WO2011051709A1

WO2011051709A1 - A proximity ligation assay involving generation of catalytic activity

Info

Publication number: WO2011051709A1
Application number: PCT/GB2010/051797
Authority: WO
Inventors: Adrian Smith; Prabhjyot Dehal; Lars Gøte Ingemar ØRNING
Original assignee: Axis-Shield Diagnostics Limited
Priority date: 2009-10-26
Filing date: 2010-10-26
Publication date: 2011-05-05
Also published as: GB0918712D0; EP2494064A1; CN102695803A

Abstract

A homogenous assay comprising the steps: 1) contacting at least two molecules each comprising a recognition component and a partial message with a sample wherein the contact of the recognition components with the sample results in the partial messages forming a continuous message; 2) amplifying the continuous message to form a catalytic entity which either directly or indirectly generates catalytic activity; 3) providing a substrate which allows detection of a recognition event between the at least two molecules and the sample via the catalytic activity.

Description

A PROXIMITY LIGATION ASSAY

INVOLVING GENERATION OF CATALYTIC ACTIVITY

[001] The present invention relates to an assay which detects a recognition event through generation of catalytic activity.

[002] Assays, for example, immunoassays are well known in the art as methods for detecting and measuring the concentration of analytes in biological samples and other samples. Immunoassays utilise the specific binding of an antibody or other recognition molecule to its antigen to detect an analyte, wherein the analyte may comprise either the antibody or antigen or both. The presence of one or both of the antigen or antibody may then be measured to quantitatively determine the concentration of analyte. In this way it is possible to detect and measure extremely low concentrations of analytes.

[003] Immunoassays may be conducted in various manners and formats. Of all the different formats for the application of immunoassays, probably the most widely used are those that employ solid phase separation using immobilised antibodies and/or antigens. This is known in the art as a heterogeneous immunoassay. Separation of immobilised antibodies and/or antigens is needed to effectively measure only the material recognised as the analyte of interest, wherein the solid phase serves as a physical binder which allows washing steps to be performed to remove non-target material. This washing procedure separates the analyte to be measured from the other constituents of complex mixtures that comprise the samples.

[004] In order to detect and measure the concentration of analyte in an immunoassay, a detection system is required. For example, an enzymatic assay may be used, wherein enzymatic action turns an undetectable bound analyte into a detectable and quantitatively measurable signal. Other means for detecting antibody and/or antigen may be achieved by labelling the antibody and/or antigen with detectable labels. Such labels could include colloidal gold labels, radioisotope labels, magnetic labels and fluorescent labels.

[005] Heterogeneous immunoassay systems have been, and continue to be, extremely valuable methods for detecting and measuring analytes. This is reflected by the significant number of these tests carried out annually. However, while these methods are both established and are typically reliable as a routine method of measurement, they also have disadvantages. For example, the use of a solid phase separation technique requires multiple steps and physical manipulations. Much of this process complexity can be reduced by the application of computer-driven instrumentation, but certain problems still remain. As process complexity increases, issues such as reliability also become more of a challenge. Specifically, the levels of imprecision become particularly significant with high sensitivity assays, wherein precision suffers due to the multi-stage nature of the existing processes, including liquid additions and removals and multiple wash steps. Accumulation of error over these multiple stages leads to this imprecision. A further consequence of this complexity and disadvantage of these processes is that increasingly complex instrumentation and software is required to perform the tests, which increases cost, reduces reliability, and increases development time and difficulty encountered in attempting to add new assays to work on such a system. [006] An alternative method, used to overcome some of the problems of complexity associated with heterogenous immunoassays, is so-called homogeneous immunoassays. These homogeneous immunoassays may be performed in aqueous solution using one or more additions of reagents but without the need for separation and wash steps. These homogeneous immunoassays have been seen to benefit from higher precision and sensitivity of measurement and make a valuable contribution to the field of analyte measurement.

[007] However, in order to detect analytes of interest, homogeneous immunoassays require the self-reporting of binding events of antibodies to their specific antigens, known as biomolecular recognition events. Such self- reporting or transduction of binding events may be achieved with various methods. For example, Enzyme Multiplied Immunoassay Technique technology (EMIT) relies on analyte- mediated inhibition of solution enzyme activity, while the Cloned Enzyme Donor Immunoassay (CEDIA) approach measures the presence of analyte in the sample mixture by the formation of enzyme activity by reconstruction of a working enzyme from inactive components. Other more recent technologies useful in this regard include Forster Resonance Energy Transfer (FRET) and other fluorescence amplification approaches such as time resolved fluorescence. Additionally there are successful methods using chemiluminescent or bioluminescent reporting which have the potential to be very sensitive.

