WO2018208234A1 - Detection probe for detecting and quantifying multimeric target proteins and methods of using the same - Google Patents

Abstract

Disclosed herein are probes comprising a plasmonic nanoparticle, wherein the nanoparticle comprises a target protein binding layer comprising multiple copies of nucleic acid molecules capable of binding to one or more types of target proteins capable of oligomerisation, wherein the target protein is a multimeric protein comprising at least four binding partners. Also disclosed are methods of using the probes as described herein for identification and diagnosis of diseases and drug screening, and a kit for the same. In particular, the invention relates to colorimetric detection of p53 protein function using gold nanoparticles conjugated with canonical DNA response elements.

Description

DETECTION PROBE FOR DETECTING AND QUANTIFYING MULTIMERIC TARGET PROTEINS AND METHODS OF USING THE SAME

CROSS-REFERENCE TO RELATED APPLICATIONS

[001] This application claims the benefit of priority of Singapore provisional application No. 10201703919T, filed 12 May 2017, the contents of it being hereby incorporated by reference in its entirety for all purposes.

FIELD OF THE INVENTION

[002] The present invention relates generally to the field of molecular biology. In particular, the present invention relates to the use of biomarkers for the detection and diagnosis of cancer.

BACKGROUND OF THE INVENTION

[003] Traditional approaches to study protein-DNA interactions such as electrophoretic mobility shift assay (EMS A) and DNA footprinting are labour-intensive, time-consuming and only semi-quantitative. Methods such as fluorescence anisotropy and Surface Plasmon Resonance (SPR) require expensive instrumentation. Immunochemical approaches like enzyme linked immunosorbent assay (ELISA), and chromatin immunoprecipitation Assay (ChIP) are heavily reliant on the quality of antibodies. In comparison, although Forster resonance energy transfer or fluorescence resonance energy transfer (FRET) based methods are considered to be more convenient compared traditional methods the limitations of FRET-based methods lie in the complicated and expensive DNA fluorescence labelling and half-sited DNA molecular beacon design required. Thus, there is an unmet need for a rapid, sensitiveassay to study protein-DNA interactions.

SUMMARY OF THE INVENTION

[004] In one aspect, the present invention refers to a probe comprising a plasmonic nanoparticle, wherein the nanoparticle comprises a target protein binding layer, wherein the target protein binding layer is selected from the group consisting of multiple copies of single stranded nucleic acids, multiple copies of double stranded nucleic acids and combinations thereof , wherein the target protein binding layer is capable of binding to one or more types of target proteins capable of oligomerisation; wherein the target protein is a multimeric protein comprising at least four binding partners. [005] In another aspect, the present invention refers to a method of detecting oligomerisation of multimeric target proteins having at least four binding partners, the method comprising contacting the probe of the present invention with a sample suspected to comprise the target protein, wherein oligomerisation of the target proteins with the probe is indicated by a colour change.

[006] In yet another aspect, the present invention refers to a method of evaluating the binding affinity of multimeric target proteins having at least four binding partners, the method comprising mixing a sample comprising unbound response elements with the multimeric target proteins, and allow binding of the response elements to the multimeric target proteins, to obtain a first mixture, contacting the probe as disclosed herein with the first mixture to obtain a second mixture, determining the presence or absence of a colour change in the second mixture, wherein the absence of colour change indicates that the multimeric target proteins have a higher affinity to the unbound response elements than to the probe.

[007] In a further aspect, the present invention refers to a method for screening drugs capable of restoring oligomerisation ability of multimeric target proteins, the method comprising contacting a sample comprising multimeric target proteins lacking, or thought to lack, oligomerisation ability with the drug to obtain a first mixture, contacting the first mixture with the probe as disclosed herein to obtain a second mixture, determining the presence or absence of a colour change in the second mixture, wherein the absence of colour change indicates that the drug is not capable of restoring oligomerisation ability of the multimeric target protein; wherein the multimeric protein comprising at least four binding partners.

[008] In another aspect, the present invention refers to a method of detecting the presence of absence of a disease in a subject, the method comprising contacting a sample obtained from the subject with the probe as disclosed herein; determining the presence or absence of a colour change in the mixture, wherein the absence of colour change indicates the presence of the disease; wherein the disease is caused or caused in part by lack of oligomerisation of one or more multimeric target proteins.

[009] In yet another aspect, the present invention refers to a kit comprising the probe as disclosed herein.

BRIEF DESCRIPTION OF THE DRAWINGS

[0010] The invention will be better understood with reference to the detailed description when considered in conjunction with the non-limiting examples and the accompanying drawings, in which: [0011] FIG. 1 shows a schematic illustration of the sensing principle of the colorimetric bioassay for sequence specific p53-DNA binding detection using AuNPs conjugated with dsDNA comprising p53 response element (RE). In more detail, Figure 1 shows an example of how the probes described herein can be used. In the example illustrated in Figure 1, a colour shift only occurs if the wildtype p53, which forms a tetrameric complex, is bound, whereas a mutation in a subunit of the p53 tetramerizing protein disrupts oligomerisation and thus tetramerisation to form the p53 complex. However, only the tetramerised p53 complex binds to the target protein binding layer of the probe(s). The mutation disrupts tetramerisation and thus binding to the target protein binding layer of the probe. Binding of the tetramerised p53 complex results in binding to the target protein binding layer of the probe and aggregation of the different probes. Aggregation of the different probes is the result of the fact that the multimeric target protein, in this example, the tetramerised p53 complex, can bind to different target protein binding layers of different probes as illustrated in Figure 1. This results in an aggregation of the probes which results in a colour change and in the illustrated example in a colour change visible with UV-vis as well as the naked eye.

[0012] FIG. 2 shows data showing that AuNP-RES have good mono-dispersity and a single peak at 530 nm. (a) shows UV-vis absorption spectra and corresponding photograph of the AuNP-RE solutions incubated in the absence (black dash line) or presence of p53CT-WT (60 nM, dark gray line) and p53CT-R273H (60 nM, gray line); (b-c) show TEM images showing the morphology and dispersity of AuNPs (b) without p53 and in the presence of (c) p53CT- R273H (d) p53CT-WT.

[0013] FIG. 3 depicts data showing that the aggregation of AuNP-Res is due to the binding of wild type p53 proteins, which can be detected immediately by naked eye observation of colour changes and quantified by absorption spectra analysis, (a) shows absorption spectra with increasing concentrations of p53CT-WT (b) shows the results of calibration of the absorbance ratio (A650/A520) vs. p53 concentration at a fixed amount of AuNP-RE probe (10 nM), which was applied in all measurements; insert shows the linear regression line for the range of 0-30 nM. (c) The colour photographs were taken at 5 minutes upon adding the AuNP-RE to the respective p53CT-WT samples of different concentrations.

[0014] FIG. 4 depicts data generated from subsequent experiments, (a) shows absorption spectra with increasing concentrations of p53CT-WT (b) shows the calibration of the absorbance ratio (A650/A520) vs. p53 concentration; a fixed amount of AuNP-RE probe (5 nM) was applied in all measurements, (c) shows column graphs representing statistical analysis of the generated data, indicating that AuNP-REs exhibit excellent selectivity towards p53CT-WT, while p53CT- R273H and nonspecific proteins such as bovine serum albumin (BSA), human serum albumin (HSA) shows negligible effect. p53CT-WT in a high background of BSA does not significantly affect the relative aggregation.

[0015] FIG. 5 shows data generated to show that the tetramerisation of p53 induces aggregation of AuNP-REs. (a) shown an UV absorption spectra and (b) shows column graphs representing the statistics of relative aggregation (A650/A520) of AuNP-RE upon mixing with pre-incubated p53CT-WT (60 nM) and p53CT-R273H mixture at increasing p53CT-R273H: p53CT-WT ratio.

[0016] FIG. 6 shows the UV absorption spectra of ERa- specific AuNP-REs before and after mixing with 40 nM ERa. The absence of any absorption peaks around 650 nm indicates the inability of the dimeric ERa to cause AuNP-REs aggregation.

[0017] FIG. 7 shows the results of the evaluation of DNA binding affinity using a competition assay, (a) shows a schematic diagram illustrating the principle of competitive assay, in which reduced AuNP-RE aggregation is expected in the presence of other response elements with lower K_D values. As described in the present disclosure, competitive binding assays (competition assays) rely on the difference in binding affinity of target proteins to different nucleic acid molecules. Such difference can be utilised to assay target proteins, as shown in (a), (b) Statistics of absorbance ratios (A650/A520) after pre-incubating wild type p53 proteins with different competing DNA promoter sequences including ConA, GADD45, Bax, WRNC and scrambled DNA (scrDNA). The underlying principle disclosed herein is that transcription factor tetramerization induces DNA-AuNPs aggregation and colour change. With regard to the competition assay (for example DNA sequence selectivity), low p53-DNA binding affinity results in a blue/purple colour, whereas a high p53-DNA binding ability results in a red colour.

[0018] FIG. 8 shows results response element (RE) binding p53 in vitro and drug screening. As described in the present disclosure, such drug screening assays rely on the capability of drugs and chemical compounds to reinstate the oligomerisation capability in proteins which have lost their ability to oligomerises. Reasons for such loss of oligomerisation ability can be, for example, the presence of mutations. Thus, drug screening methods, as shown in the schematic shown in the present figure, rely on the change in oligomerisation ability of target proteins based on the exposure of the target proteins to drugs and/or chemical compounds. Such difference can be utilised in drug screening, as shown in (a), (a) shows an illustration of the drug reactivation of mutant p53 protein, (b) shows a graph depicting the relative aggregation of AuNR-RE with wild type p53 in in vitro transcription and translation samples (IVT-WT) (c) shows column graphs depicting the results of a drug screening of inactive (PhikanXOOl), weakly activating (Phikan083) and strong activating (PK5176) drugs for reactivation of mutant p53 (Y220C) expressed in IVT and R273H as a negative control (n = 3, *p < 0.05).

[0019] FIG. 9 shows a histogram depicting the relative aggregation of AuNP-RE after enhancement of p53 levels by Nutlin-3 at indicated concentrations in HCT116 cell lysate.

[0020] FIG. 10 shows a schematic of a tetrameric transcription factor (represented in a ribbon diagram) and that the tetramerisation of transcription factors is crucial for their normal functioning. In this example, a mutation in the Tet domain of the protein is shown.

[0021] FIG. 11 shows a flow chart depicting how tetrameric transcription factors can function as biomarkers for specific disease, for example but not limited to diseases dependent on the p53 family (for example but not limited to, p53-related cancers, p63, p73-related developmental diseases), the STAT family (for example but not limited to, STAT3 and STAT5-related immune diseases), and estrogen receptor related diseases, such as but not limited to, cancer.

[0022] FIG. 12 shows a schematic drawing of an example of a direct assay for tetramer detection. The underlying principle disclosed herein is that transcription factor tetramerization induces DNA-AuNPs aggregation and colour change. With regard to the direct assay (for example protein detection), presence of wild type p53 results in a blue/purple colour (indicating tetramerisation of the peptide), whereas presence of mutant p53 results in a red colour (indication a lack of tetramerisation of the peptide).

[0023] FIG. 13 shows a schematic drawing of an example of a direct assay for drug screening using the claimed method. The underlying principle disclosed herein is that transcription factor tetramerization induces DNA-AuNPs aggregation and colour change. With regard to the direct assay (for example drug screening), presence of mutant p53 which is drug activated results in a blue/purple colour (indicating tetramerisation of the peptide), whereas failure of the drug to activate tetramerisation results in a red colour.

[0024] FIG. 14 shows column graphs depicting the binding of various nanoparticles conjugated with different transcription factors and different proteins. It is can be seen that nonspecific proteins, such as bovine serum albumin (BSA) and human serum albumin (HSA), have negligible interaction with the gold nanoparticle-conjugated response elements.

[0025] FIG. 15 shows column graphs depicting the different binding affinities (K_D) of DNA response elements based on the examples ConA, GADD45, Bax, and scrambled DNA (scrDNA). This competition assay had been designed to evaluate the binding affinity (K_D) of wild type p53 proteins to various promoter sequences. Their sequences and respective K_D values are shown in Table 1. Growth arrest and DNA Damage (GADD45) and Bel-associated X (Bax) are physiological promoter motifs involved in DNA repair, apoptosis. Additionally, a strong-binding artificial RE (ConA), non-responsive WRNC and scrambled DNA (scrDNA) were included as positive control and negative controls, respectively.

