WO2013178844A1 - Specific aptamers against gluten and associated method for detecting gluten - Google Patents

Abstract

The invention relates to single-stranded DNA molecules, also called aptamers, that are able to recognise and bind specifically to gluten. The invention also relates to a method for recognising, capturing and/or detecting gluten using said aptamers, and to a kit comprising said aptamers.

Description

 SPECIFIC APTÁMERQS AGAINST GLUTEN AND METHOD OF

 DETECTION OF ASSOCIATED GLUTEN

DESCRIPTION

The present invention relates to single-stranded DNA molecules, also called aptamers, capable of specifically recognizing and binding to gluten, and a method of recognition, capture and / or detection of gluten using said aptamers. Therefore, the invention could be framed in the field of biotechnology.

STATE OF THE TECHNIQUE

Celiac disease is an autoimmune disease of genetic origin, which is characterized by a permanent inflammatory process of the small intestine induced by the ingestion of the storage proteins (gluten) of wheat (all species called Triticum such as durum wheat, spelled and kamut ), barley, rye, hybrid varieties and probably oats. According to current data, this disease has a prevalence of approximately 1 percent of the population (S.K. Lee et al. 2006 Curr Opin Rheumatol 18, 101-107). The only known effective therapy is based on following a gluten-free diet for life, which is complex and expensive given the massive use of these cereals in the current diet. Although the causal relationship between gluten intake and celiac disease is perfectly established, the relationship between the amount of gluten ingested and the severity of clinical manifestations is not defined. The individual variation as well as the clinical heterogeneity of the patients is a problem to establish threshold values that allow the protection of all susceptible individuals, since even traces of gluten-contaminated foods can be detrimental to some celiacs.

To meet the needs of this sector of the population, the food industry has developed a range of products with limited quantities of gluten, since its total suppression in cereals that contain it naturally is technically difficult and expensive. In addition, there are other products that, although they do not contain gluten naturally, can be contaminated during collection, storage, processing and / or transport. The European Union has homogenized the rules on the composition and labeling of food products intended for this group, based on the most recent guidelines of the Codex Alimentarius Commission (Codex Alimentaríus ALINORM 08/31 / REP, Appendix VII, p 107), which they indicate a threshold of 20 ppm for foods called "gluten-free", so that they find foods adapted to their needs and level of sensitivity.

This value is a compromise between the detectability of current analytical methods based on affinity tests with enzyme-labeled antibodies and the technological capacity to reduce pollution without increasing economic and environmental costs. Unfortunately, this value is not safe enough for many celiac patients. In 1999, the Federation of Celiac Associations of Spain (FACE) created the guarantee brand "Controlled by FACE" for those products that guarantee a maximum value of 10 ppm, a value very close to the detection limits of current methods.

The methods of gluten analysis are based on the direct detection of the allergenic protein. Gluten is made up of hundreds of proteins characterized by their high proline and glutamine content and low amino acid content with loaded side chains. Traditionally it is subdivided into two large fractions according to its solubility in alcohol: water (60%): the soluble prolamines (gliadin in wheat, hordein in barley, secalin in rye, avenin in oats) and the insoluble glutenins. Various toxic or immunogenic fragments have been identified in prolamines that are resistant to digestion by human proteases (R. Ciccocioppo et al. 2005 Clin Exp Immunol 140, 408-416), although recent studies suggest that glutenins are also toxic ( DH Dewar et al. 2006 Eur. J. Gastroenterol. Hepatol. 18, 483-491). Shan et al. identified a 33 amino acid peptide in α-2-gliadin (L. Shan et al. 2002 Science 297, 2275-2279) which proved to be immunodominant (L. Shan et al. 2005 J Proteome Res 4, 1732-1741). Today, the standard methods for gluten detection are based on ELISA type assays, using different antibodies that recognize different epitopes of prolamins. However, the results obtained depend strongly on the antibody as well as the reference material used (R. van Eckert et al. 2010 J. Cereal Sci. 51, 198-204). The antibody called MAb 401.21 developed in 1990 by Skerrit (JH Skerritt et al. 1990 J. Agrie. Food Chem. 38, 1771-1778) and described in Australian patent AU572955, is marketed in a sandwich test approved by the AOAC International ("the scientific association dedicated to analytical excelence®", (JH Skerritt et al. 1991 J. AOAC 74, 257-264) described in patent applications GB2207921, CA1294903 and AU1891788, although it is not able to quantify hordeins (detects only 4-5%), underestimate durum wheat and overestimate triticale and secalins (T. Thompson et al. 2008 J. Am. Diet. Assoc. 108, 1682-1687). Nor does it detect partially hydrolyzed gluten due to the type format sandwich Recent studies show that it also reacts against glutenins (R. van Eckert et al. 2010 J. Cereal Sci. 51, 198-204) Subsequently, the antibody called R5 has been developed, which recognizes the potentially toxic epitope QQPFP and other sequences similar pre Seats in prolamines, including 33mer (L. Sorell et al. Febs Lett 439, 46-50; I. Valdes et al. 2003 Eur. J. Gastroenterol. Hepatol 15, 465-474). The sandwich test with R5 has been designated as type I method by the Codex Alimentarius Analysis and Sampling Method Committee (Codex Alimentarius ALINORM 06/29/23, Appendix II, p. 40) and allows the detection of gluten in samples natural or heat processed, after extraction with a solution containing reducers to remove intercatenary disulfide bridges that form after heating (E. García et al. 2005 Eur. J. Gastroenterol. Hepatol. 17, 529-539) described in Spanish patent with publication number ES2182698 and patent family (EP1424345, US7585529). However, this method does not serve to measure hydrolyzed prolamines. Other antibodies have been developed such as PN3 (HJ Ellis et al. 1998 Gut 43, 190-195), CDC5 (HM Nassef et al. 2008 Anal. Chem. 80, 9265-9271) and G12 (B. Morón et al. 2008 Am. J. Clin. Nutr. 87 405-414; B. Morón et al. 2008 Pios One 3) against toxic or immunogenic epitopes comprised between amino acids 31-49, 56-75 and 57-89 of a-gliadin, respectively.