[008] Another technique used in the self reporting of biorecognition events in homogeneous immunoassays is the system of Proximity Ligation Assay (PLA) . In this system, at least two antibodies having affinity for different epitopes on the same analyte are labelled with specific sequences of single-stranded DNA. Upon binding to the analyte of interest, the two antibodies are brought together in close proximity to each other. The proximal nature of the attached DNA sequences allows the complementary binding of an overlapping ss-DNA probe or probes. With the assistance of added specific enzyme, the ends of such probes may be joined or ligated to form a continuous circular single-strand of DNA. By careful design of this sequence, and with added polymerase enzyme reagents, there can follow an amplification of the DNA sequence according to the isothermal Rolling Circle Amplification process. The product of this operation is a growing strand of DNA of repetitive sequence emanating from each of the assembled analyte- antibody complexes. The incorporation of fluorescent tags into the growing DNA polymer allows for the quantitative measurement of the amount of ligated primer and hence also the quantity of analyte present in the sample.

[009] Homogeneous immunoassays could have the potential to be useful in clinical immunoassays. Homogeneous immunoassays can have advantages over heterogeneous immunoassays. However, they also have limitations.

[0010] One limitation of homogeneous immunoassays is the time to first result. Significant assay times are typically needed to generate sufficient detectable analytes for a reliable test whereas to be of optimum use in a modern clinical laboratory, assay times ideally need to be as quick as may be practicable.

[0011] A further, and arguably more significant, limitation of this method concerns the instrumentation commonly available in clinical laboratories, which is not readily capable of measuring fluorescent signals. Existing clinical laboratory instrumentation which is validated for use in diagnosis tends either to be for use for heterogeneous immunoassays, or is in the form of clinical chemistry analysers for spectrophotometric detection. These analysers have extremely limited spectral absorbance measurement capability and are usually unsuitable for reporter systems currently required by homogeneous immunoassays. [0012] There is therefore a need to provide a method for detecting analytes which benefits from both the advantages of homogeneous immunoassay technology together with the accessibility and ease of spectrophotometric detection while allowing for a suitably short procedure duration.

[0013] The present invention provides a homogenous assay comprising :

[0014] a) contacting at least two molecules each comprising a recognition component and a partial message with a sample wherein the contact of the recognition components with the sample results in the partial messages forming a continuous message ; [0015] b) amplifying the continuous message to form a catalytic entity which either directly or indirectly generates catalytic activity;

[0016] c) providing a substrate which allows detection of a recognition event between the at least two molecules and the sample via the catalytic activity.

[0017] The correlation between the catalytic entity/catalytic activity and the recognition event results in a precise and sensitive assay.

[0018] The present invention also enables disadvantages associated with systems that rely on transcription of a nucleic acid sequence followed by translation to form working enzyme protein through in-vitro protein synthesis to be addressed, wherein the likely complexity in terms of numbers of reagents, timings and potential reagent incompatibilities would be impractical. In addition, a coloured product may be produced by the assay which may be measured on clinical chemistry analysers that at the moment are readily available in clinics. [0019] The homogenous nature of the assay provides a relatively quick assay because it does not require removal or separation of any substrates. Further this assay, unlike PLA and other methods that rely on nucleotide amplification and fluoroprobes for signal generation (which can require 20- 40 cycles of up to two minutes, as in real time polymerase chain reaction (PCR), to reach a predetermined background signal), has an extra enzyme step whereby it is considered to enable an additional several fold increase in sensitivity, and thus requires fewer amplification cycles to reach the background signal.

[0020] Preferably the assay comprises only two molecules each comprising a recognition component and a partial message. In this way the materials required to perform the assay may be minimised. It will be apparent that the appropriate number of such molecules may be dependent on the nature of the sample that is being assayed and what it is desired to detect. [0021] Preferably the recognition component is selected from a protein, an antibody, an antibody fragment, molecular imprinted polymer, a complementarity determining region, oligopeptide, olgionucleotide, small organic chemical, peptide, polypeptide, polynucleotide or aptamer.

[0022] It will be apparent that the recognition component on each molecule may be the same or different. For example, they may be the same antibody to the same protein, e.g. one that is part of a multimeric complex or a cell surface component. Or, if different, they could as an example be an antigen for, and antibody directed to, a target antibody of interest .