[0026] FIG. 16 shows data depicting the canonical DNA interaction between wild type p53 and RE-AuNP. (a) shows a schematic diagram showing working principle of the colorimetric assay in sensing sequence-specific DNA interactions with wild type p53. (b) shows an UV- visible absorption spectra of RE-AuNP probes in the (i) absence of p53 protein (black dashed line), (ii) presence of wild type p53 protein (p53-WT) (blue line) and (iii) presence of mutant p53 protein (p53-R273H) (red line), (c) Calculated aggregation ratio, and (d) shows TEM images for samples (i) to (iii) and their corresponding colour photographs (inset of Fig 16(d)).

[0027] FIG. 17 shows data depicting the sensitivity and selectivity of RE-AuNPs for detecting wild type p53-DNA binding, (a) shows a line graph depicting the aggregation extent (A650/A520) and colour differentiation (inset) of p53-WT and p53-R273H at different protein concentrations in the presence of RE-AuNP probes, (b) shows a Calibration plot showing a linear correlation of p53-WT and RE-AuNP interaction, (c) shows a column graph depicting the specificity of RE-AuNP probe response in the presence of different protein samples, (d) shows column graphs depicing the results of a competition assay for DNA selectivity based on the aggregation extent of RE-AuNP after pre-incubating wild type p53 protein with competing DNA-RE sequences of different dissociation constants: K_D of Con A (consensus sequence) = 1.0 + 0.2 nM, GADD45 sequence (Growth Arrest and DNA Damage) = 7.7 + 1.2 nM, Bax sequence (bcl-2-like protein 4) = 73.7 + 33 nM, and non-binding or scrambled DNA sequence (scrDNA).

[0028] FIG. 18 shows data depicting the aggregation extent of RE-AuNP probes for the evaluation of drug-dependent p53-Y220C reactivation. p53 proteins were IVT-synthesized and exposed to various compounds including DMSO vehicle control, PK-X001 (inactive analogue), PK-083 (weak) and PK-5176 (strong) before examining RE-AuNP binding activity. IVT- expressed wild type p53 (red bar) and p53-R273H (green bars) exposed to the same drug panel shows control signals for probe-binding and drug- specificity, respectively. Error bars represent mean + S.D. of three independent binding experiments. Two-tailed student's t-tests were performed to assess statistical significance (**p<0.01).

[0029] FIG. 19 shows data illustrating the effect of a loss in p53 protein canonical binding function to RE-AuNP probes in tetramerization mutants L344P and L344A. (a) shows an UV- visible absorption spectra and (b) shows column graphs depicting aggregation extent (A650/A520) of RE-AuNP in the presence of p53-WT and tetramerization-compromised mutant p53 proteins including monomeric mutant L344A and dimeric mutant L344P. Fig. 19b inset shows the western blot results for p53-WT, L344P and L344A, respectively. Error bars represent mean + S.D. of three independent binding experiments. Two-tailed student's t-tests were performed to assess statistical significance (**p<0.01).

[0030] FIG. 20 shows the results of harnessing the dominant-negative effects of mutant p53 proteins in sample diagnostics, (a) shows a schematic diagram showing the concept of exploiting mutant p53 protein dominant-negative effects as a strategy for clinical diagnosis, (b) shows an UV-visible absorption spectra of loss in p53-WT DNA-binding function in the presence of increasing mutant p53 proteins, (c-e) shows column graphs depicting the aggregation extent of RE-AuNP from purified p53-WT spiked-in prior to incubating with either (c) purified R273H mutant p53 proteins, or (d-e) whole-cell lysate from samples (from a fixed 40 000 cells) containing increasing numbers of HT29 (p53-R273H) cells. RE-AuNP aggregation response was tested by spiking in p53-WT at either (d) 60 nM or (e) 30 nM. Error bars represent mean + S.D. of three independent binding experiments. Two-tailed student's t-tests were performed to assess statistical significance (*p<0.05, ***p<0.001).

[0031] FIG. 21 shows data depicting the characterization of gold nanoparticle conjugated response elements (RE- AuNP) versus gold nanoparticles (AuNP). (a) shows an UV-visible absorption spectra, while (b) shows a TEM micrograph of the respective nanoparticles; and (c) hydrodynamic size and zeta potential (in table form).

[0032] FIG. 22 shows a line graph depicting a calibration curve of thiazole orange (TO) with known concentrations of DNA-RE. The number of DNA-RE strands per AuNP was calculated by X/Y.

[0033] FIG. 23 shows a line graph depicting the stability of DNA- response element conjugated gold nanoparticle (RE-AuNPs). Shows is a UV-visible absorption spectra showing the salt stability of RE-AuNPs in the absence and presence of 1 M NaCl. A control sample with bare AuNP was unstable in the presence 1 M NaCl (gray line with triangular marking).

[0034] FIG. 24 shows a line graph depicting the stability of the proposed RE-AuNP assay. The graph shown in FIG. 24 shows an aggregation profile of RE-AuNP with different concentrations of p53-WT protein (0 to 70 nM) over 60 minutes.

[0035] FIG. 25 shows line graphs depicting the DLS measurement of RE-AuNPs size change in IVT samples. DLS measurement of the hydrodynamic size change of RE-AuNPs upon addition of p53-WT of different concentration (0 to 70 nM) produced by in vitro transcription/translation is shown. The inset shows the corresponding linear relationship (R = 0.93). [0036] FIG. 26 shows line and column graphs with data showing mutant p53 protein dominant- negative activity against purified wildtype p53 protein (p53-WT) in crude HT29 cell lysate. UV-visible absorption spectra of RE-AuNPs after incubating p53-WT (60 nM) in (a) phosphate buffer (black line), and crude cell lysate (gray line), respectively, (b) shows line graphs generated from data where p53-WT (60 nM) was spiked into mutant p53-expressing HT29 cell lysates (derived from indicated cell numbers, with total cell number kept constant by addition of p53-null H1299 cells), (c) shows a column graph depicting the aggregation extent of the corresponding samples calculated from the absorption spectra in FIG. 26 (a) and (b). DETAILED DESCRIPTION OF THE PRESENT INVENTION

[0037] Proteins interact with nucleic acid molecules at various stages during mammalian development. One stage is during processes known as transcription and translation, which are stages of gene expression. As an example, transcription factors (TFs) represent a class of proteins that bind sequence-specific DNA sequences in order to manipulate the rate of gene transcription.

[0038] The nanoparticles and the method disclosed herein are based on the principle of interparticle properties of the nanoparticles, which change during binding of, for example, oligomerising proteins to the nucleic acid molecules bound to the nanoparticles. This change in interparticle properties results in a shift in the optical spectrum, which translates into a change of colour of the nanoparticles. In one example, this change in colour does not require any equipment to be detected, as it can be detected with instruments detecting changes in the visible or near visible (such as UV light) spectrum and/or with the naked eye.

[0039] By way of explanation, the presently described method and the particles used in the present disclosure are the result of the aggregation of multiple nucleic-acid conjugated particles due to the binding of oligomerising proteins. Without being bound by theory, the binding of said oligomerising proteins to the nucleic acid molecules results in the aggregation of the nanoparticles and the oligomerising proteins. This underlying concept results in large-scale aggregation of the nanoparticles in a sample, thereby enabling detection of the change of colour on the visible spectrum that can be detected, in one example, without any equipment, that is, for example, using the naked eye.

[0040] Thus, the principle of the presently disclosed subject matter requires the presence of an oligomerising protein that binds to nucleic acids.

[0041] In one example, there is disclosed a probe comprising a plasmonic nanoparticle, wherein the nanoparticle comprises a target protein binding layer. In one example, the target protein binding layer is, but is not limited to, multiple copies of single stranded nucleic acids, multiple copies of double stranded nucleic acids or combinations thereof. In one example, the target protein binding layer is capable of binding to one or more types of target proteins capable of oligomerisation. In another example, the target protein is a multimeric protein comprising at least four binding partners. In a further example, the probe as disclosed herein comprises a plasmonic nanoparticle, wherein the nanoparticle comprises a target protein binding layer, wherein the target protein binding layer is selected from the group consisting of multiple copies of single stranded nucleic acids, multiple copies of double stranded nucleic acids and combinations thereof , wherein the target protein binding layer is capable of binding to one or more types of target proteins capable of oligomerisation; wherein the target protein is a multimeric protein comprising at least four binding partners.

Target proteins

[0042] As used herein, the terms "oligomerising", "oligomerisation", "oligomer" and "oligomeric" refer to the assembly of proteins, whereby the proteins are capable of binding to multiples of themselves to form a complex, or binding with other proteins to form a (multi- protein) complex. Thus, in one example, these oligomerising proteins can be referred to by the number of oligomerisation partners required to form the final complex, for example, dimeric (that is the binding of two proteins which can be the same or different), trimeric (that is the binding of three proteins which can be the same or different), tetrameric (that is the binding of four proteins which can be the same or different), pentameric (that is the binding of five proteins which can be the same or different), hexameric (that is the binding of six proteins which can be the same or different), septameric (that is the binding of seven proteins which can be the same or different), octameric (that is the binding of eight proteins which can be the same or different), heptameric (that is the binding of nine proteins which can be the same or different), decameric (that is the binding of ten proteins which can be the same or different), or multimeric or oligomeric (that is the binding of at least four or more proteins which can be the same or different). In one example, the subunits forming the oligomerising proteins are the same. In one example, the tetrameric proteins comprise identical subunits. As used herein, the term "oligomerisation" can also be used to describe the assembling of proteins which are more than tetrameric. In other words, the resulting complexes ("oligomers") can comprise of more than four components.

[0043] As used herein, the term "type" or "kind" refers to different target proteins which can bind to the nucleic acid sequences disclosed herein. For example, different types of target proteins can be transcription factors and cof actors. In this example, transcription factors and cofactors are proteins, albeit different types of proteins in terms of their designated function. Thus, in one example, it is possible that both transcription factors and cofactors both bind the nucleic acid molecules of the target protein binding layer surrounding the plasmonic nanoparticles as disclosed herein. In another example, both transcription factors and cofactors can bind and be found in the complex of assembled proteins.

[0044] As used herein, the term "multimeric" can be used synonymously with "oligomeric".

[0045] Most transcription factors do not work alone. Many large transcription factor families form, for example, complex homotypic or heterotypic interactions through oligomerisation. In the example of gene transcription, in order for it to occur, a number of transcription factors must bind to the DNA regulatory sequences. This collection of transcription factors, in turn, recruits intermediary proteins such as cofactors that allow efficient recruitment of the pre-initiation complex and RNA polymerase, in the example of gene transcription. Thus, for a single transcription factor to initiate transcription, all of these other proteins need also be present, and the transcription factor need be in a state where it can bind to these cofactors if necessary.

[0046] As used herein, the term "cofactor" refers to proteins that modulate the effects of transcription factors. Cofactors are interchangeable between specific gene promoter, whereby the protein complex that occupies the promoter region on the DNA and the amino acid sequence of the cofactor determine its spatial conformation.

[0047] In one example, the tetrameric or multimeric protein is a transcription factor. In another example, the tetrameric or multimeric protein can be a transcription factor and one or more cofactors. In yet another example, the tetrameric or multimeric protein is at least one transcription factor and one or more cofactors.

Target proteins - example: transcription factors

[0048] The methods and nanoparticles disclosed herein are used to detect the binding of target proteins to the nucleic acid sequences disclosed herein. One example of such target proteins is, but is not limited to, multimeric proteins, or oligomerising or multimeric transcription factors.

[0049] As used herein, the term "transcription factor", also known as a sequence-specific DNA-binding factor, refers to a protein that controls the rate of transcription of genetic information from DNA to messenger RNA, by binding to a specific DNA sequence. In other words, transcription factors are involved in the process of converting, or transcribing, DNA to RNA, and include a wide number of proteins, excluding, for example, RNA polymerases (which initiate and regulate gene transcription). The function of transcription factors is to regulate genes (that is, turn genes on and off) in order to ensure that they are expressed in the right cell at the right time and in the right amount throughout the life of the cell and the organism. Groups of transcription factors function in a coordinated manner in order to direct, for example, cell division, cell growth, and cell death throughout life; cell migration and organization during embryonic development; and intermittently in response to signals from outside the cell, such as a hormone.