In order to detect hydrolyzed gluten, competitive assays have been proposed, which require a single epitope, and which have been marketed using the R5 antibodies described in the Spanish patent with publication number ES2304874 (see also WO2008110655) and G12. However, these methods are not compatible with the aforementioned extraction procedure because they denature the antibodies and enzymatic markers during the inevitable incubation between the sample and the only detection antibody used. Aptamers are a type of non-protein molecular receptor, they are thermally and chemically stable under extreme conditions and, therefore, constitute a promising alternative to antibodies. Aptamers are oligonucleotides that are selected in vitro by a combinatorial method called SELEX (systematic evolution of exponential enrichment ligands) and are characterized by having a high affinity and specificity towards a given ligand (AD Ellington et al. 1990 Nature 346, 818- 822; C. Tuerk et al. 1990 Science 249, 505-510). The presence of the ligand can induce a conformational change in the oligonucleotide that allows molecular recognition. Once the oligonucleotide sequence is known, its synthesis is chemical (does not require the use of animals), very reproducible and cheap.

SELEX is to find the sequence of higher affinity for a ligand in the desired experimental conditions for the greatest possible number of different strands (~ ^{13 October} -10 ¹⁵⁾ by an iterative system contacting between nucleic acid strands and the ligand, separation of the strands with affinity for the ligand and PCR amplification thereof. The selection of aptamers against proteins is usually carried out using as a target the complete protein However, obtaining aptamers against specific protein epitopes is also possible following several strategies (AV Kulbachinsky et al. 2007 Biochemistry-Moscow 72, 1505-1518). One of them is to use the specific peptide sequence, much shorter than the complete protein. It has been shown that the aptamers thus obtained are capable of recognizing the peptide within the complete protein (D. Proske et al. 2002 Chembiochem 3, 717-725), although in some cases with lower affinity (W. Xu et al. 1996 Proc. Nati. Acad. Sci. USA 93, 7475-7480). Although a priori one might think that aptamer selection is possible for any ligand, there are a number of prerequisites to have a high probability of finding a high affinity and specificity aptamer. Ligands with positively charged groups, capable of forming hydrogen bonds or flat aromatic structures, are more favorable than those with negatively charged groups and, especially, those with a strong hydrophobic nature (R. Stoltenburg et al. 2007 Biomolecular Engineering 24 , 381-403). Although the recognition of RNA / DNA from hydrophobic structures is known, it is quite rare (BA Gilbert et al. 1997 Bioorgan ed Chem 5, 1115-1122). The strategy to facilitate the interaction of nucleic acids with hydrophobic molecules could be the use of modified nucleotides with groups that confer hydrophobicity, compatible with amplification enzymes (SD Jayasena 1999 Clin. Chem. 45, 1628-1650). Therefore, it is not evident that the selection of aptamers against highly hydrophobic ligands, such as the 33-mer peptide employed in this invention, using nucleotides unmodified with hydrophobic groups, results in high affinity consensus sequences.

DESCRIPTION OF THE INVENTION

The present invention relates in a first aspect to a single-stranded DNA molecule characterized in that said molecule specifically recognizes and binds to a peptide comprising the amino acid sequence SEQ ID NO: 1. In a second aspect, the present invention relates to a kit for the detection of gluten characterized in that it comprises a single stranded DNA molecule of the first aspect of the invention.