[0023] It will be understood that a continuous message results from the partial messages becoming linked or connected in some way. As will be appreciated, the connection/linkage of the partial messages may be direct or there may be (a) further, intervening message molecule or molecules. In this way the continuous message whose formation is enabled by the partial messages of the molecules comprising recognition components may further comprise one or more linking partial messages. Therefore, optionally the forming of the continuous message is by virtue of the addition of one or more linking partial messages.

[0024] Conveniently the partial messages comprise or may be formed from nucleic acid (e.g. DNA or RNA) . They may be of the same or different form.

[0025] Conveniently the partial messages are formed from ssDNA but could also be an analogue such as PNA (peptide nucleic acid) .

[0026] In this way, partial messages formed from polynucleotides (particularly ssDNA) can be induced to anneal/hybridise to each other under appropriate conditions to enable formation of a continuous message.

[0027] Preferably the continuous message is amplified by isothermal amplification. This form of amplification, through avoiding thermal cycling, reduces the steps required to be taken by the person performing the assay and means that the assay may be much more easily conducted without expensive specialised equipment. [0028] Conveniently the isothermal amplification is rolling circle amplification. Rolling circle amplification can be more sensitive and faster then other forms of amplification. Further, it does not require thermal cycling which is not possible in most current clinical chemistry analysers.

[0029] Optionally the assay further comprises a ligation step prior to amplification of the continuous message. In this way a message comprising a nicked sequence to be amplified may be more easily used in the assay. For example, if the continuous message is ssDNA comprising a non- circularised message component, a standard DNA ligation reaction may be used to circularise the relevant component. As a further example, in embodiments wherein linking partial messages are added to form the continuous message and the amplification is to be by rolling circle amplification, a ligation step may be used to circularise the continuous message and enable amplification. It would be apparent to a person skilled in the art how ligation may be performed. [0030] Preferably the effect of the catalytic entity directly or indirectly on the substrate results in a visible detection product. This visible detection product can then either be viewed without the aid of a machine by the person performing the assay or it can be analysed by a suitable machine (e.g. a spectrophotometer or a colorimetric assay machine) . Alternatively, depending upon the specific capability of the instrumentation, catalytic formation of fluorescent or chemiluminescent products may also be included, by the use of appropriate substrates.

[0031] Optionally the detection product is luminescent, either by fluorescence, phosphorescence or chemiluminescence .

[0032] Conveniently the visible detection product is coloured. Enzymes that have been found to be useful in generation of coloured products include β-galactosidase and β-lactamase, where an appropriate chromogenic substrate is added to reveal the extent of catalytic activity, which in turn will relate to the quantity of analyte in solution.

[0033] Preferably the catalytic entity comprises DNA (either ssDNA or dsDNA) , RNA or a polypeptide.

[0034] Preferably the catalytic entity can be DNA (either ssDNA or dsDNA) , RNA or a polypeptide. If the catalytic entity is a polypeptide translation of the continuous message if it is DNA or RNA, will need to take place.

[0035] In the same or another embodiment the assay further comprises at least two complementary catalytic activity subunits which are not in functioning proximity. The subunits might not be in functioning proximity because they are kept spaced apart by an appropriate spacing component (e.g. polypeptide or polynucelotide ) or because they require a suitable linking component or have limited natural affinity for each other.

[0036] Conveniently the at least two complementary catalytic activity subunits are brought into functioning proximity by the catalytic entity. If the subunits are kept apart by an appropriate spacing component, the catalytic entity acts as a ligature thereby drawing the subunits together. If the subunits require a suitable linking component, the catalytic entity acts as a linking component. [0037] As an example, catalytic activity generation may occur by the labelling of the inactive subunits with lengths of complementary ssDNA such that the required subunits affix to the catalytic entity. In this manner, each catalytic entity will trigger the possibility of a new enzyme unit through a proximity binding occurrence (see figure 4 for illustration) .

[0038] Preferably, the intrinsic mutual affinity of each of the complementary catalytic activity subunits of the catalyst is low, so that the new generation of catalytic activity is driven, in the limit, entirely by analyte presence rather than by a default background recombination of the individual catalytic subunits. [0039] Accordingly, this embodiment results in guided assembly of incomplete catalytic units to generate de novo catalytic activity. [0040] Preferably the at least two complementary catalytic activity subunits are an apoenzyme and cofactor.

[0041] In one embodiment the catalytic entity is a DNAzyme. A DNAzyme would require no translation or transcription to produce a catalytic entity thereby reducing the substrates required in the assay and the time taken to first result.