[0050] Transcription factors work alone or with other proteins in a complex (by oligomerisation), by promoting (as an activator), or blocking (as a repressor) the recruitment of RNA polymerase, which is the enzyme that performs the transcription of genetic information from DNA to RNA, to specific genes.

[0051] One defining feature of transcription factors is that they contain at least one DNA- binding domain (DBD), which binds to a specific sequence of DNA adjacent to the genes that they regulate. Transcription factors are grouped into classes based on their DNA-binding domains. Other proteins such as co-activators, chromatin remodelers, histone acetyltransferases, histone deacetylases, kinases, and methylases are also essential to gene regulation, but lack DNA-binding domains, and therefore are not classed along with transcription factors.

[0052] Transcription factors bind to either so-called enhancer or promoter regions of DNA. These enhancer or promoter regions as usually adjacent to the genes they regulate. Depending on the transcription factor, the transcription of the adjacent gene is either up- or down-regulated. Transcription factors use a variety of mechanisms for the regulation of gene expression. These mechanisms include, but are not limited to, stabilising or blocking binding of RNA polymerase to DNA; catalysing the acetylation or deacetylation of histone proteins; acetylating histone proteins (also known as histone acetyltransferase (HAT) activity), which weakens the association of DNA with histones, making the DNA more accessible to transcription, thereby up -regulating transcription; deacetylating histone proteins (also known as histone deacetylase (HDAC) activity) which strengthens the association of DNA with histones, making the DNA less accessible to transcription, thereby down-regulating transcription; and recruiting co-activator or co-repressor proteins to the transcription factor DNA complex.

[0053] Transcription factors can be characterised, among others, by their tertiary structures. Thus, in one example, the transcription factor is, but is not limited to, a leucine zipper, a helix - loop-helix, a zinc-coordinated DNA binding domain, a helix-turn-helix, a beta-scaffold factor with one or more minor groove contacts. For example, p53 transcription factors generally fall within the so-called signal transducer and activator of transcription (STAT) protein family, which are primarily activated by membrane receptor-associated Janus kinases (JAK). Both the STAT proteins and the Janus kinases are involved in the so-called JAK-STAT pathway. [0054] Transcription factors work by, for example, oligomerising at or around the promoter region (also known as transcription factor-binding site, or binding site). Thus, in one example, the transcription factors are not monomeric transcription factors. That is to say that the transcription factor alone is not capable of binding to the promoter region. This means that in order to be effective in, for example, initiation transcription of a gene coupled to a promoter region, the transcription factor can bind with another transcription factor (dimeric), or two other transcription factors (trimeric), or three other transcription factors (tetrameric). These transcription factors can be multiple copies of the same transcription factor, or can be different types of transcription factors which then form a multi-protein complex upon binding. Thus, in another example, the transcription factors are dimeric, trimeric, tetrameric or multimeric transcription factors. In a further example, the transcription factor is tetrameric or multimeric. In the example of p53, the oligomerisation of p53 is the assembly of four identical wild type p53 protein monomers.

[0055] Specifically, oligomerisation of transcription factors controls the translocation of these transcription factors into the nucleus and increases DNA binding affinity, making it crucial for the normal functioning of transcription factors.

[0056] Although some transcription factors are dimeric and trimeric, tetramerization is crucial for several important superfamilies of transcription factors, including the p53 family, the signal transducers and activators of transcription (STAT) family. Thus, in on example, the target protein is a tetrameric transcription factor. In another example, the tetrameric transcription factor is, but is not limited to, p53- specific transcription factors, p73-specific transcription factors or STAT- specific transcription factors.

[0057] In one example, the p53 tumour suppressor is a transcription factor that binds sequence- specifically to DNA response elements (REs) to regulate cell fate in response to stress signals that can compromise genome integrity. p53 binding to response elements leads to the transcription of target genes that induce specific cellular programs such as apoptosis, DNA repair or cell-cycle arrest. p53 recognizes DNA-binding motifs traditionally defined as two palindromic canonical half-sites, 5'-RRRCWWGYYY-3' (where R = purine; W = A/T; Y = pyrimidine; SEQ ID NO: 1), each contacting a p53 dimer which further stabilizes upon tetramerization. The requirement for p53 in safeguarding against tumourignesis is strongly demonstrated in cancer- predisposed Li-Fraumeni patients lacking functional p53 alleles, and in orthologous p53-null animal models. Dysfunctional mutant p53 proteins, inactivated frequently through missense mutations, are strongly associated with cancer development and maintenance, and constitute a clinically viable target for both diagnostics and therapeutics. In particular, 'hotspot' mutant p53 proteins have been linked to higher penetrance and allelic frequency in cancers.

Target protein binding layer

[0058] As mentioned above, the nanoparticles disclosed herein comprise a target protein binding layer. This target protein binding layer, but is not limited to, ions, inorganic and organic molecules, such as, for example, organic ligands comprising polypeptides, nucleic acid molecules, chemical ligands, DNA-binding partners; and combinations thereof.

[0059] Organic molecules coating inorganic nanoparticles are also known as stabilizers, capping and surface ligands, or passivating agents. Non-limiting examples of organic molecules used as an interfacial layer are, but are not limited to, proteins or polypeptides, amino acids, ligands, nucleic acid molecules (for example, single- stranded DNA, double- stranded DNA, RNA), biological receptors, receptor-associated ligands, DNA-binding domains, response elements, promoter regions, genes or fragments thereof, and combinations thereof.

[0060] A person skilled in the art will appreciate that the protein binding layer can be modulated according to the target protein of interest. Therefore, if the protein to be detected binds to single stranded DNA, for example, the protein binding layer of the nanoparticle as disclosed herein comprises single stranded DNA. In another example, if the binding partner of the protein to be detected is a further protein, then the protein binding layer comprises these further proteins. Thus, in one example, the nucleic acid molecules as disclosed herein are, bit are not limited to, single-stranded DNA molecules, double-stranded DNA molecules, single- stranded RNA molecules, double- stranded RNA molecules, or combinations thereof. In another example, the nucleic acid molecules are double- stranded DNA molecules.

[0061] In one example, the nanoparticles comprise protein binding layer comprising double stranded DNA. In another example, the double-stranded DNA used herein is without sticky ends, in other words, sticky-end free double- stranded nucleic acid molecules or DNA. As used herein, the term "sticky ends" refers to overhang at the end of the nucleic acid sequences, usually the result of the type of restriction enzyme used to generate the double- stranded nucleic acid sequences used herein. These sticky ends are also called cohesive ends. As a collective term, these sticky or cohesive ends can be called "non-blunt" ends, to differentiate them from blunt ends, which are nucleic acid sequences with even, non-overhanging ends.

[0062] In another example, the protein binding layer comprises multiples of nucleic acid molecules which target one particular multimeric protein. In a further example, the protein binding layer comprises double stranded DNA molecules. In yet another example, the protein binding layer comprises response elements. In a further example, the protein binding layer comprises double- stranded DNA molecules (response elements) to which transcription factors from the families of, for example, but not limited to, p53, p73, NF-kB, STAT, bind. Thus, in one example, the nucleic acid molecules comprise a response element.

Response elements

[0063] As used herein, the term "response element", refer to short sequences of DNA within a gene promoter region that are able to bind specific transcription factors and regulate transcription of genes. Using the example of transcription factors, transcription factors interact with their respective response elements using a combination of electrostatic and Van der Waals forces. Due to the nature of these chemical interactions, most transcription factors bind DNA in a sequence- specific manner. However, it is possible that not all bases in the transcription factor-binding site interact with the transcription factor. In addition, some of these interactions may be weaker than others. Thus, transcription factors do not bind just one sequence but are capable of binding a subset of closely related sequences, each with a different strength of interaction. Additional recognition specificity can be obtained by using more than one DNA-binding domain that bind to two or more adjacent sequences of DNA, for example, the presence of tandem DNA-binding domains in the same transcription factor, or through dimerization of two transcription factors.

[0064] Thus, in one example, the response element is, but is not limited, to a double-stranded DNA molecule or sequence which allows binding of transcription factors. These transcription factors are, but are not limited to multimeric proteins of the p53, p73, NF-kB, or STAT family.

[0065] When contemplating use of, or when using a specific target protein binding sequence to generate the protein binding layer as disclosed herein, a person skilled in the art would appreciate that the nucleic acid molecule used to generate the target protein binding layer needs to be longer than the specific target protein binding sequence itself. Otherwise, the multimeric proteins would not have sufficient space away from the surface of the nanoparticle to bind and to aggregate with other, similarly functionalised nanoparticles. That is to say, in order to prevent steric hindrance of the binding of multimeric proteins to the target protein binding layer, the nucleic acid molecules that comprise the target protein binding layer must be long enough to allow binding of the multimeric proteins target to the nanoparticle, as well as be long enough allow assembly of the multimeric proteins attached to their nanoparticles to form aggregates, as dictated according to the method disclosed herein. In other words, the term "steric hindrance", as used herein, refers to the possible blocking of the binding interaction between the target protein and the nanoparticle probe if the size or structure of the probe which is too bulky or being limited by its surface area. [0066] For example, although the consensus binding site for the TATA-binding protein (TBP) is TATAAAA (SEQ ID NO: 2), the TATA-binding protein transcription factor can also bind similar sequences such as TATATAT (SEQ ID NO: 3) or TATATAA (SEQ ID NO: 4). This means that transcription factors are capable of binding a set of related sequences.

[0067] In one example, the p53 tumour suppressor is a master transcription factors that binds sequence- specifically to DNA response elements (RE) to regulate cell fate. p53 binding to response elements leads to the transcription of target genes and ultimately the induction of specific cellular programs such as apoptosis, DNA repair or cell cycle arrest. The p53protein is active when it is tetrameric, and in this conformation it binds with high affinity to DNA or interacts more efficiently with various other proteins. In general, p53 tetramerisation is essential for stable DNA binding, protein-protein interactions, post-translational modifications, and degradations. Without being bound by theory, it is understood that a single mutation in the tetramerisation domain will render the wild type p53 protein inactive in a manner similar to a mutation in the DNA-binding domain, which reference a potential use of cancer screening and developing anticancer drugs targeting this domain. Similarly for p73, a p53 superfamily member, its tetramerization is involved in cell cycle regulation and induction of apoptosis. Absence of tetramerization domain abolishes the ability of p73 in transactivation and growth suppression. Thus, in one example, the response element is a p53- and/or p73-specific response element.

[0068] STAT proteins play vital roles in modulating gene expression in response to cytokines, interferons, and various growth factors; they are critical for development and cellular functions. The tetramerisation of STAT5 or STAT3 may amplify or repress gene expression, which is critical for cytokine responses and to maintain normal immune functions. Since tetramerisation of these transcription factors play crucial roles in the human body, the detection and evaluation of transcription factor tetramerisation is an important target for disease screening and drug development. The importance of transcription factor tetramerisation underscores the usefulness of a robust, sensitive protein-DNA interrogation technique. Furthermore, such technology will be invaluable to discover novel cancer therapeutics targeted at restoring wild type functions to inactivated p53 mutants frequently over-expressed in cancer. Thus, in another example, the response element is a STAT-specific response element.

Nanoparticles

[0069] As used herein, the term "nanoparticle" (also known as ultrafine particles) refers to particles between 1 nanometre to 100 nanometres (nm) in size. As disclosed herein, nanoparticles can comprise a surrounding target protein binding layer. The interfacial surface of the nanoparticle is understood to be an integral part of nanoscale matter, and fundamentally affects all of nanoparticles' properties.

[0070] In nanotechnology, the term "particle" is defined as a small object that behaves as a whole unit with respect to its transport and properties. Nanoclusters, representing a group of nanoparticles, for example, have at least one dimension between 1 nm and 10 nm, along with a narrow size distribution. Nanopowders, for example, can be used to describe agglomerates of nanoparticles, or nanoclusters.

