in Fig. 2b. Both geometries display an approximately step-like response, with dimerization probability essentially equal to one for separations below ~10 nm for geometry 1 (left hand side plot symbols) and ~20 nm for geometry 2 (right hand side plot symbols). At larger separation distances, dimerization rapidly becomes essentially impossible, and the probability falls to zero at ~16 nm (geometry 1) and ~25 nm (geometry 2).

For the presence and location of S1-S2 dimers to be positively identifiable via DNA-PAINT, binding of the imager to closed S1 loops must be practically undetectable, yet the imager must have a sufficient affinity for the exposed docking site when the S1 loop is open. We verify this by estimating the interaction free-energy between the P1 imager and S1 in its closed and open loop states, shown in Fig. 2c as a function of the number of formed base-pairing bonds between the two strands. The free energy barrier for the formation of the first bond is similar between the open and closed loop configurations but while in the closed-loop case further base pairing does not result in a significant drop in free energy, a steep monotonic decrease is observed for the open-loop configuration. As a result, we estimate the average duration of binding events between the imager to the closed S1 loop as ~2 ps, well below the detection threshold for a typical DNA PAINT experiment (see Supporting Information and Table S5). In turn, for the open loop configuration we predict the binding events to last -0.2 s, ideal for DNA-PAINT at typical frame integration times of 50-300 ms. Comparison between the binding free energy of P1 to the open S1 loop of a S1-S2 dimers to that towards a conventional single-stranded (ss) DNA docking strand reveals no perceptible difference (Fig. 2c), and, consistent with this observation, the predicted bond lifetime in the two cases was found to be comparable.

Example 2

As a well-studied model that is expected to yield a positive PD-PAINT signal, biotin-binding sites of streptavidin were imaged (Figure 3). Streptavidin binds up to four biotin molecules, with two sites each located on each side of the protein tetramer, at a distance of -2 nm from each other. The very high affinity of biotin ensures an effectively permanent attachment of strands S1 and S2. The present inventors used streptavidin attached to the surface of polystyrene colloidal particles. Note that only two of the four biotin-binding sites are typically to be available for surface-attached streptavidin. The prominent ring structure seen in an optical section generated by the PD-PAINT signal provides a positive confirmation that binding of DNA-PAINT imagers is observed as expected. Biotinylated strands S1 and S2 were mixed thoroughly before streptavidin-coated nanoparticles were dispersed with them to ensure a stochastically distributed attachment of S1 and S2 to the biotin-binding sites. Assuming biotinylated S1 and S2 bind with about equal affinity, on average approx. 50% of biotin site pairs should contain S1/S2 pairs., which enable the S1 loop to be opened up. Figure 3B and C show a rendered image of the strong DNA-PAINT signal, indicating an opened stem loop structure. If imagers were added at concentrations much higher (-100 times) than the one used for PAINT, as done in Figure 3d, nearly all docking sites are be occupied by imagers resulting in a fluorescence signal visible in widefield fluorescence mode.

To confirm the negligible interaction between imagers and isolated S1 with a closed loop, streptavidin was imaged without addition of S2 (Figure 3E-H). No DNA-PAINT signal is detected, indicating that imager-S1 binding events have a duration in the region of the microsecond and are thus undetectable. Hardly any binding is observed even at high imager concentrations (Figure 3H).

This example confirms that the method of the present invention can be utilised to detect an analyte of interest, in this case streptavidin, using first and second probes. The method of the present invention is sensitive in the nanometer range resulting in high resolution results.

Example 3 Figure 4 demonstrates the method of the present invention (PD-PAINT) in biological samples, namely fixed cardiac tissue. The primary antibody, of mouse origin, is targeted against the cardiac ryanodine receptor (RyR2). This primary antibody has been used for DNA-PAINT imaging of RyRs previously (Jayasinghe et ai, 2018). By applying the S1 and S2 secondaries simultaneously, at equal concentration, we achieved a high proportion of primary antibodies being occupied by both S1 and S2 secondaries. This effectively recreates the scenario validated in Figure 3 with streptavidin (the adjacent bindings sites are here the epitopes on the same primary antibody) albeit now in a biological tissue.