[0042] Preferably the DNAzyme comprises a G-quadruplex or is G-quadruplex based. Accordingly, preferably the continuous message comprises an anti-sense message for G-quadruplex DNA.

[0043] Preferably the DNAzyme is G-quadruplex. Various other DNAzymes may be used, but sequences forming G-quadruplex units are especially suitable as these are of low molecular weight and can therefore easily be accommodated within the limited confines of the circular DNA of the RCA (rolling circle amplification) template. The repetitive formation of the said sequence should result in repeated self-assembly of G-quadruplex units, interspersed with linking sections. It has recently been found that the G-quadruplex units actively bind added hemin molecules, the product of which is an efficient peroxidase activity. [0044] A DNAzyme that comprises one or more G-quadruplexes that bind hemin to give peroxidase activity may consequently alternatively be thought of as an apo-DNAzyme, and hence hemin may be thought of as a co-factor. [0045] Preferably the catalytic activity is peroxidase activity .

[0046] Conveniently hemin is introduced to the assay. [0047] Conveniently hemin and substrate solution may be exposed to the G-quadruplex . G-quadruplex in the presence of hemin has a peroxidase activity and a substrate such as ABTS (2,2 azinobis [ 3-ethylbenzothiazoline-6 sulfonic acid]) or TMB ( tetramethylbenzidine) will produce a visible colour which can be detected. Preferably, the detected colour will have a linear relationship to the recognition event. [0048] Preferably the substrate comprises ABTS and/or TMB.

[0049] Conveniently the partial messages comprise any one or more of the nucleotide sequences of SEQ ID Nos 1, 2 and 3 or a homologue or functional fragment thereof.

[0050] Optionally one partial message may comprise the nucelotide sequence of SEQ ID No 1 while another partial message comprises the nucelotide sequence of SEQ ID No 3 or SEQ ID Nos 2 and 3, or a homologue or functional fragment thereof.

[0051] Optionally one partial message may consist of the nucelotide sequence of SEQ ID No 1 while another partial message consists of the nucelotide sequence of SEQ ID No 3 or SEQ ID Nos 2 and 3, or a homologue or functional fragment thereof .

[0052] By a combination specifically of Rolling Circle Amplification and the sequence of message encoding G- quadruplex units, it is possible to generate multiplied peroxidise activity from each biorecognition event. This peroxidise activity can then be detected by application of any of a number of chromogenic peroxidise substrates (for example, but not exclusively, ABTS, TMB) , as well as other novel substrates. The resultant coloured product may then be measured by optical methods, and the ultimate concentration of analyte be determined by calculation by reference to a suitable calibration curve.

[0053] In this way, by directly incorporating an enzymatic step and hence amplifying the component that is detected, the assay system of the present invention enables greater sensitivity and hence shorter assay processing times than current protocols, e.g. proximity ligation assays, whereby, for example, in measuring incorporation of a fluorophore there is not a latter additional enzymatic amplification step .

[0054] According to another aspect of the present invention there is provided a method for detecting an analyte in a sample, the method comprising an assay as described herein wherein the recognition event occurs, either directly or indirectly, between the at least two molecules and the analyte .

[0055] Optionally the analyte is a protein, peptide, polypeptide, antigen antibody, antibody fragment or metabolite of interest.

[0056] It will be understood by those skilled in the art that the assay and method described herein could be performed in a great variety of ways and relying upon different principles.

[0057] The present invention will now be illustrated with reference to the following Examples, none of which is intended to limit to scope of the invention, and the accompanying drawings, in which:

[0058] Figure 1 illustrates an example target (analyte) /recognition molecule/continuous message structure. In the case exemplified in Figure 1 the target (analyte) molecule for measurement in the sample is represented by Streptavidin, the recognition components of the (at least) two molecules are each represented by Biotin, one of the partial messages is represented by the anchor and template complex (or could otherwise be thought of as the template or relevant part of the template) and the other partial message is represented by the primer (or could otherwise be thought of as the relevant part of the primer) .

[0059] In Figure la the partial message (component) that is represented by the template is not circularised (it is nicked) . Figure lb depicts a slightly different situation wherein the template is joined (circularised) instead of nicked.