[0071] Thus, in the size of a plasmonic nanoparticle can be between 1 nm to 100 nm, between 1 nm to 10 nm, between 7 nm to 15 nm, between 10 nm to 30 nm, between 25 nm to 50 nm, between 60 nm to 100 nm, about 8 nm, about 9 nm, about 10 nm, about 11 nm, about 11.5 nm, about 12 nm, about 12.5 nm, about 12.75 nm, about 13 nm, about 13.25 nm, about 13.5 nm, about 13.75 nm, about 14 nm, about 14.5 nm, about 15 nm, about 16 nm, about 17 nm, about 18 nm, about 19 nm, about 20 nm, or about 25 nm. In one example, the size of the plasmonic nanoparticle is between 1 nm to 100 nm. In another example, the size of the plasmonic nanoparticle is between 7 nm and 15 nm. In another example, the size of the plasmonic nanoparticle is 13 nm.

[0072] When referring to three dimensional structures, the sizes mentioned herein refer to the longest edge of the nanoparticle, or, if referring to circular structures, the diameter of said circular structure.

[0073] In order to be able to be used in the method as disclosed herein, the plasmonic nanoparticles need to be made materials with plasmonic properties, in other words, plasmonic metals. Without being bound by theory, plasmons are produced from interactions of light with metal-dielectric materials. Thus, the plasmonic nanoparticles can be made from materials such as, but not limited to, noble metals such as ruthenium (Ru), rhodium (Rh), palladium (Pd), silver (Ag), osmium (Os), iridium (Ir), platinum (Pt), and gold (Au); other plasmonic metals and metal alloys, such as but not limited to gold-silver alloy, copper, molybdenum, cobalt, nickel, tantalum, zinc, copper zinc, iron nickel, molybdenum(IV) sulphide, silver-copper alloy, silver-tin alloy and combinations thereof. Thus, in one example, the plasmonic nanoparticle is made of a noble metal. In a further example, the plasmonic nanoparticle is made gold, silver, or gold-silver alloy. In another example, the plasmonic nanoparticle is made of gold or silver. In yet another example, the plasmonic nanoparticle is a gold particle.

[0074] Nanoparticles often possess unexpected optical properties as they are small enough to confine their electrons and produce quantum effects. The colour which a nanoparticle exhibits depends on the material from which it is made. For example, gold nanoparticles appear deep-red to black in solution, whereby nanoparticles of yellow gold are usually red in colour. Without being bound by theory, it is understood that the colour of nanoparticles in solution is caused by localised surface plasmon resonances (LSPRs).

[0075] Localised surface plasmon resonances (LSPRs) are collective electron charge oscillations in metallic nanoparticles that are excited by light. They exhibit enhanced near-field amplitude at the resonance wavelength. This field is highly localized at the nanoparticle and decays rapidly away from the nanoparticle/dielectric interface into the dielectric background, though far-field scattering by the particle is also enhanced by the resonance. An important aspect of LSPRs is light intensity enhancement, and localization means the localised surface plasmon resonance has very high spatial resolution (sub-wavelength), limited only by the size of, for example, nanoparticles. Because of the enhanced field amplitude, effects that depend on the amplitude, such as magneto-optical effect, are also enhanced by localised surface plasmon resonances.

[0076] For nanoparticles, for example, localized surface plasmon oscillations can give rise to the intense colours of suspensions or sols containing said nanoparticles. Nanoparticles or nanowires of noble metals have been shown to exhibit strong absorption bands in the ultraviolet- visible light regime that are not present in the bulk metal. The energy (colour) of this absorption differs when the light is polarized along or perpendicular to the nanowire. Shifts in this resonance due to changes in the local index of refraction upon adsorption to the nanoparticles can also be used to detect biopolymers such as DNA or proteins, as disclosed in the present description. In one example, the nanoparticles used herein allow detection with instruments detecting colour changes in the visible or near visible range of the spectrum, such as UV-vis or even colour changes visible with the naked eye.

[0077] In view of the above, a person skilled in the art will be able to adjust the size and shape of the nanoparticle depending on the particular material used, in order to ensure that the nanoparticle retains the property of localised surface plasmon resonance to be used in the presently described method.

[0078] Noble metal nanoparticles such as silver and gold have been utilized widely in biological detection and clinical diagnostics due to their unique optical properties which support localized surface plasmon resonance (LSPR). The basic principle of the AuNPs solution colour change is due to the change of their interparticle distance. However, there are different ways to manipulate the interparticle distance through biomolecular interactions for the colorimetric assay design. [0079] As disclosed herein, the nanoparticles can be of an irregular and/or regular, three dimensional shape, such as, but not limited to, nanosphere, nanorod, nanocube, dodecahedron, truncated dodecahedron, icosahedron, nanodisc and nanowires. In one example, the nanoparticle is a sphere.

[0080] It will also be appreciated that the nanoparticles as disclosed herein can also be of an irregular shape, that is three dimensional shape that is not symmetrical, so long as the particle exhibits the required localised surface plasmon resonance as required by the method disclosed herein.

[0081] Thus, in one example, the nanoparticle disclosed herein is a spherical gold nanoparticle. In another example, the nanoparticle is a spherical silver nanoparticle. In yet another example, the nanoparticle is a gold nanorod. In a further example, the nanoparticle is a spherical gold-silver alloy nanoparticle. In another example, the nanoparticle is a gold-silver alloy nanorod.

Methods

[0082] The probe, as disclosed herein, and the presence or absence of a colour change of said probe can be detected using various methods. These methods can be quantitative or qualitative. A non-limiting example of a qualitative method is using eye sight. Non-limiting examples of quantitative methods include, but are not limited to, spectral analysis, measuring optical density, ultraviolet-visible spectroscopy or ultraviolet-visible spectrophotometry, methods based on absorption spectroscopy and combinations thereof. In one example, the change in colour of the probe as described herein is measured using ultraviolet-visible spectroscopy or ultraviolet- visible spectrophotometry. In another example, the change in colour is identified using the naked eye. Thus, in one example, the change in colour is visible to the naked eye, and/or wherein the change is detectable and quantifiable using UV-Vis, spectral analysis, optical density-based methods or combinations thereof.

[0083] Based on the principle described herein, the method and the nanoparticles disclosed herein can be used to detect any oligomerising proteins which bind to nucleic acid molecules. Thus, in one example, the nanoparticles disclosed herein are used to detect oligomerising proteins which bind to double stranded nucleic acid molecules. In a further example, the nanoparticles disclosed herein are used to detect oligomerising proteins which bind to double stranded nucleic DNA molecules. In yet another example, the nanoparticles disclosed herein are used to detect oligomerising proteins which bind to double stranded RNA molecules.

[0084] In another example, the nanoparticles disclosed herein are used to detect the binding of tetrameric or multimeric proteins to double stranded nucleic acid molecules. In another example, the nanoparticles disclosed herein are used to detect tetrameric or multimeric proteins which bind to double stranded DNA molecules. In yet another example, the nanoparticles disclosed herein is used to detect tetrameric or multimeric proteins which bind to double stranded RNA molecules. In a further example, disclosed herein is a method of detecting oligomerisation of multimeric target proteins having at least four binding partners, the method comprising contacting the probe as disclosed herein with a sample suspected to comprise the target protein, wherein oligomerisation of the target proteins with the probe is indicated by a colour change.

[0085] Described herein is a label-free colorimetric method to study tetrameric transcription factor-DNA interactions using double- stranded (ds)-DNA conjugated gold nanoparticles (AuNPs). In one example, the target proteins to be detected according to the method disclosed herein are p53-specific, STAT-specific, or p57-specific transcription factors. In another example, the target proteins to be detected are p53- specific or p57-specific transcription factors.

[0086] As used herein, the term "label-free" refers to the fact that no further signal enhancers are required in order to detect the resulting colour change that occurs according to the methods disclosed herein. That is to say, in the conventional meaning of the term, a label refers to a compound used to enhance, amplify or multiply a signal resulting from a reaction in an assay. Depending on the signal to be amplified, various labels or signal enhancers can be used. The method disclosed herein does not require the use of such labels or signal enhancers.

[0087] In one example, the assay and method disclosed herein relies on the specific binding of wild type p53 protein to the DNA sequence in the gold nanoparticle-conjugated response elements (AuNP-REs), which alter the interparticle distance of gold nanoparticle-conjugated response elements (AuNP-RE), resulting in a distinct change in solution colour, as well as UV- vis absorption spectra. In one example, the colour of the response element-conjugated gold nanoparticles changes from red to purple after wild type p53 binding, which is different from the other reported gold nanoparticle -based assays in that protein binding in those previously reported assays leads to stabilization of probes, which in turn does not result in a change in colour. In other words, previously disclosed methods do not result in a shift in the visible spectrum. This can be attributed to previously reported gold nanoparticles being fundamentally different in structure, difference in conjugation between the nanoparticles. The presently disclosed nanoparticles and method discloses the use of single nanoparticles, to which multiple nucleic acid molecules are bound on its surface. This is one of the factors which enables large-scale aggregation of the nanoparticles in solution in the presence of oligomerising proteins; and it is this large-scale aggregation of nanoparticles which results in the shift of absorption wave length on the optical/visible spectrum. The absorbance ratios can be calculated to quantify the wild type p53 concentration. Thus, in one example, the method as disclosed herein involves only one set of nanoparticle-conjugated nucleic acid molecules, whereby the target oligomerising protein is added to the nanoparticles, which then aggregate upon oligomeric protein binding to result in colorimetric, instrument-free detection.

[0088] The time frame within which the colour change due to the aggregation of the proteins, as disclosed herein, takes place is almost immediate. For measuring purposes, it is preferable to wait between 2 minutes to 1 hour. To allow comparability of different measurements, the measurements need to be made at the same point of time after starting the reaction due to the kinetic nature of the continuously ongoing chemical reaction. With the method disclosed herein, the colour change (shift in the visual spectrum) to be viewed/detected is distinct at 5 minutes. Without being bound by theory, the colour change is considered to remain stable for a minimum of 60 minutes. Thus, in one example, the colour change is present within 5 minutes of the probe being added.

[0089] The stability of the reaction is based on the kinetics of the protein and the nucleic acid molecules to which the proteins bind. Therefore, the time point at which the detection of the shift in the visible spectrum is at its strongest depends on the kinetics of the oligomerisation of the protein in question to its nucleic acid molecule. As previously mentioned, when measuring multiple samples (for example, when comparing a positive and a negative control sample), it is important to note that the time at which the colour change is viewed is to be the same across all samples, as it would otherwise not be possible to make an accurate and correct comparison between the samples.

[0090] The high variability observed in physiological p53 response elements mandates a critical understanding of p53-DNA interactions, and underscores the importance of robust, sensitive protein-DNA interrogation techniques. Such technologies are also invaluable in discovering cancer therapeutics targeted at restoring wild type p53 protein functions to inactivated mutant p53 protein in cancer. Traditional approaches to study protein-DNA interactions such as electrophoretic mobility shift assay (EMSA) and DNA foot printing are labour-intensive, time-consuming and only semi-quantitative. Fluorescence anisotropy and surface plasmon resonance (SPR) require expensive instrumentation and cannot be adapted to the high-throughput formats. Immunochemical approaches such as, but not limited to, enzyme- linked immunosorbent assay (ELISA), and chromatin assay (ChIP) are heavily reliant on antibody quality, while Forster resonance energy transfer (FRET) -based methods offer sensitivity at the expense of costly synthetic labelling. Microsphere-immobilised response elements have been previously developed for p53 detection, but do not afford direct colorimetric visualization. Development of a rapid, sensitive and instrument-free assay for p53-DNA binding will be invaluable in understanding p53 biochemistry.

[0091] As used herein, the term "rapid" is used to describe an effect which happens in a short amount of time, at great speed, or at an increased rate. For example, rapid methods or assays are methods or assays in which the (final) incubation period prior to the read out of the method or assay result is short, compared to the time taken to perform the assay. In one example, the term rapid refer to the presence of the assay result immediately after addition of the final assay component. The term rapid can also refer to the presence of a detectable signal up to 10 minutes after additional of the final assay component. A person skilled in the art would appreciate the use of the term rapid in when describing methods or assays to indicate that the referenced method is one or more factors faster than conventional or comparable methods. Thus, in one example, the method disclosed herein is a rapid assay or a rapid method.

[0092] Competition assay with different free response element sequences enables the evaluation of the binding affinity of wild type p53 to various promoter sequences. Due to specific conjugation of transcription factor and response element on the AuNPs probe, the assay is applicable to complex biological samples such as cell lysates. It has been successfully applied to screen drugs, that is reactivation compounds for mutant p53 proteins generated from in vitro transcription/translation (IVT) samples. This fast, simple and instrument-free, visual detection method for transcription factor tetramerization is technology with application in cancer screening and drug development.