In order to allow the sequential imaging of all targets involved and to also study the dynamics of PD-PAINT while imaging, S1 and S2 were not directly conjugated to the antibodies. Instead, extended docking sequences P1d and P5d were attached to the secondary antibodies that would later carry the S1 and S2 strands, respectively. P1d and P5d were designed to respectively bind the modified S1 and S2 oligonucleotides with high affinity, resulting in an effectively irreversible labelling. The two secondary antibodies were initially imaged (Figure 4A i+ii) in conventional Exchange-PAINT mode (Jungmann et ai, 2014), with P1 and P5 imagers interacting with P1d and P5d which also host docking domains for these imagers. The good agreement of P1d and P5d images (Figure 4A i+ii bottom panels) suggests a high proportion of primary antibodies labelled with both types of secondary antibodies.

When strand S1 was added at sufficient concentration, while imager P1 was still present, the binding rate for imager P1 was eventually reduced to background levels, because S1 permanently hybridises with P1d, blocking the docking site (Figure 4a iii). Notably, during this step the P1 docking site of S1 remains inaccessible due to the closed stem loop structure. The suppression of DNA-PAINT binding occurred rapidly, within <5 min, which indicates rapidly saturating binding of P1d and S1 strands. Added S2 attached with a similarly high rate to P5d (observed with imager P5, data not shown). If a pair of secondary antibodies labelled with P1d and P5d are located within close distance of each other, the proximity of S2 allows the S1 stem loop structure to open up and a DNA-PAINT signal to be detected (Figure 4A iv). A constant DNA-PAINT binding rate was reached after approximately 30 min, indicating that an equilibrium state of S1/S2 complex formation was reached.

PD-PAINT signals are only expected in areas of co-localisation of the two conventional DNA- PAINT channels, as shown in the overlay of the three channels in Figure 4B (inset). The limited resolution of conventional DNA-PAINT in tissue samples, especially along the optical axis (up to several 100 nm in regular 2D SMLM imaging), leads to a non-negligible number of false positive co-localisation signals. This difference and an indication of the degree of co-localisation can be estimated from differences between the conventional DNA-PAINT signals and PD-PAINT (Figure 4B, cross-section through several RyR2).

Figure 4C and Figure 5 demonstrate the application of PD-PAINT to detect the proximity of two populations of primary antibody proteins, which bind to different epitopes of RyR2. With the nanoscale resolution enabled by PD-PAINT, the density of these antibody-pairs can be detected as well as their distribution into clusters. The secondary antibodies and PD-PAINT scheme were similar to that described above, with the exception that the P1d docking/attachment sequence was linked to an anti-mouse secondary antibody that binds to the RyR primary raised in mouse. The P5d docking/attachment strand in turn, was linked to an anti-rabbit antibody against the second RyR primary antibody which was raised in rabbit. PD-PAINT observations were broadly consistent with those obtained with S1 and S2 attached to the same primary, however, a lower imager binding rate was detected. With the binding rate being proportional to the number of available docking sites, the reduced rate indicates that a lower number of S1/S2 strand pairs have interacted, likely due to an increased average distance between S1 and S2.

Example 4

The steep dependency of PD-PAINT signals on the proximity of strands S1 and S2 is further demonstrated in a negative control experiment (Figure 4). In this experiment, collagen VI and RyR2 were labelled with primary and secondary antibodies so that strands S1 and S2 were targeted against collagen VI and RyR primary antibodies, respectively. RyR and collagen VI follow a similar pattern oriented along the t-tubules, with some areas in conventional DNA- PAINT imaging suggesting co-localisation. A lack of corresponding PD-PAINT signal, however, suggests that these are false positives of conventional imaging and that no epitopes of Col VI and RyR2 are located within high proximity to each other. This is to be expected as, although relative distances can often be smaller than 100 nm, Col VI and RyR2 are located in distinct compartments within a cardiac cell.

In general, the reduction of PD-PAINT imager binding rates with increasing distances of the imaged epitopes confirms the distance dependency of S2 to S1 strand displacement and confirms that S1 and S2 do not interact if either S1 or S2 are not bound to an analyte.