[0060] Figures 2 and 3 show typical results from an assay of the present invention measuring the presence of Streptavidin in a sample. The figures show that different concentrations of Hemin (in these cases ΙμΜ in figure 2 and 47μΜ in figure 3) can result in different assay sensitivities. In Figures 2 and 3 the X-axes show the relevant streptavidin concentrations studied (in pmol) and the Y-axes show the % change in absorbance. The change in absorbances shown in Figures 2 and 3 relate to a mean relative change across the relevant samples in comparison to the mean absorbance obtained for controls where no Streptavidin was present. [0061] Figure 4 illustrates the guided assembly of inactive subunits to give an active de novo enzyme. [0062] Example 1: Demonstration of Homogeneous Assay Format

[0063] Materials:

Primer conjugated Biotin:

Biotin/TEG5 ' GT GGA GGG TC 3'

(Primer sequence, SEQ ID No 1: TGT GGA GGG TC)

Anchor DNA conjugated Biotin:

5'CTG ACA GGG ACG GGA AGA AAA GG3 ' C6/Biotin

(Anchor sequence, SEQ ID No 2: CTG ACA GGG ACG GGA AGA AAA GG)

Template DNA:

5 'CCA CAT TTT AAG GAT TTT CCT TTT CTT CCC GTC CCT GTC AGC TGA ATT CGT GTG AGAC CCT3 ' phosphate

(Template sequence, SEQ ID No 3: CCA CAT TTT AAG GAT TTT CCT TTT CTT CCC GTC CCT GTC AGC TGA ATT CGT GTG AGAC CCT)

[0064] Primer and anchor DNA conjugated to Biotin were designed for the purposes of this example and can be obtained from commercial sources from companies such as IDT.

[0065] The template DNA was designed/selected for the purposes of this example. The template sequence comprises an anti-sense message for G-quadruplex DNA.

[0066] The template DNA in this example had a 3' phosphate to minimise spontaneous circularisation . Those skilled in the art would realise that the phosphorylation is not always necessary and, further, it may be desirable to have a circularised template, as discussed below. [0067] Products having a structure akin to the above primer conjugated Biotin and anchor conjugated Biotin molecules may in themselves each represent a molecule comprising a recognition component (e.g. biotin) and a partial message (e.g. anchor/primer). Alternatively, either of the molecules comprising a recognition component and a partial message may be more extensive (as described herein in relation to anchor/template-complex conjugated biotin), whereby the partial message may be thought of as comprising more than one component, e.g. the anchor and template. Alternatively it will be apparent from this instance that the template could be thought of as a partial message itself.

[0068] Accordingly, to form one of the subject molecules comprising a recognition component and a partial message for the example, template DNA diluted to 2pmol/ l in water was mixed with equal volumes of biotin conjugated anchor and incubated at 70°C for a minute (an alternative suitable temperature to reduce potential secondary structure formation but not denature the recognition element can be used) . The mixture was then cooled to facilitate template and biotin conjugated anchor binding/hybridistion . This resulted in formation of a stable biotin anchor-template DNA complex.

[0069] Method:

[0070] Formation of a continuous message in the presence of a sample [0071] Streptavidin, representing a target within a sample to be detected, was diluted to various concentrations between 0 to 50pmol. [0072] ΙΟμΙ aliquots of streptavidin were added to reaction tubes. To these reaction tubes was also added 20μ1 of the biotin-anchor/template DNA complex. The reaction mixtures were then incubated at room temperature for 5 minutes to allow the biotin-anchor/template DNA complex to bind the streptavidin.

[0073] ΙΟμΙ of biotinylated primer was added to each tube and mixed well before incubating at room temperature for 5 minutes to enable binding of the biotin to the streptavidin and hence formation of a continuous message complex such as that represented in Figure la/b.

[0074] As discussed above, though for the purposes of the present example the procedure to form the continuous message was carried out as described, it will be apparent to the skilled person that a range of alternative methods could equally be exploited. For example, messages conjugated directly to the two biotin recognition components (e.g. as represented by the anchor and primer features in figures la and b) could interact directly, or more extensive/removed partial messages, including, e.g., a template such as is depicted in figures la and b, could be used.

[0075] Accordingly, it can be understood from this example that when the DNA strands are brought into close proximity by binding of the two biotin molecules to streptavidin a piece of overlapping DNA (template) , being part of one of the partial messages, can bind to the other partial message to form a continuous message. In this case, the primer, representing one of the partial messages, binds to the template ( (part of) the other partial message) to form a continuous message.

[0076] The template DNA could be circularised by the ligation of its 3' and 5' ends, or not (as is represented by figure la/b) . In view of this the person skilled in the art will appreciate that, prior to amplification of the continuous message, a ligation step (such as that described below) may be appropriate, depending on the nature of the partial messages selected. For a circularised template such as that of figure lb and other relevant messages, it will be appreciated that a ligation step could be avoided. In this example however a ligation step was used.