[0093] Thus, in one example, a sample, as disclosed herein, can be a sample obtained from a subject. In one example, the subject can be a diseased subject or a disease-free subject. In another example, the samples is, but is not limited to, protein samples, tissue samples, cell lysate samples, purified samples, in vitro transcription, in vitro translation samples (IVT) and combinations thereof. In one example, the tissue sample can be, but is not limited to, resection samples or biopsy samples. The high specificity and sensitivity of method as disclosed herein allows the unambiguous detection of tetrameric transcription factors and, for example, its mutant reactivating drugs in the complex biological samples, such as but not limited to in vitro transcription/translation (IVT) proteins or cell lysates.

[0094] If a control sample is required, a person skilled in the art would be able to determine the appreciate control sample (be it as a negative control or a positive control) based on the target protein to be analysed. [0095] Furthermore, as shown in the examples section below, the method as disclosed herein does not require large sample volumes in order to be able to show the desired results or the resulting effect of the probe as disclosed herein. The sample size used with the presently described method is at least 0.5 μΐ. The sample size can be between 0.5 μΐ and 100 μΐ, for example. In one example, the sample size is between 1 μΐ to 5 μΐ, between 8 μΐ to 15 μΐ, between 12 μΐ to 20 μΐ, between 18 μΐ to 25 μΐ, between 22 μΐ to 35 μΐ, between 30 μΐ to 45 μΐ, between 37 μΐ to 50 μΐ, between 48 μΐ to 55 μΐ, between 54 μΐ to 65 μΐ, between 60 μΐ to 78 μΐ, between 75 μΐ to 88 μΐ, between 80 μΐ to 98 μΐ, between 95 μΐ to 100 μΐ, about 5 μΐ, about 10 μΐ, about 15 μΐ about 20 μΐ, about 25 μΐ, about 30 μΐ, about 35 μΐ, about 40 μΐ, about 45 μΐ, about 50 μΐ, about 55 μΐ, about 60 μΐ, about 65 μΐ, about 70 μΐ, about 75 μΐ, about 80 μΐ, about 85 μΐ, about 90 μΐ, about 95 μΐ, or about 100 μΐ.

[0096] The tumour suppressor protein p53 plays a central role in preventing cancer through interaction with DNA response elements (REs) to regulate target gene expression in cells. Due to its significance in cancer biology, efforts have been directed toward understanding p53-DNA interactions for the development of cancer therapeutics and diagnostics. Thus, disclosed herein is a rapid, label-free and versatile colorimetric assay to detect, for example, wild type p53 DNA- binding function in complex solutions. The method is based on a concept that alters interparticle- distances between RE-AuNPs from a crosslinking effect induced through tetramerization of wild type p53 protein (p53-WT) upon binding to canonical DNA motifs modified on gold nanoparticles (RE-AuNPs). This leads to a visible solution colour change from red to blue, which is quantifiable by the UV- visible absorption spectra with a detection limit of 5 nM. Contrastingly, no colour change was observed for the binding -deficient p53 mutants and nonspecific proteins due to their inability to crosslink RE-AuNPs. Based on this sensing principle, its utility for fast detection of drug-induced nucleic acid binding function (for example, but not limited to DNA) to cancer-associated Y220C mutant p53 protein is further illustrated using well- established reactivating compounds. By exploiting the dominant-negative property of mutant p53 over p53-WT and interactions with RE-AuNPs, this method is configurable to detect low numbers of mutant p53 expressing cells in minuscule sample fractions obtained from typical core needle biopsy-sized tissues without signal attrition, alluding to the potential for biopsy sampling in cancer diagnostics or for defining cancer margins. This nanoparticle, for example nanogold, enabled colorimetric assay provides a facile yet robust method for studying important parameters influencing p53-DNA interactions with implementation in clinically pertinent applications.

[0097] In the present application, a assay design or method exploits two different forms of interrelated biomolecules interactions to crosslink double- stranded (ds) DNA functionalised AuNPs containing DNA response element (RE) for, for example, p53 protein binding in a homogenous solution without salt addition is shown. That is to say that the method disclosed herein does not require the use of a salt (for example, potassium chloride (KC1)) in order to promote aggregation of the nanoparticles in a sample. In other words, the binding and the oligomerisation of the proteins is sufficient to induce aggregation of the nanoparticles in the methods as disclosed herein. It can further be said that the method disclosed herein does not rely on the principle of dynamic light scattering (for example, due to the change in nanoparticle size) in order to detect aggregation of the nanoparticles due to oligomeric protein binding.

[0098] The interparticle crosslinking of response element-conjugated gold nanoparticles (RE- AuNPs) is driven by the intrinsic tetramerisation property of p53 protein after sequence- specific response element (RE) binding. The absence of either interaction modules does not lead to the efficient crosslinking of the gold nanoparticles. It has also been shown that the aggregation was not due to the salt-induced stability change of RE- AuNPs.

[0099] Furthermore, control experiments prove that mutated p53 lacking a functional tetramerisation domain will not induce RE-AuNPs aggregation even when in the presence of DNA carrying correct recognition element for protein binding. Thus, this assay provides a direct assessment of the p53 protein function itself. Based on this sensing principle, the application of this assay to detect and discern activity of specific drug compounds (PhiKan) designed to target and restore DNA-binding functions to the Y220C cancer-associated mutant p53 protein has been demonstrated. Furthermore, mutant p53 protein expressing cells are often found in early stages of cancer and mark tumour margins of certain cancer types, a feasibility study was conducted to configure the assay as a diagnostic tool for detecting small numbers of mutant p53 expressing cells within a bigger cell mass. This fast, label-free, visually detectable colorimetric assay with a low detection-limit shows utility as a new technology platform for use in drug discovery, disease discovery and cancer diagnostics. Thus, in one example, the method as disclosed herein can be used for detecting the presence or the absence of a specific disease, whereby the disease is modulated by, or is the result of, the absence or presence of oligomerisation of a protein.

[00100] Such drug screening assays rely on the capability of drugs and chemical compounds to reinstate the oligomerisation capability in proteins which have lost their ability to oligomerises. Reasons for such loss of oligomerisation ability can be, for example but not limited to, the presence of mutations within the target proteins. Thus, drug screening methods, as shown in the schematic shown in Figure 8(a), rely on the change in oligomerisation ability of target proteins based on the exposure of the target proteins to drugs and/or chemical compounds. Probe design and colour sensing principles for colorimetric assay to detect tetrameric transcription factors

[00101] The design of the nanoparticle (AuNP) and the sensing principle are schematically illustrated in Figure 1. Particularly, gold nanoparticles (diameters of roughly 13 nm) are first conjugated with double- stranded DNA comprising a p53-specific DNA response element (RE) to form response element-conjugated gold nanoparticles (AuNP-REs). It is determined that approximately 100 stands of DNA duplexes were functionalised into a protein binding layer on the surface of each gold nanoparticle. This dense negatively charged monolayer of double- stranded DNA on the gold nanoparticles, result in an electrostatic repulsion that inhibits the strong van der Waals attraction between gold nanoparticles to maintain a stable colloidal system, and increase assay sensitivity. Thus, in one example, a method of detecting oligomerisation of multimeric target proteins having at least four binding partners, the method comprising contacting the probe as disclosed herein with a sample suspected to comprise the target protein, wherein oligomerisation of the target proteins with the probe is indicated by a colour change. In another example, there is disclosed a method of detecting oligomerisation of multimeric transcription factors having at least four binding partners, the method comprising contacting the probe as disclosed herein with a sample suspected to comprise the multimeric transcription factor, wherein oligomerisation of the multimeric transcription factor with the probe is indicated by a colour change.

[00102] The number of DNA-RE strands conjugated on AuNPs was determined using fluorescence spectroscopy. Here, thiazole orange dye (TO) was used as a DNA-RE intercalator and a fluorescence label. TO is quenched when it is in close proximity with AuNPs through surface energy transfer mechanism. Therefore, to investigate the number of DNA-RE, AuNP (5 nM) was first dissolved using 1 mM of NaCN followed by staining 'free' DNA-REs with TO. The concentration of DNA-RE (X) released from AuNP (Y) can be evaluated from the linear correlation between the fluorescence intensity of intercalated thiazole orange dye and the concentration of DNA-RE. The concentration of DNA-RE (0.498 x 10 μΜ) corresponds to the fluorescence intensity of TO (257 nm) dye emission at 535 nm wavelength. The number of nucleic acids conjugated to each particle can be calculated according to the following formula, which has been shown for the example of DNA:

-concentration of DNA-RE (Α')

Number of DMA per particle— (- concentration of AuNPs ( )

[00103] Further characterisation of the nanoparticles as disclosed herein can be found in FIG.

22. [00104] This colorimetric assay is designed based on the interparticle-distance dependent (LSPR) properties of gold nanoparticles. The binding of wild type p53 proteins to response element-conjugated gold nanoparticles (AuNP-REs) as tetramers cross-links the response element-conjugated gold nanoparticles leading to distinct colour change from red to purple as shown in the insert. On the contrary, mutant p53 which loses its binding ability to response elements has minimal interaction with the response element-conjugated gold nanoparticles, thus causing negligible colour change. In addition, non-specific proteins such as bovine serum albumin (BSA) and human serum albumin (HSA) have no interaction with the response element- conjugated gold nanoparticles.

[00105] A person skilled in the art, based on the requirements as disclosed in the present application, would be able to attach the nucleic acid molecules as disclosed herein to the surface of the nanoparticles as disclosed herein. Non-limiting examples of methods used to attach nucleic acid molecules to nanoparticles are, but are not limited to, "aging-salting" processes or low-pH method, where either Na⁺ or H⁺ is used to minimize charge repulsion and facilitate attachment of thiolated nucleic acid molecules (for example, DNA) onto gold nanoparticles.

[00106] In one example, the nucleic acid molecules are thiolated at one end, and are therefore able to be conjugated to the surface of the gold nanoparticles through strong gold-thiol interactions.

[00107] In the presence of wild type p53 (wtp53), the AuNP-REs aggregate as p53 tetramerises on the response element causing the change in colour from red to blue, whereas the addition of mutant p53 (mutp53) proteins do not interact with the AuNP-REs which remain dispersed (red colour) in solution. Top and bottom panels display colour photographs of AuNP- RE incubated with increasing concentrations of p53CT-WT and p53CT-R273H (0-80nM), respectively.

[00108] As shown in Figure 2a, gold nanoparticle-conjugated response elements (AuNP-REs) have a single peak at 530 nm wavelength with good monodispersity as shown in TEM (Figure 2b). This is comparable to unconjugated AuNPs having a signature peak at 520 nm. Incubation with purified wild type p53 proteins (p53CT-WT) introduces a second peak at 650 nm wavelength and changes the originally red colloidal solution to blue/purple colour. AuNP-RE aggregation upon wild type p53 addition is evidenced by the AuNP clusters observed in transmission electron microscope (TEM) images (Figure 2d). In contrast, the DNA -binding deficient purified mutant p53 proteins (p53CT-R273H) only causes a slight increase in absorbance around 650 nm due to the non-specific electrostatic interactions between the negatively charged DNA-AuNPs and positively charged protein. It can be seen that majority of AuNP-REs with the p35CT-R273H samples remain well-dispersed in TEM (Figure 2c).

Colorimetric detection of tetrameric p53-DNA interactions

[00109] In Figure 3, it is shown that the aggregation of AuNP-REs due to the binding of wild type p53 proteins can be detected immediately by naked eye observation of colour changes and quantified by the absorption spectra measurement. The well-dispersed AuNP-REs exhibit a LSPR peak at around 520 nm, which decreases with increasing wild type p53 concentration. Another peak at a longer wavelength, i.e., 650 nm appears and increases with increasing wild type p53 addition. The degree of aggregation can be calculated from the absorbance ratio (A650/A520) which is correlated to the concentration of purified recombinant p53 proteins (p53CT-WT) with good linearity (R = 0.95) between 0 - 30 nM at 10 nM probe concentration. The detection limit calculated by the absorbance ratio is 0.5 nM, while the colorimetric detection limit is 5 nM. In the subsequent experiments, 5 nM of probe was used, which offers similar dynamic range (0-40 nM) and detection limit (0.5 nM) (Figure 4).