This negative control experiment further highlights the high resolution results which can be obtained using the method of the present invention. Unlike conventional co-localisation techniques, in the method of the present invention proximity detection is decoupled from imaging resolution which results in a highly sensitive method. This is especially important in biological cell and tissue samples where the achievable resolution can vary greatly due to refractive index inhomogeneities and related optical challenges.

Example 5

Figure 7 demonstrates the possibility to obtain quantitative information on the number of docking sites, and thus on the number of protein pairs from PD-PAINT by applying a method previously demonstrated for conventional DNA-PAINT (“qPAINT”). Biotin-binding sites on streptavidin-coated particles are images imaged as discussed above. A single biotin binding site labelled with a docking strand is used as calibration. Binding of a single docking strand was achieved by adding docking strands in solution at very low concentration (0.5 nM). The number of docking sites is calculated via the imager-docking association rate k_on, which in turn depends on the mean dark time. The mean dark time is calculated from an exponential fit to the dark time distribution. With an imager concentration of c, = 5 nM, the fitted mean dark time gives an imager-docking association rate k_on = (T_D,meanCi) ¹ = 3.5x10⁶ s ¹M ¹ , which is in agreement with previously reported values. The association rate then allows to calculate the number of imaged docking sites of the particles. The measured number of accessible binding sites is in agreement with the particle’s binding capacity as stated by the supplier and shows an expected reduction for PD-PAINT imaging due to two major reasons, (1) 50% of biotinylated strands do not contain a docking sequence (i.e. strand S2, corrected values shown in grey in figure 7D), (2) a stochastic distribution of strands S1 and S2 reduces the number of S1 -S2 pairs to approx. 50%. Correcting for the stochastic pairing of S1-S2 pairs yields binding site numbers very close to those of conventional DNA-PAINT, which indicates that in this case a majority of S1 loops in proximity to S2 strands are in an open state.

Example 6

Figure 8 demonstrates the possibility to obtain multiplexed information by PD-PAINT. Two examples include (1) spectral multiplexing, i.e. different imager sequences are labelled with dyes of different emission spectra. They are simultaneously excited by a single laser source but the detection path is chromatically split, and (2) Temporal multiplexing (“Exchange-PAINT”), both imagers are labelled with Atto 655 and imaged sequentially. The concept can easily be generalised for the imaging of nanoscale distribution of different protein pair populations.

Example 7

Having established the viability of the platform in-silico, the predictions of the simulations were tested by establishing the PD-PAINT scheme using a DNA-origami platform. DNA origami tiles were produced. The tiles each featured 6 pairs of single stranded D1 and D2 overhangs, to which S1 and S2 can hybridize as illustrated in Fig. 9, thus realizing the conditions of geometry 2 in Fig. 1c. Three types of tiles were tested, with nominal distances between S1 and S2 tethering points d=5, 10 or 15 nm, as shown in panels 3d-f. Strand D1 contains a P1* docking domain, complementary to imager P1 (Fig. 9a), which allowed to directly image the origami in the absence of S1 and S2, and assess the quality of the tiles. In the following, this experimental stage is referred to as the“D1 control phase”. An average of 5 out of 6 spots were counted on the origami, with no substantial variation between tiles with different binding-site distances. The absence of a few spots was expected, and ascribed to imperfections in the synthesized origami. Subsequently, S1 was added, which as it binds to the entire D1 strand, blocks access to the previously imaged docking domain on D1 (Fig. 9b). In the following step, the protected S2 (S2- B) nanostructure is added, and it binds to D2 (Fig. 9b). At this stage no increase in event rate was observed, demonstrating the successful hinderance of direct S1-S2 dimerization. Finally, the shield strand B was removed by adding an excess of remover strand R, exposing the S1-S2 toehold domain, enabling their dimerization and making the docking domain on S1 accessible for imaging with P1 (Fig. 9c). This latter stage of the experiment is referred to as“S1 imaging phase”.