[0077] Ligation reaction

[0078] ΙΟμΙ ligase mix (5μ1 ligase buffer + 0.5μ1 (1000 units) T4 DNA ligase (e.g. New England Biolabs M0202) + 4.5μ1 water) was added to each reaction tube and the reaction mixtures were incubated at room temperature for 2 to 10 minutes to allow nicks between relevant polynucleotide ends

(in this instance of the template) to close.

[0079] Clearly the streptividin/biotin binding partnership is used as an exemplar for the purposes of the present example where detection of streptavidin as a target analyte is to be shown, but it will be understood by the skilled person that the recognition event to be detected may occur equally between any other binding partnership, such as antibodies (or fragments etc of antibodies) and proteins/peptides etc, so that a target analyte of interest is detected. Further, the recognition components involved in an assay may be different such that there are different binding interactions occurring. [0080] Amplifying the continuous message to form a catalytic entity

[0081] ΙΟμΙ of polymerase master mix (note heat inactivated polymerase was used as a control) was added to each reaction tube. The reaction mixture was then incubated at 30 °C for 30 to 270 minutes to allow the message to amplify. The polymerase master mix consisted of Ιμΐ (10 units) phi29 DNA polymerase (e.g. as supplied by New England Biolabs; M0269), Ιμΐ polymerase buffer, 5μ1 dNTPs and 3μ1 water.

[0082] In this example the template DNA sequence comprised an anti-sense message for G-quadruplex DNA. Accordingly, the continuous message of a complex of the form of that shown in figure la/b was amplified by rolling circle amplification to generate a continuous DNA polynucleotide which spontaneously folds to form tandem G-quadruplex structures. The incorporation of added hemin into the G-quadruplexes results in a structure with peroxidase activity. [0083] It will be apparent to a person skilled in the art that partial messages of varying sequences to those indicated in this example could be used. For example, the template message component could be varied to produce different G- quadruplex DNA.

[0084] Detecting the recognition event

[0085] Hemin stock solution was prepared by dilution in DMSO to 0.5mM. A further dilution in hemin solution ( 0.1M Tris-HCl, 20mM MgCl₂, 0.05% Triton X-100) to a concentration of 330μΜ was then prepared. An appropriate volume of 330μΜ hemin was then added to the reaction tube to give the final concentration of hemin as desired (in this instance ΙμΜ or 47μΜ) . Experiments using different concentrations of hemin were performed to alter the assay range. The reaction mixture was then incubated at room temperature for up to 2 hours before transferring to a 96 well microtitre plate and adding ΙΟΟμΙ; TMB substrate solution. The reaction was allowed to develop by incubating at room temperature for between 5 and 45 minutes before stopping by addition of ΙΟΟμΙ of 0.25M sulphuric acid. Absorbance was read at 450nm. [0086] From the data shown it can been seen that the final concentration of hemin in the reaction mix can be varied to enhance the sensitivity of the assay (see figures 2 and 3, figure 2 relates to an assay using ΙμΜ hemin and figure 3 to one using 47μΜ hemin) .

[0087] Typical results of the experiment described above are shown in figures 2 and 3. The absorbance data given represent mean relative changes across the relevant samples in comparison to the mean absorbance obtained for controls where no Streptavidin was added.

[0088] It can therefore be understood that in this example the assay principle is that G-quadruplex complexes formed by amplification, that is enabled when a continuous message comprising an anti-sense G-quadruplex sequence is formed, give rise, in the presence of hemin, to peroxidase activity that metabolises TMB to produce a coloured product that can be detected by measuring absorbance. [0089] Though for the purpose of the present example the method has been set out in a staged manner, it will be apparent to the skilled person that various steps could be performed concurrently in order to reduce the time taken to first result. Further, the assay may be performed in the same vessel used for detecting the recognition event.

[0090] Example 2: Immunocomplex formation with ssDNA-tagged binding partners

[0091] A cyclic peptide (according to Structure 1 below) known to be immunoreactive against autoantibodies, the presence of which is diagnostic of early rheumatoid arthritis, and possessing a modified ssDNA sequence on the N-terminus was used as one of the two binding partners in this work. Addition of a 22 pM solution of this peptide to a serum sample containing the antibody, from a known positive sample, caused binding to the antibody of the sample as measured by immunoassay after incubation for 10-12 minutes.

[0092] A further binding partner, this time a solution containing a large molar excess of a modified anti-human IgG specific for the Fc region (obtained from Serotec) was added to the mixture, and allowed to incubate for 15-20 minutes at room temperature. This antibody was modified by covalent linkage with a ssDNA strand on its Fc portion.