[00110] The method as disclosed herein allows for instrument-free visual detection with a limit of detection (LOD) of about 5 nM, and/or a quantitative measurement using, for example, a simple UV-vis spectrophotometer with a limit of detection (LOD) of about 0.5 nM.

[00111] While the method disclosed herein is capable to being detected using the naked eye, a person skilled in the art would appreciate that the detection limit will vary depending on the method used to detect the colour change. Therefore, the more sensitive the detection method used, the closer the detection limit will come to the sensitivity of the method disclosed herein. While terms "sensitivity" and "detection limit" can be used to describe the same effect, that is the lowest possible target protein concentration that can be detected using the method as disclosed herein, a person skilled in the art would understand that two aspects of the same concepts are being described, from different points of view. That is to say, the sensitivity of an assay dictates what minimum concentration of a target analyte or protein needs to be present in a sample in order for the method to produce a statistically relevant result (in other words, to ensure that the result being produced is not a false-positive result). The term detection limit refers to the capability of the detection method to detect the result being produced by the method described. Thus, the detection limit is limited in itself by the sensitivity of the method or assay applied. Thus, in one example, the method as disclosed herein has a sensitivity of between 0.01 nM to 30 nM target concentration. In another example, the sensitivity of the assay or method is between 0.25 nM and 0.8 nM target concentration. In one example, the sensitivity of the assay or method is 0.5 nM. In yet another example, the detection limit is 0.5 nM target concentration. In one example, the detection limit using UV-vis is about 0.3 nM target concentration. In another example, the detection limit using visual detection is about 5 nM target concentration.

[00112] In addition, it is shown that the use of a typical mutant p53 in human cancers (p53CT- R273H) resulted in negligible increase in absorbance ratio across the entire concentration range tested. This result suggests a differentiation between wild type p53 and mutant p53 in terms of their DNA binding activity. As shown in Figure 4c, non-specific proteins (BSA and HSA) at the same concentrations showed no binding activity. Furthermore, it is noted that wild type p53 protein (60 nM) in a background of high level of non-binding BSA (1 μΜ) gives similar signal as the wild type p53 protein alone, suggesting the potential to apply this method for complex sample testing.

Evaluation of transcription factor tetramerization

[00113] To validate the hypothesis that the tetramerisation of p53 is important to induce the aggregation of AuNP-REs, p53CT-R273H was mixed with a fixed concentration of p53CT-WT (60 nM) at increasing ratios and pre-incubated for 10 minutes before introducing the AuNP-RE probe. As shown in Figure 5, p53CT-R273H addition hinders the effective tetramerisation of p53CT-WT on the AuNP-REs which results in more than 50% decrease in AuNP aggregation at 1: 1 (p53CT-WT:p53CT-R273H) ratio as compared to p53CT-WT only (1:0). The aggregation ratio decreases as the ratio of mutant to wild type increase, and it subsequently drops to 25% when the ratio of p53CT-R273H to p53CT-WT is 5: 1. This result suggests that current colorimetric assay can be used to detect the tetramerization of p53, which lead to the AuNP-RE aggregation. This observation is consistent with the fact that single mutation in the tetramerisation domain can inactivate wild type p53 protein in a same manner as mutation in DNA-binding domain.

[00114] To further support this claim, it is also shown that estrogen receptors a (ERa), which are dimeric in nature, are not able to induce any aggregation of AuNP-RE probes with estrogen response elements (EREs) (Figure 6). This result again confirms the applicability of this simple and fast colorimetric assay to detect transcription factor tetramerization.

Evaluation of DNA binding affinity with competition assay

[00115] A competition assay had been designed to evaluate the binding affinity (K_D) of wild type p53 proteins to various promoter sequences. Their sequences and respective K_D values are shown in Table 1. Growth arrest and DNA Damage (GADD45) and Bel-associated X (Bax) are physiological promoter motifs involved in DNA repair, apoptosis. Additionally, a strong-binding artificial RE (ConA), non-responsive WRNC and scrambled DNA (scrDNA) were included as positive control and negative controls, respectively. Response

Function K_D References

Element

Jordan, J. J., Menendez, D., Sharav, J., Beno, I., Rosenthal, K., Resnick, M. A., & Haran, T. E. (2012). Low-level p53

ConA - 3.1 expression changes transactivation rules and reveals superactivating sequences. Proceedings of the National Academy of Sciences, 109(36), 14387-14392

Jordan, J. J., Menendez, D., Sharav, J., Beno, I., Rosenthal, K., Resnick, M. A., & Haran, T. E. (2012). Low-level p53

GADD45 DNA repair 5.0 expression changes transactivation rules and reveals superactivating sequences. Proceedings of the National Academy of Sciences, 109(36), 14387-14392

Jordan, J. J., Menendez, D., Sharav, J., Beno, I., Rosenthal, K., Resnick, M. A., & Haran, T. E. (2012). Low-level p53

Bax Apoptosis 14 expression changes transactivation rules and reveals superactivating sequences. Proceedings of the National Academy of Sciences, 109(36), 14387-14392

Noureddine, M. A., Menendez, D., Campbell, M. R., Bandele, O. J., Horvath, M. M., Wang, X., & Bell, D. A. (2009). Probing the functional impact of sequence

WRNC NA NA

variation on p53-DNA interactions using a novel microsphere assay for protein-DNA binding with human cell extracts. PLoS genetics, 5(5), el000462.

scrDNA NA NA -

Table 1. REs with their corresponding functions and binding affinities (K_DS)

[00116] The method as disclosed herein describes a competition assay format which allows for the simple determination of relative binding affinity of tetrameric transcription factors to various DNA promoter sequences using single detection probe, without the need to conjugate different DNA sequences to AuNPs for a single binding affinity study. Thus, in one example, there is disclosed a method of evaluating the binding affinity of multimeric target proteins having at least four binding partners, the method comprising mixing a sample comprising unbound response elements with the multimeric target proteins, and allow binding of the response elements to the multimeric target proteins, to obtain a first mixture, contacting the probe disclosed herein with the first mixture to obtain a second mixture, determining the presence or absence of a colour change in the second mixture, wherein the absence of colour change indicates that the multimeric target proteins have a higher affinity to the unbound response elements than to the probe.

[00117] As used herein, the term "free" or "unbound" refers to nucleic acid molecules which are not attached to any surface or any other molecule. That is to say that the nucleic acid molecules are free of any anchor or free of any conjugation to a surface.

[00118] As shown in Figure 7a, a ten-fold excess free DNA sequences was pre-incubated with p53CT-WT (60 nM) for 10 minutes followed by the addition of AuNP-REs. In this process, AuNP-REs compete with the free response elements already present in solution for binding with wild type p53 proteins. The DNA sequences with high affinity will bind with wild type p53 proteins more strongly thus disrupting the binding of wild type p53 proteins to AuNP-REs, resulting in reduction in the degree of aggregation; whereas DNA sequences with low affinity will not affect assay outcome. The degree of aggregation (A650/A520) obtained from the competition assay indicate preferential binding towards each RE in the order ConA > GADD45 > Bax > WRNC > scrDNA (Figure 7b), consistent with reported K_D values.

Drug screening in complex samples

[00119] Based on the principles and other methods disclosed herein, in one example, there is disclosed a method for screening drugs capable of restoring oligomerisation ability of multimeric target proteins, the method comprising contacting a sample comprising multimeric target proteins lacking, or thought to lack, oligomerisation ability with the drug to obtain a first mixture, contacting the first mixture with the probe described herein to obtain a second mixture, determining the presence or absence of a colour change in the second mixture, wherein the absence of colour change indicates that the drug is not capable of restoring oligomerisation ability of the multimeric target protein; wherein the multimeric protein comprising at least four binding partners. In one example, the the multimeric target protein is a transcription factor.

[00120] Most mutant p53 proteins lose their ability to bind response element sequences. Intense research is underway to search for chemical compounds that can restore the binding and consequently reactivate the p53 function for cancer therapy. The application of this colorimetric assay for detecting specific TFDNA binding activity has been demonstrated in complex samples, such as, but not limited to, cell lysate and those produced from in vitro transcription/translation (IVT) synthesis, and is further applied it to evaluate the efficiency of mutant reactivation by measuring the binding affinity upon drug treatment. Screening ofPhiKan drugs in in vitro translation samples

[00121] Figure 8a illustrates the underlining principle for screening mutant p53 reactivation drugs that are able to restore DNA-binding activity to mutant proteins. In the presence of p53 reactivating compounds, they can restore p53 conformation and then rescue its binding to response elements, thus causing the AuNP-REs to aggregate. In contrast, inactive drugs are unable to restore native p53 conformation, thus the mutant p53 would not be able to bind to AuNP-REs even after exposure to these inactive drugs, and therefore no AuNP-REs aggregation is observed.

[00122] As disclosed herein, in vitro transcription and translation p53 protein samples (IVTp53) were synthesised de novo using cDNA gene templates. A C-terminal truncated p53 variant was used (IVTp53A22) to increase the DNA binding activity. Titrating increasing amounts of rVTp53A22-WT proteins against a fixed concentration of AuNP-REs showed a linear correlation (R = 0.99) between absorbance ratio and p53 protein concentration (Figure 8b).

[00123] Next, sequence-specific DNA binding activity of IVTp53A22-Y220C mutant proteins was studied in the presence of different PhiKan compounds. The PhiKan compounds are known to target the p53-Y220C mutant at the mutation-induced cleft to restore wild type-p53 structure and DNA-binding activity. PK083 (KD=150 μΜ) is an earlier generation compound significantly weaker at binding IVTp53A22-Y220C mutant than its later iteration PK5176 with strong binding (KD =20.6 μΜ). PKX001 is an inactive analogue. p53-DNA binding was tested by mixing IVT-expressed protein with AuNP-REs, except that IVTp53 proteins were pre- incubated with Phikan compounds. The results showed an increase in canonical DNA-binding only when IVTp53A22-Y220C was tested in the presence of active Phikan compounds. The trend is well-correlated with compound activity (PK5176 > PK083 > PKX001), with the control analog being completely inactive (Figure 8c). Additionally, both weakly and strongly active PhiKan compounds had negligible effects when tested on the R273H contact mutant under the same conditions, alluding to mutant specific nature of the drug as expected.

Disease detection

[00124] The lack of protein oligomerisation can cause or in part cause the occurrence of one or more diseases in a subject. These diseases can be, but are not limited to diseases which are dependent on the oligomerisation of proteins such as, but not limited to p53, p73 and STAT. Thus, in one example, the disease is a STAT -dependent, a p53-dependent or a p73-dependent disease, or combinations thereof. In another example, the disease is a p53-dependent or a p73- dependent disease. Other non-limiting examples of such disease are cancer, Li-Fraumeni syndrome, and Beckwith-Wiedemann syndrome. In yet another example, the disease is cancer.

[00125] Therefore, in one example, there is disclosed a method of detecting the presence of absence of a disease in a subject, the method comprising contacting a sample obtained from the subject with the probe as disclosed herein; determining the presence or absence of a colour change in the mixture, wherein the absence of colour change indicates the presence of the disease; wherein the disease is caused or caused in part by lack of oligomerisation of one or more multimeric target proteins.

[00126] In addition to the detection of drug-reactivated mutant p53 in IVT samples, the colorimetric assay was next tested using cell lysates. The MDM2 protein is over-expressed in many human malignancies, and often results in an attenuated p53 response. Identifying compounds that inhibit p53-MDM2 interaction have been a successful strategy in treating wild type p53 expressing cancers through augmenting cellular p53 levels and activity. Lysates from HCT116 (human colon carcinoma) cells treated with a well-known MDM2 inhibitor, nutlin, resulted in aggregation when mixed with AuNP-Res (Figure 9). Due to the increase in p53 protein levels with increasing nutlin concentration, the relative aggregation also increases, further demonstrating the disclosed technique's utility in drug screening with cell lysates.

[00127] Thus, in one example, the disease is a disease based on lack of protein oligomerisation. In another example, the disease to be detected is a p53 -dependent disease. In yet another example, the disease is an MDM2-dependent disease. In a further example, the disease is selected from the group consisting of cancer, Li-Fraumeni syndrome, and Beckwith-Wiedemann syndrome. In one example, the disease is cancer.