After removal of the shield and allowing sufficient time for S1-S2 dimerization to occur (up to 10,000 s), the fraction f_PD of sites which first appeared in the D1 control phase was determined and then detected again during the S1 imaging phase (as shown in Fig. 9a). For all three tile configurations, a reduction in the number of detected sites per tile was observed, as demonstrated in Fig. 9d-f. The fraction f_PD decreased with increasing S1-S2 tethering distance from -52% for d=5 nm, to -28% at d=10 nm and -18% at d=15 nm.

This observation is in apparent contrast with the simulation results of Fig, 2b (right hand side plot symbols), predicting a high S1-S2 dimerization probability with little dependence on d, up to a nominal threshold distance beyond which S1-S2 interaction becomes unlikely, largely due to geometrical constraints.

This discrepancy may be partially accounted for by defects in origami assembly (e.g. missing D2), incomplete saturation of the available D2 with S2, or incomplete displacement of B upon adding the shield remover R. In fact, it has been reported in various instances that reactions mediated by toeholding do not normally progress to 100%, nor does hybridization between fully complementary DNA strands. However, none of these effects would be expected to depend on the distance between D1 and D2, and therefore cannot account for the drop of f_PD with increasing d. Another possibility is that differences in S1-S2 dimerization kinetics for different distances d could account for the unexpected distance dependence of f_PD. While the free energy calculations in Fig. 2 indicate that the equilibrium dimerization probability should be independent of d within the studied range, they do not offer direct insight into the kinetics of the process. Dimerization times increasing with increasing d could result in a reduced fraction of S1 sites opening over the duration of the observations, in a distance-dependent manner.

To verify this hypothesis and gain further insight into the properties of S1-S2 dimerization we analyzed the“blinking” behavior of individual origami sites, both during the D1 control phase (Fig. 10a) and the S1 imaging phase (Fig. 10b). In Fig. 10c, each horizontal line of dots indicates a timeline of events in which P1 binds to a docking domain (on D1 or S1) at a single origami binding site, with each dot representing a single binding event. The left-hand-side of Fig. 10c shows several typical binding sequences during the D1 control phase. As expected, the duration of dark times measured between subsequent bright events follows an exponential distribution, from which we can estimate a P1 on-rate k_on of 2.3- 10⁶ s ¹ M ¹, a value in good agreement with previous observations for the P1 imager binding to its docking site.

The second set of traces on the right-hand side of Fig, 10c is representative of the events recorded in the S1 imaging phase, after removal of the shield strand. The addition of remover R is indicated by a dashed line. For each site the first passage time³⁰ f_fpt was measured, as the time interval between the addition of the remover and the first observed bright event. The distribution of f_fpt is shown in Fig. 10d for an origami design with d= 5 nm, with its cumulative distribution function displayed in the inset. Under the reasonable assumption that the timescales of shield displacement are much faster than S1-S2 binding that leads to loop opening, the distribution of f_fpt should depend on the imager binding on-rate k_on and the S1-S2 dimerization rate k₀p_en .

_

r^tOn L^r -^LJ ^K-open

A fit of the cumulative distribution function derived from Eq (1) to the experimental data yields an estimate k_open = 7.7 10^-4 s^-1. We can then estimate the expected mean first passage time for the fitted k_open and k_on as (t_fpt) =—— h -— ^— , which yields a prediction of the expected kopen [Pl]fc_on

timescale of binding-site“activation” after the addition of the remover strand R. For d=5nm we find (tf_Pt)= 1500 s. However, no monotonic trend is observed at increasing d, as we determined (tf_pt) (10 nm) = 4400 s and (t_rpt) (15 nm) = 2300 s. For all explored values of d, the predicted activation times were so short as compared to the overall experimental timeframe (10,000 s) that we would expect all sites featuring S1 and S2 constructs to become active at least once. However, a subset of sites first detected in the D1 control phases did not show any bright events when imaging S1 , suggesting that these sites were not available for S1-S2 interaction, consistent with the observations in Fig. 9. The fact that the fraction of apparently unavailable sites increases with increasing d.