[0093] Verification of the ternary complex was obtained by immunoassay utilising anti-murine IgG specific antibodies bound to the surface of magnetic beads, which when separated from the mixture and allowed to react with alkaline phosphatase bearing covalent ssDNA strand complementary to that of the ssDNA on the cyclic peptide. With subsequent exposure after washing with 1.5 mM p-nitrophenylphosphate in 100 mM diethanolamine buffer, coloured product was developed and the signal read at 450 nm, showing positive binding against a control containing no positive serum.

X = NH₂, CH₃, HCH₃ or N(CH₃)₂;

\ / Y = 0, NH, HCH₃ or N(CH₃)₂; condition that when X = NH₂ the modified arginine residue residue. For citrulline, X =

S HQE S TXGRS RGRS GRS G S

Structure 1: The top structure is a representation of the modified side chain of the arginine (X) seen in the amino acid sequence.

[0094] Example 3: Demonstration of proximal placement of ssDNA sequences and ligation.

[0095] Detection of the ternary immune complex via ligase- generated ssDNA sequence.

[0096] To determine the presence of the ligatable sequences within the ternary complex the ligation mixture was added to the mixture containing the complex, the latter as verified by immunoassay.

[0097] The ligation mixture contained ligase enzyme, a ligation template, and ATP. (Ligation mix: volume 5 L, 50 mM KC1, 10 mM Tris-HCl pH 8,3, 3 mM MgCl₂, 0.8 mM ATP, 20 nM ligation template, 3 Weiss units T4 DNA ligase.) This was added to the immunocomplex mixture and allowed to react for 5 minutes after which it was inactivated by heat treatment at 85°C for a further 10 minutes. [0098] For the purpose of this example the ligated sequence was detected by PCR (PCR mix(TaqMan PCR) : volume 30 L, 50 mM KC1, 10 mM Tris-HCl pH 8.3, 0.4 mM MgCl₂, 0.33 mM dNTPs , lx buffer A, 1 μΜ forward primer, 1 μΜ reverse primer, 83 nM TaqMan probe, 1.56 Units AmpliTaq Gold polymerase (Perkin- Elmer) . The amplification/detection mix contains PCR primers, TaqMan probe, dNTPs and DNA polymerase.

[0099] Amplification detection mix Probe Ale: (SEQ ID NO:4)

TACTCAGGGCACTGCAAGCAATTGTGGTCCCAATGGGCTGAGTATGTGGTCTATGTCGT CGTTCGCTAGTAGTTCCTGGGCTGCAC

Probe A2c (SEQ ID NO: 5)

TCGAGGCGTAGAATTCCCCCGATGCGCGCTGTTCTTACTCAGGGCACTGCAAGCAATTG TGGTCCCAATGGGCTGAGTAT

Splint template for ligation (6+20): (SEQ ID NO: 6)

GGGGGAATTCTACGCCTCGAGTGCAG

Fwd primer (SEQ ID NO: 7; nucleotides 44-65 of SEQ ID NO:4): ATGTGGTCTATGTCGTCGTTCG

Rev primer (SEQ ID NO: 8; reverse complement of nucleotides 22-41 of SEQ ID NO : 5 ) :

TGAGTAAGAACAGCGCGCAT Taq Man probe Al+2c (SEQ ID NO 9; reverse complement of SEQ ID NO: 6): Fluor-CTGCACTCGAGGCGTAGAATTCCCC-Tamra

PCR cycles: hold 10 min 95, cycle 95 (15 sec) -60 (1 min) 45 times Combined Ligation Mix and Per Mix for Higher Sensitivity

[00100] Example 4: Competitive Proximity-probing for Analytes with Only One Binding Site [00101] To be generally useful, there is a need to provide a practical solution where an analyte only has one binding moiety. This can be achieved by using a competitive assay. Herein, a purified amount of the analyte itself is conjugated to a nucleic acid and the one existing binding moiety is conjugated with the other reactive nucleic acid. When these two conjugates are permitted to react in a sample mixture containing an unknown amount of the analyte, the non- conjugated analyte of unknown amount in the sample will compete for binding to the binding moiety of the proximity- probe thereby decreasing the probability of the conjugated nucleic acids reacting. The signal from the reaction is in this case inversely proportional to the analyte concentration . [00102] Example 5: Alternative material preparation and procedural methods .