[00128] The method disclosed herein is a homogeneous assay which is able to mimic physiological conditions and which does not result in steric hindrance

[00129] Also disclosed herein is a kit comprising the probe as disclosed herein. In one example, the kit comprises the probe as disclosed herein, an assay buffer, a calibration standard (for example, a positive control), and a negative control.

[00130] The invention illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms "comprising", "including", "containing", etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the inventions embodied therein herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.

[00131] As used in this application, the singular form "a," "an," and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a genetic marker" includes a plurality of genetic markers, including mixtures and combinations thereof.

[00132] As used herein, the term "about", in the context of concentrations of components of the formulations, typically means +/- 5% of the stated value, more typically +/- 4% of the stated value, more typically +/- 3% of the stated value, more typically, +/- 2% of the stated value, even more typically +/- 1% of the stated value, and even more typically +/- 0.5% of the stated value.

[00133] Throughout this disclosure, certain embodiments may be disclosed in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosed ranges. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[00134] Certain embodiments may also be described broadly and generically herein. Each of the narrower species and sub-generic groupings falling within the generic disclosure also form part of the disclosure. This includes the generic description of the embodiments with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

[00135] The invention has been described broadly and generically herein. Each of the narrower species and sub-generic groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

[00136] Other embodiments are within the following claims and non- limiting examples. In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.

EXPERIMENTAL SECTION

Materials

[00137] HAuC14.3H20 (99%), trisodium citrate dehydrate, sodium chloride (NaCl), potassium chloride (KC1), sodium monophosphate (Na2HP04), potassium phosphate (KH2P04) ethylene diamine tetra acetic acid (EDTA), hydrochloric acid (HCl), sodium hydroxide (NaOH), human serum albumin (HSA) and bovine serum albumin (BSA) were purchased from Sigma Aldrich, Singapore. Oligonucleotides listed in Table 2 were purchased from Integrated DNA technologies (IDT), Singapore. Sense and antisense oligonucleotides were annealed to make double stranded (ds) DNA fragments by mixing equimolar concentrations in DNA buffer (10 mM Tris-HCl, pH 7.4; 100 mM NaCl), before heating at 95 °C for 5 minutes and slowly cooled to room temperature. All chemicals were used without further purification. Ultrapure water (18 M were prepared from Millipore Elix 3 purification system) was used throughout the experiment as a solvent unless otherwise stated.

Names Oligonucleotide sequences SEQ ID NO:

(denoted as)

(ds) 5 ' -TAGAGATGCGAGAGTTCAGTAAGCGGGGCAGA- 7

Scrambled 3'

ConA 3 ' -ATCTCTACGCTCTCAAGTCATTCGCCCCGTCT-5 ' 8

(ds)BAX 5 ' -TCATTC ACAAGTTAGAGAC AAGCCTAGCTC-3 ' 11

3 ' -AGTAAGTGTTCAATCTCTGTTCGGATCGAG' -5⁷ 12

Table 2. Sequences of p53 response elements Apparatus

[00138] UV- visible absorption spectra was obtained with the aid of a TECAN infinite M200 plate reader (Tecan Tradition AG, Switzerland) using 384-well transparent flat bottom UVtransparent microplates (Corning Incorporated, USA). Dynamic light scattering (DLS) measurement was carried out using DynaPro Plate Reader II from Wyatt technology with the aid of 384 microplate reader to record hydrodynamic size. The morphology of the AuNP was studied under transmission electron microscope (TEM) using JOEL 2100 transmission electron microscope operating at 200 kV.

Synthesis of gold nanoparticles (AuNPs)

[00139] Gold nanoparticles (AuNPs) of approximately 13 nm in diameter were synthesised based on citrate reduction of HAuC14 approach as previously reported. The concentration of

8 -1 -1

AuNPs was calculated using Beer's Law, A = eel (where ε = 2.7 X 10 M^" cm^" ).

Preparation of RE-conjugated AuNP (RE-AuNP)

[00140] DNA molecules were functionalized onto gold nanoparticles (AuNP) as earlier described. 1 mL of AuNP (5 nM) was mixed with 10 \J thiolated ssDNA (100 μΜ) at 100: 1 molar ratio by vortexing. The mixture was incubated at room temperature for 10 minutes and adjusted to pH 3.0 by adding 9 μΐ. 1 M HC1, followed by the addition of 30 μΐ. 1 M NaCl. The mixture was further incubated another 20 minutes before neutralizing with 9 μΐ_^ 1M NaOH. ssDN A- conjugated AuNPs were annealed to unmodified complementary DNA (10 μΐ,, 100 μΜ) by heating at 95 °C for 5 minutes and slowly cooled to room temperature. The mixture was then washed with 0.5X PBS (0.01% Tween 20) and centrifuged (15000 rpm, 15 minutes) twice. The resulting deep red and oily pellet was suspended in p53 phosphate buffer (25 mM NaPi, pH 7.2, 100 mM KC1), characterized (FIG. 21) and stored at 4°C. Characterisation of the nanoparticles as disclosed herein can be seen in FIG. 22.

Colorimetric assay for p53-DNA binding

[00141] For binding experiments using purified p53 proteins, wild type (p53-WT) or mutant p53 proteins (p53-R273H) consisting only the DNA binding core and tetramerisation domain (amino acid residues 94 - 360) in p53 phosphate buffer (25 mM NaPi, pH 7.2, 100 mM KC1, 4mM DTT) were used. p53 containing samples are mixed with RE-AuNP and incubated at room temperature for 10 minutes before UV-visible absorption spectra are measured. Unless otherwise stated, all reaction mixtures contained a final concentration of 60 nM p53-WT proteins and 5 nM RE-AuNP in 50 volumes. For p53 titrated response, p53-WT proteins were added to achieve a final concentration between 0 - 80 nM in each reaction. As negative controls, p53-R273H and non-relevant (BSA or HSA) proteins were used respectively at a fixed 60 nM, or in excess of p53-WT. For the competition assay, 6 μΜ dsDNA oligonucleotides (GADD45, Bax, or scrambled DNA) were pre-incubated with p53-WT for 30 minutes at room temperature before RE-AuNP were added to evaluate competitive binding. These oligonucleotides have beencharacterised in previous reports. [00142] Experiments on mutant p53 protein detection were conducted by adding either purified p53-R273H, or whole-cell lysates from p53-R273H-expressing HT29 cells, to a fixed concentration of p53-WT proteins first, RE-AuNP probes are then mixed and assayed as before. For proof-of-principle experiments involving purified p53-WT, p53-R273H were titrated at increasing molar excess (30, 60, 120, 180, 240 or 300 nM) over wild type proteins before RE- AuNP probes were added and assayed. In experiments involving celllysates,

[00143] HT29 or H1299 cells were separately quantified (4 million cells) and lysed (200 passes on ice using a dounce homogenizer) in 2 mL p53 phosphate buffer (with addition of 4 mM DTT and cOmplete™ protease inhibitor, Sigma Aldrich). Resulting whole-cell lysates (2000 cells^L concentration) were combined to contain varying proportions of either lysate (HT29 or H1299) in a 20 μL· mixture comprising of a total of 40 000 cells.

[00144] Experiments were conducted using both debris-removed supernatant (20 minutes centrifugation at 18 000 r.c.f.) or crude lysates. Lysate samples were then mixed with p53-WT first, before adding RE-AuNP and assayed as described earlier.

RE-AuNP binding with I VI synthesized p53

[00145] p53 protein variants (wild type, Y220C, L344P or L344A mutants) carrying a C- terminal Domain (CTD) truncation (ρ53Δ22) were synthesized using an in-vitro cell-free transcription/translation (IVT) reaction (PURExpress®, NEB) through the use of cDNA templates as described previously. The IVT-translated p53 proteins were tested for RE-AuNP binding as described above. For experiments on mutant p53 protein reactivation, Y220C ρ53Δ22 mutant proteins were first IVT-translated, exposed to 20 μΜ of respective phikan compounds (PK-X001, PK-083, PK-5176) for 20 minutes on ice, before binding to RE-AuNP is assayed. Cell culture

[00146] HT29 and HI 299 cells (ATCC, USA) were cultured, respectively in HyCloneTM MyCoy's 5 A modified medium or HyCloneTM High glucose DMEM (L-glutamine), and supplemented with 10% Fetal Calf Serum (GE Healthcare Life Science, USA). Cells were grown at 37 °C in a humidified incubator with 5% atmospheric C0₂.

Sensing principle for colorimetric detection ofp53-DNA interactions using RE-AuNP

[00147] Design of RE-AuNP probe and colorimetric sensing principle are illustrated in Fig. 16a. The spherical AuNPs of -18 nm in hydrodynamic size (with absorption peak at 520 nm wavelength) are first conjugated with DNA duplexes comprised of a p53 response element (RE) to form RE-AuNP probes. The as-conjugated RE-AuNPs consisting -100 strands of REs per AuNP were stable at high salt concentration up to 1M NaCl (Fig. 23). This bioassay was designed based on interparticle-distance dependent colorimetric properties of AuNPs induced by tetramerization of p53 onto specific RE sequences. Specifically, binding of wild type p53 proteins to RE-AuNP as tetramers will cross link the nanocojugates leading to distinct colour changes. On the contrary, inactive mutant p53 proteins unable to recognize and bind DNA sequences remain in solution, and have minimal effects on the stability of RE-AuNP probes (Fig. 16a).

[00148] Fig. 16b shows the UV-visible absorption spectra of RE-AuNPs in the absence (control) and presence of p53-WT and p53-R273H after 5 minutes incubation. It was noted that the presence of p53-WT shifts the absorption spectrum to a longer wavelength, with a new LSPR peak observed at 650 nm (curve iii, Fig. 16b). In contrast, the DNA-binding deficient p53- R273H shows negligible changes (curve ii) as compared to control sample (curve i, Fig. 16b). The slight shift in spectrum might be due to the non-specific electrostatic interactions between free mutant p53 protein and RE-AuNPs. The red-shift of AuNPs spectrum is an indication of particle aggregation, the extent of which can be calculated from the ratios of absorbance peaks at 650 nm and 520 nm wavelength (A650/A520).

[00149] Fig. 16c shows the calculated aggregation extent of RE-AuNPs (A650/A520) in the presence of different protein samples (including control without p53), which corresponds well with RE-AuNPs images (Fig. 16d) acquired using transmission electron microscope (TEM). Selectivity and quantitative measurement of p53 -DNA binding complex

[00150] To quantify the measurement, the extent of p53-DNA binding induced aggregation was examined by adding increasing concentrations of p53-WT (Fig. 17a) against a fixed amount of RE-AuNP probe (5 nM). It has been shown that a probe concentration of 5 nM gave an improved signal to noise ratio and was thus adopted for all subsequent experiments.

[00151] The extent of aggregation correlates linearly to p53-WT concentration (R2 = 0.98) with a detection limit of 5 nM (Fig. 17b). The use of a DNA-binding compromised mutant p53 protein, common in human cancers (p53-R273H), in the same binding experiment resulted in negligible signal across all concentrations tested, suggesting a good signal-to-noise differentiation. Non-specific control proteins tested (BSA and HSA) at the same concentrations showed no binding activity (Fig. 17c). Furthermore, similar levels of p53-WT binding signals were detected even in the presence of excess BSA, suggesting high assay specificity and suitability for use in complex biological samples.

[00152] Thus, in one example, the method as disclosed herein enables a high detection specificity with negligible background interference.

[00153] A panel of DNA-REs with different wild-type p53 protein binding affinities, including physiological p53 RE involved in DNA repair (Growth Arrest and DNA Damage - GADD45 sequence) and apoptosis (bcl-2-like protein 4 - Bax sequence), a consensus sequence (Con A) that carries the high-affinity CWWG motif, and a nonbinding scrambled DNA (scrDNA) as control sequence, were next assessed through a competition assay. An excess of each free, competing DNA-RE was incubated with p53-WT for 30 minutes before the addition of RE- AuNPs (Fig. 17d). As a result, p53 molecules available for binding with RE-AuNP probes changes depending on its affinity for binding free DNA-RE, and returns a colorimetric signal correlating to the relative binding affinity. scrDNA has no p53 binding motif and therefore results in maximum RE-AuNP aggregation, unlike Bax, which displays an intermediate response owing to its moderate binding affinity (Fig. 17d). Contrastingly, Con A sequence, having the lowest dissociation constant of 1.0 + 0.2 nM, binds p53 strongly and leaves the RE-AuNP dispersed in solution (Fig. 17d). Results from the competition assay displayed preferential binding towards DNA-RE in the order Con A > GADD45 > Bax > scrDNA, which is consistent with their reported Kd values.