The DNA origami platform also enabled several control experiments to confirm that the number of false positives, i.e. signals detected from S1 without the presence of S2 in proximity, is low and that shielding of S2 worked as expected. In an experiment where only S1 was present, 1.89% of the origami binding sites imaged during the D1 control period were detectable in the S1 imaging period, indicating a very low false-positive rate. Similarly, protection of S2 with the shield B avoided interaction with S1 when applied at a concentration used during attachment whereas unprotected S2 is found to dimerize to tethered S1 strands. Finally, we confirmed that adding the shield remover R does not affect closed S1 loops in the absence of S2.

Fig. 11 demonstrates the applicability of PD-PAINT to study the proximity of two proteins in a biological sample. The cardiac ryanodine receptor (RyR) was labeled and the junctional protein junctophilin (JPH) in isolated and PFA fixed cardiac ventricular muscle cells, which had been previously shown to be in close proximity, a subset of JPH is within £50 nm of RyRs.

The two epitopes were first targeted by two distinct primary ABs, one rabbit-raised and the other mouse-raised. Secondary ABs conjugated to the D1 and D2 strands, respectively, were then applied, targeting the primary ABs for JPH and RyR, respectively.

Prior to adding S1 , the JPH epitopes were directly imaged using the docking site on D1 with imager P1 , as shown in Fig. 11a. A clear signal and the distinct patterns expected from these receptors were found. Upon addition of S1 , blocking of the P1 docking site on D1 resulted in a rapid drop in event rate to background levels, just as observed with the origami platform. Following an exchange-PAINT step to wash out P1 imagers, then directly imaged RyR receptors using newly added P5 imagers, complementary to a domain on the D2 strands. A broadly similar pattern to the one found for JPH (Fig, 11 b) was detected, as previously observed in dual-color super-resolution studies of RyR-JPH distribution. As expected, addition of the complex S2-B, capping D2, rapidly suppressed the P5 imager signal indicating successful attachment of S2-B. Finally, upon re-introducing P1 and adding the remover strand R, bright events resumed following the dimerization of S1 and S2, as expected, given the proximity of RyR and JPH (Fig. 11c). The rate of events increases over time, with a transient of a few minutes, in line with the dimerization kinetics observed for the origami platform.

Figure 11 d demonstrates that although JPH and RyR often appear co-localized within the same molecular cluster, the PD-PAINT signal is not always present, indicating that in some cases the JPH-RyR proximity is not sufficient to allow direct interaction and S1-S2 dimerization.

A second pair of cardiac proteins, RyRs and the intrinsic sarcoplasmic reticulum membrane protein SERCA, were imaged in a similar way with PD-PAINT in cardiac tissue sections, which provide an environment very challenging for optical super-resolution imaging.

The method of the present invention is advantageous as it can be modified to detect a range of analyte types using a range of analyte-binding domains. It will be appreciated that numerous modifications to the above described method may be made without departing from the scope of the invention as defined in the appended claims. For example, although in the examples given above the analytes detected are proteins, it will be appreciated by the skilled person, on reading the specification, that any other analyte of interest can be detected using the method of the invention. For example, the method of the present invention may be used to detect a nucleic acid (RNA, DNA or any modification thereof), lipid, small molecule, a cell, microorganism or fragment thereof. Furthermore, although the examples described are used in the detection of one or two analytes, the skilled person will appreciate that the method is not limited to the detection of one or two analytes, and proximity (or co-localisation) of three or four analytes could be detected using the method described herein.

References

Baddeley, D., Cannell, M.B., and Soeller, C. (2010). Visualization of Localization Microscopy Data. Microscopy and Microanalysis 16, 64-72.

Baddeley, D., Crossman, D., Rossberger, S., Cheyne, J.E., Montgomery, J.M., Jayasinghe, I.D., Cremer, C., Cannell, M.B., Soeller, C. (2011). PLoS ONE 6:e20645.

Crossman, D.J., Hou, Y., Jayasinghe, I., Baddeley, D., and Soeller, C. (2015). Combining confocal and single molecule localisation microscopy: A correlative approach to multi-scale tissue imaging. Methods 88, 98-108.

Jayasinghe, I., Clowsley, A.H., Lin, R., Lutz, T., Harrison, C., Green, E., Baddeley, D., Michele, L.D., and Soeller, C. (2018). True Molecular Scale Visualization of Variable Clustering

Properties of Ryanodine Receptors. Cell Reports 22, 557-567.