[00103] Circular DNA template preparation: A circular DNA template was prepared as follows: First, the linear DNA ( 5 ' -GATCCTAACCCAACCCGCCCTACCCAAAACCCA

ACCCGCCCTACCCAAAACCCAACCCGCCCTACCCAACCACAC-3' (SEQ ID NO: 10)), 6 x 10^"6 M, was phosphorylated using T4 polynucleotide kinase, 0.4 units/μΐ, the ligation template (5'- TTAGGATCGTGTGGTT-3 ' (SEQ ID NO: 11)), 3.6 x 10^"5 M, in the Quick Ligation Kit buffer, at 37° C for 30 min. The synthesis was completed by the Quick Ligation Kit, using the manufacturer-supplied protocol. The enzymes were denatured by heating at 90° C for 10 min. The ligated circular DNA was then purified and separated from the ligation template by urea, 8 M, using a Centricon filtration device (10,000 cutoff, Millipore Inc.). [00104] RCA assay: Fixed concentrations of the hairpin 4, 2 x 10^"7 M and the circular DNA 2, 2 x 10^"8 M were employed. Polymerase Klenow exo-, 0.4 units/μΐ and dNTPs, 0.2 mM were included. The RCA process was performed in a buffer solution consisting of 50 mM Tris-HCl, pH 7.5, 10 mM MgCl₂, 1 mM DTT and 50 g/ml BSA.

[00105] The conditions for the absorbance studies are optionally as follows: The experiment was performed in a solution consisting of the products; hemin, 4 x 10^"7 M; H₂0₂, 4.4 x 10^"5 M; ABTS2-, 1.82 x 10^"4 M in a buffer solution consisting of 25 mM HEPES, 20 mM KC1 and 200 mM NaCl, pH 7.4, 25°C. Absorbance changes were followed at 415 nm to characterize the rate of the oxidation of ABTS2-.

Claims

A homogenous assay comprising: a) contacting at least two molecules each comprising a recognition component and a partial message with a sample wherein the contact of the recognition components with the sample results in the partial messages forming a continuous message;

b) amplifying the continuous message to form a catalytic entity which either directly or indirectly generates catalytic activity;

c) providing a substrate which allows detection of a recognition event between the at least two molecules and the sample via the catalytic activity.

An assay according to claim 1 wherein the recognition components each comprise any of an antibody, an antibody fragment, protein, molecular imprinted polymer, a complementarity determining region, oligopeptide, olgionucleotide, small organic chemical, peptide, polypeptide, polynucleotide or aptamer .

An assay according to claim 1 or 2 wherein the forming of the continuous message is by virtue of the addition of one or more linking partial messages . 4. An assay according to any preceding claim wherein the partial messages comprise or are formed from ssDNA or an analogue thereof.

5. An assay according to any preceding claim wherein the continuous message is amplified by isothermal amplification .

5 6. An assay according to claim 5 wherein the isothermal amplification is rolling circle amplification.

7. An assay according to any preceding claim further comprising a ligation step prior to amplification of

10 the continuous message.

8. An assay according to any preceding claim wherein the effect of the catalytic entity directly or indirectly on the substrate results in a visible

15 detection product.

9. An assay according to claim 8 wherein the visible detection product is coloured.

20 10. An assay according to claim any of claims 1 to 7 wherein the effect of the catalytic entity directly or indirectly on the substrate results in a detection product that is luminescent, by fluorescence, phosphorescence or chemiluminescense .

25

11. An assay according to any preceding claim wherein the catalytic entity comprises, or is selected from, DNA, RNA or a polypeptide.

30 12. An assay according to any preceding claim wherein the catalytic entity is a DNAzyme.

13. An assay according to claim 12 wherein the DNAzyme comprises a G-quadruplex or is G-quadruplex based.

14. An assay according to any of claims 4 to 12 wherein the partial messages comprise any one or more of the nucleotide sequences of SEQ ID Nos 1, 2 and 3 or a homologue or functional fragment thereof.

15. An assay according to either claim 13 or 14 further comprising introducing hemin.

16. An assay according to any of claims 1 to 11 further comprising at least two complementary catalytic activity subunits which are not in functioning proximity.

17. An assay according to claim 16 wherein the at least two complementary catalytic activity subunits are brought into functioning proximity by the catalytic entity.

18. An assay according to either claim 16 or 17 wherein the at least two complementary catalytic activity subunits are an apoenzyme and cofactor.

19. A method for detecting an analyte in a sample, the method comprising an assay according to any preceding claim wherein the recognition event occurs between the at least two molecules, either directly or indirectly, and the analyte.