In-vitro detection of drug-induced DNA-binding activity on Y220C mutant p53 protein using RE- AuNPs

[00154] Restoring wild type transcriptional function to mutation-inactivated p53 protein is a longstanding yet challenging strategy for cancer treatment. Using the well-characterized Phikan compounds developed to reactivate the Y220C mutant p53 protein (p53-Y220C), it was examined if the described method can be utilized to identify small molecule reactivators of mutant p53 proteins. Without being bound by theory The PhiKan compounds target the p53- Y220C at the mutation-induced cleft to restore wild type p53 protein structure and DNA-binding activity. p53 proteins were synthesized de novo using an in vitro transcription and translation (IVT) cell-free extract. Titrating increasing amounts of IVT-expressed p53-WT proteins against a fixed concentration of RE-AuNP showed a good linear correlation as before (R2 = 0.93) with a commensurate increase in hydrodynamic size (Fig. S4). Functional activity of Phikan compounds were next tested by treating IVT-translated p53-Y220C with either active (PK-083 and PK-5176) or control (PK-X001) compounds, before assessing RE-AuNP binding. PK-5176 (Kd = 20.6 μΜ ) is a stronger binding and more active compound compared to the earlier derived PK-083 (Kd = 150 μΜ), while PK-X001 is an inactive analogue. The results show a drug- dependent restoration in canonical DNA-binding to p53-Y220C, but not in vehicle (DMSO) or control-treated samples. p53 reactivation activity correlated with the binding affinities of each compound (PK-5176 > PK-083 > PK-X001) towards p53-Y220C, while control analogue remained inactive (Fig. 18). Additionally, active Phikan compounds were completely inactive towards the p53-R273H contact mutant due to the absence of p53-Y220C mutation-induced groove required for drug activity, demonstrating a robust assay for the sensitive assessment of mutant- specific and drug-dependent restoration of DNA-binding function to mutant p53 protein. Exploiting mutant p53 protein dominant -negative activity for cancer diagnostics

[00155] It was hypothesized that the sensitivity and robust colour change in sensing canonical DNAbinding activity is acutely dependent on p53's natural proclivity to oligomerise, and it was sought to exploit this for further assay development. As a proof-of-principle, p53 tetramerization mutants were cloned, IVT-translated and assayed for RE-AuNP binding. The leucine residue at position 344 within the tetramerization domain of p53 protein has been reported to be critical for p53 oligomerization, and mutations to either Arginine (L344A) or Proline (L344P) results in the respective accumulation of dimeric or monomeric subunits. Although both tetramerization mutants were synthesized at similar levels to p53-WT (Fig. 19. western blot inset), DNA-binding function was severely compromised, with the dimeric variant (L344A) binding expectedly stronger than the monomeric form (L344P; Fig. 19). This strongly suggests that the p53- dependent crosslinking effect observed for RE-AuNPs and solution colour change is critically dependent on the successful oligomerization of DNA binding-proficient p53 protein molecules. Somatic missense mutations that inactivate p53 accumulate frequently during tumourignesis and contribute to cancer initiation, poor clinical prognosis and increased metastasis. More than half of human cancers harbour full-length, mutant p53 proteins that will abrogate DNA-binding function of p53-WT when concurrently present, due to the formation of mutant-wild type heterotetramers. It was rationalized that a diagnostic tool for mutant p53 protein detection (acting as a proxy for cellular transformation or incipient tumourignesis) can be developed by first mixing p53-WT with a sample in question before looking for a loss in RE-AuNP binding signal, as illustrated in the schematic diagram in Figure 20a. DNA-binding competent p53-WT acts as a 'bait protein' for mutant p53 protein present, to which it oligomerises with and loses DNA- binding function in the process.

[00156] As a proof-of-concept, decreasing amounts of purified p53-R273H were titrated into a solution containing fixed levels of p53-WT before RE-AuNP probes are added. The resulting mutant-dependent loss in DNA-binding function against RE-AuNP probes is readily detectable (Fig. 20b and 20c). p53-R273H induced dominant-negative effects on p53-WT DNA-binding is detectable at equi-molar concentrations and saturates when present at 2-3-fold molar excess over p53-WT (Fig. 20c). To better demonstrate clinical applicability, cancer cells harbouring mutant p53 proteins were used. Whole-cell lysates from a fixed total number of 40,000 cells containing varying proportions of HT29 (expressing endogenous p53-R273H) and H1299 (p53-null) cells were used to mimic a biopsy sample containing different amounts of mutant p53 protein- expressing cells (1.5- 30% mutant cells). Lysate mixtures were preincubated briefly with p53- WT (60 nM) before RE-AuNP probes are added and assayed. A significant loss in DNA-binding function can be observed which intensifies with increasing HT29 cell numbers (Fig. 20d). Additionally, a similar trend is also observed when using crude whole-cell lysates (Fig. 25), although a decrease in sensitivity was observed. For enhanced sensitivity, p53-WT 'bait protein' and RE-AuNPs should be present at optimal proportions, allowing discernible p53-WT DNA- binding activity while maintaining sensitivity to dominant-negative effects of low mutant p53 protein levels. By decreasing p53-WT levels (60 nM to 30 nM), further improvements in assay sensitivity were shown, where detection limit falls from 2000 (2.5% of cell mass) to 600 (1.5% of cell mass) cells (Fig. 20e). It is also notable that the activity of the 'detection module', represented by interacting p53-WT 'bait protein' and RE-AuNP probes, returned identical signals when measured in p53 phosphate buffer or in the presence of both crude and whole-cell lysate samples (Fig. 20d, 20e and 25), strongly alluding to assay robustness and clinical compatibility.

[00157] In conclusion, a rapid, label-free and versatile colorimetric assay to detect canonical p53 DNA-binding function has been developed for use in clinical diagnostics and cancer drug discovery. The assay is designed based on sequence-specific binding and tetramerization of wild type p53 proteins (p53-WT) to the DNA response elements (RE) conjugated onto gold nanoparticles (AuNPs), which leads to crosslinking and aggregation of RE-AuNPs.

[00158] This results in a simple visual readout by distinct solution colour change from red to blue, and a sensitive detection limit of 5 nM p53-WT by quantitative UV-visible spectra measurement. Contrastingly, no colour change was observed for the binding-deficient mutant p53 proteins and non-specific proteins due to their inability to cross-link REAuNPs. The competition assay performed using physiological competitors resulted in K_d-correlating RE- AuNP response, alluding to possible applications in p53-DNA binding platforms or screens through simple colorimetric visualization. Critically, this assay is amenable to detect p53 protein function in complex solutions (IVT cell-free extracts and crude whole-cell lysates) without signal attrition, as well as mutant p53 protein reactivation in a drug activity -specific manner. Given the critical contributions of p53 somatic mutations and loss-of-heterozygosity in the development of numerous human cancers, it is also noteworthy to highlight the assay's potential and configurability for clinical diagnosis in cancer, in particular for the rapid and visually- enabled detection of mutant p53 expressing cells in tissue biopsies. Several clinical studies performed on head and neck squamous cell carcinoma (HNSCC) have demonstrated a strong association between local cancer recurrence with the presence of residual mutant p53 expressing cancer cells residing at the tumour margin. The major challenges in defining a clean and mutant p53 negative margin during surgical resection using classic immune -histopathological methods include (i) the variability in p53 mutations (further compounded by availability of mutation- specific antibodies) and (ii) the difficulty in sampling entire resection surfaces due to limited throughput. However, these challenges have been addressed in the disclosed technique, and is achieved by exploiting the natural tendency of wild type p53 protein to lose canonical functions in the presence of mutant p53 protein with compromised DNA -binding activity through hetero- oligomerization, independent of the mutated amino-acid residue. Furthermore, the low detection limit of 600 cells within a 40 000 cell mass (1.5% cell mass), as well as robust and sensitive signal generated from p53-AuNP probe interaction supports clinical applicability, and provides a step forward in targeting p53 proteins in the clinics.

Claims

1. A probe comprising a plasmonic nanoparticle, wherein the nanoparticle comprises a target protein binding layer, wherein the target protein binding layer is selected from the group consisting of multiple copies of single stranded nucleic acids, multiple copies of double stranded nucleic acids and combinations thereof, wherein the target protein binding layer is capable of binding to one or more types of target proteins capable of oligomerisation; wherein the target protein is a multimeric protein comprising at least four binding partners.

2. The probe of any of the proceeding claims, wherein the nucleic acid molecules are selected from the group consisting of single- stranded DNA molecules, double-stranded DNA molecules, single-stranded RNA molecules and double- stranded RNA molecules. 3. The probe of claim 2, wherein the nucleic acid molecule comprises a response element.

4. The probe of claim 3, wherein the response element is a p53- and/or p73-specific response element. 5. The probe of any one of the preceding claims, wherein the protein is a tetrameric transcription factor.

6. The probe of claim 5, wherein the tetrameric transcription factor is p53-and/or a p73- specific transcription factor.

7. The probe of any one of the preceding claims, wherein the probe is label-free.

8. The probe of any one of the preceding claims, wherein the plasmonic nanoparticle is between 1 nm to 100 nm in size or 7 nm to 15 nm in size.

9. The probe of any one of the preceding claims, wherein the plasmonic nanoparticle is made a material selected from the group consisting of gold, silver and gold-silver alloy. 10. The probe of claim 9, wherein the plasmonic nanoparticle is a gold nanoparticle. A method of detecting oligomerisation of multimeric target proteins having at least four binding partners, the method comprising contacting the probe of any one of claims 1 to 10 with a sample suspected to comprise the target protein, wherein oligomerisation of the target proteins with the probe is indicated by a colour change.

The method of claim 14, wherein the target proteins are p53- or p57-specific transcription factors.

A method of evaluating the binding affinity of multimeric target proteins having at least four binding partners, the method comprising

^■ mixing a sample comprising unbound response elements with the multimeric target proteins, and allow binding of the response elements to the multimeric target proteins, to obtain a first mixture,

^■ contacting the probe according to any one of claims 1 to 10 with the first mixture to obtain a second mixture,

^■ determining the presence or absence of a colour change in the second mixture, wherein the absence of colour change indicates that the multimeric target proteins have a higher affinity to the unbound response elements than to the probe.

A method for screening drugs capable of restoring oligomerisation ability of multimeric target proteins, the method comprising:

^■ contacting a sample comprising multimeric target proteins lacking, or thought to lack, oligomerisation ability with the drug to obtain a first mixture,

^■ contacting the first mixture with the probe of any one of claims 1 to 10 to obtain a second mixture,

^■ determining the presence or absence of a colour change in the second mixture, wherein the absence of colour change indicates that the drug is not capable of restoring oligomerisation ability of the multimeric target protein;

wherein the multimeric protein comprising at least four binding partners.

The method of claim 14, wherein the multimeric target protein is a transcription factor.

16. A method of detecting the presence of absence of a disease in a subject, the method comprising: ^■ contacting a sample obtained from the subject with the probe of any one of claims 1 to 10;

^■ determining the presence or absence of a colour change in the mixture, wherein the absence of colour change indicates the presence of the disease;

wherein the disease is caused or caused in part by lack of oligomerisation of one or more multimeric target proteins.

The method of claim 16, wherein the disease is a p53- or a p73-dependent disease. 18. The method of claim 16 or 17, wherein the disease is cancer.

19. The method of any one of claims 11 to 18, wherein the method is a rapid assay method.

20. The method of any one of claims 11 to 19, wherein the sample is selected from the group consisting of protein samples, tissue samples, cell lysate samples, purified samples, in vitro transcription samples, in vitro translation samples and combinations thereof.

21. The method of any one of claims 11 to 20, wherein the change in colour is visible to the naked eye, and/or wherein the change is detectable and quantifiable using UV-Vis, spectral analysis, optical density-based methods or combinations thereof.

22. The method of any one of claims 11 to 21, wherein the method has a detection limit of 0.5 nM. 23. The method of any one of claims 11 to 22, wherein the colour change is present within 5 minutes of the probe being added.

24. A kit comprising the probe according to claims 1 to 10, an assay buffer, a positive control and a negative control.