Jungmann, R., Steinhauer, C., Scheible, M., Kuzyk, A., Tinnefeld, P., and Simmel, F.C. (2010). Single-Molecule Kinetics and Super-Resolution Microscopy by Fluorescence Imaging of Transient Binding on DNA Origami. Nano Lett. 10, 4756-4761.

Jungmann, R., Avendano, M.S., Woehrstein, J.B., Dai, M., Shih, W.M., and Yin, P. (2014). Multiplexed 3D cellular super-resolution imaging with DNA-PAINT and Exchange-PAINT. Nat Meth 11, 313-318.

Lutz, T., Clowsley, A.H., Lin, R., Pagliara, S., Michele, L.D., and Soeller, C. (2018). Versatile multiplexed super-resolution imaging of nanostructures by Quencher-Exchange-PAINT. Nano Res. 1-14.

McGorty, R., Kamiyama, D., and Huang, B. (2013). Active microscope stabilization in three dimensions using image correlation. Opt Nano 2, 3.

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Claims

1. A method for detecting an analyte in a sample, the method comprising:

ii) contacting the sample with an imager nucleic acid; and

2. The method according to claim 1 wherein binding of the first probe-binding region to the second probe-binding region unfolds the secondary structure.

3. The method according to claim 2 wherein unfolding of the secondary structure makes available the imager nucleic acid-binding region for binding by an imager nucleic acid.

4. The method according to any preceding claim wherein the second probe-binding region is within, or partially within, the secondary structure.

5. The method according to any preceding claim wherein the secondary structure is a stem-loop structure.

6. The method according to claim 5 wherein the imager nucleic-acid binding region is within, or partially within, the stem-loop structure.

7. The method according to any preceding claim wherein the imager nucleic acid comprises a moiety which can be detected by way of a super resolution imaging technique.

8. The method according to any preceding claim wherein the imager nucleic acid is between 7 and 20 nucleotides in length.

9. The method according to any preceding claim wherein the analyte-binding domains are selected from a protein, lectin, soluble cell surface receptor, peptide, carbohydrate, nucleic acid, fragments thereof or any combination thereof.

10. The method according to any preceding claim wherein the analyte-binding domains comprise monoclonal or polyclonal antibodies or fragments thereof.

11. The method according to any preceding claim wherein step (iii) of the method comprises detecting imager nucleic acid bound to the imager nucleic-acid binding region using a super resolution imaging technique.

12. The method according to any preceding claim wherein the super resolution imaging technique comprises DNA-PAINT.

13. The method according to any preceding claim wherein step (iii) of the method comprises detecting single imager nucleic acids bound to the imager nucleic-acid binding region using a super resolution imaging technique.

14. The method according to any one of claims 1-12 wherein step (iii) of the method comprises detecting a small group of imager nucleic acids bound to the imager nucleic- acid binding region using a super resolution imaging technique.

15. The method according to any preceding claim wherein detecting the imager nucleic acid does not include amplification of the imager nucleic acid.

16. The method according to any preceding claim wherein the sample is an environmental sample, synthetic sample, clinical sample and/or biological sample.

17. The method according to any preceding claim wherein the method is for detecting modification of an analyte.

18. The method according to any preceding claim wherein the method is for detecting co localisation or proximity of two or more analytes.

19. A kit for detecting an analyte in a sample, the kit comprising: i) first and second probes wherein each probe comprises an analyte-binding domain and a nucleic acid domain, wherein

a. the nucleic acid domain of the first probe comprises a second probe binding region and an imager nucleic acid-binding region comprised within a secondary structure, such that said imager nucleic-acid binding region is not available for binding by an imager nucleic acid;

b. the nucleic acid domain of the second probe comprises a first probe binding region capable of interacting with the second probe-binding region of the first probe when the first and second probes bind the analyte;

c. wherein binding of the first probe-binding region to the second probe binding region makes available the imager nucleic acid-binding region for binding by an imager nucleic acid; and ii) an imager nucleic acid.