In a third aspect, the present invention relates to a use of the single stranded DNA molecule of the first aspect of the invention or of the kit of the second aspect of the invention, for the detection of gluten. In a fourth aspect, the present invention relates to a method of detecting gluten based on the single stranded DNA molecule of the first aspect of the invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is a solution to the need to detect the presence of gluten in samples of food or other products that can cause intolerance in celiac individuals. The inventors of the present invention have demonstrated the usefulness of single stranded DNA sequences, also called aptamers, capable of specifically binding a peptide fraction of gliadin for the separation and / or detection of this protein. An advantage of the present invention is that aptamers are molecules that can be easily modified to bind them to marker molecules, unlike antibodies, and therefore have high versatility at the time of detection. In addition, aptamers are highly stable molecules, which is a great advantage when developing in vitro tests for gluten detection. It is important to note that aptamers are also much cheaper and easier to synthesize than antibodies, which are the molecules currently used for gluten detection. The present invention allows the detection of gluten at trace level, specifically less than 50 ppb (parts per billion) of PWG standard gliadin can be detected safely.

Therefore, in a first aspect, the present invention relates to a single stranded DNA molecule characterized in that said molecule specifically recognizes and binds to a peptide comprising the amino acid sequence SEQ ID NO: 1. Preferably, the molecule specifically recognizes and binds to gluten. More preferably, said gluten is wheat, barley, rye or oats. Even more preferably, said gluten is wheat.

The peptide with SEQ ID NO: 1 is known as the 33-mer peptide. In the present description, 33-mer peptide is understood as a wheat α-gliadin peptide, which is resistant to proteases and which has been considered as the primary initiator of the inflammatory response to gluten in celiac patients. Thus, it has been demonstrated by in vitro assays that the 33-mer peptide is the most bioactive (immunodominant) gluten peptide that is recognized by T cells from HLA-DQ2 ⁺ celiac donors. In the present invention the 33-mer peptide has been chosen as a target for the evolution of single-stranded DNA molecules (aptamers), specifically the sequence between amino acids 57 and 89 of gliadin, which corresponds to SEQ ID NO: 1 .

The term "single chain DNA molecule", as used herein, refers to a single strand of bound nucleotides, which in turn are each composed of a sugar, a nitrogen base and a Phosphate group. Unlike DNA that is presented as a double nucleotide chain, in which the two strands are linked together by connections called hydrogen bonds, single stranded DNA has a single strand. In the present description, the expression "single chain DNA molecule" and the term "aptamer" are used interchangeably. The term "specifically recognizes and binds", as used herein, refers to the ability of the aptamers of the present invention to interact with a peptide, which preferably forms part of gluten specifically, that is, , to join with a certain affinity to said protein and not to join others. This ability of aptamers of the invention to recognize and specifically bind gluten is similar to that of antibodies.

In the present description, gluten is understood as an amorphous protein found in the seed of some cereals, such as wheat, rye, barley and oats, combined with starch. Gluten represents 80% of wheat proteins. When the flour of one of these cereals is mixed with water, two grain proteins belonging to the group of prolamines, gliadins and glutenins, combine to form a protein network called gluten. This protein generates in a small part of the population a disease called celiac disease, in which the immune system responds by damaging the small intestine of people who ingest it by not being able to digest it. Gliadin appears to be the protein that presents the greatest problem in celiac disease or gluten intolerance, is modified by transgutaminase and presented by the DQ2 allele of the major histocompatibility complex. This makes every time a food with gluten is consumed an immune reaction that progressively deteriorates the intestine, can also be complicated by episodes of dermatitis herpetiformis and other neurological, rheumatic diseases, etc. In a preferred embodiment of the first aspect of the invention, the single chain DNA molecule specifically recognizes and binds gliadin, hordein, secane or avenin. Preferably, the single chain DNA molecule specifically recognizes and binds to gliadin. The authors of the present invention have demonstrated the ability of the aptamers of the present invention to specifically recognize and bind gliadin, as shown in Example 5 and Figure 4B. The term "g liad na", as used herein, refers to a prolamine that is part of the protein portion of gluten (soluble in ethanol) of wheat seeds. Prolamines are proteins that have large amounts of proline and glutamic acid. There are homologues of gliadin in barley (hordein), rye (secalin) and some varieties of oats (avenin), which explains that such cereals can also cause celiac disease.

In a preferred embodiment of the first aspect of the invention, the single chain DNA molecule is unable to recognize and specifically bind corn, soy or rice proteins. Preferably, the single chain DNA molecule is unable to recognize and specifically bind rice proteins. The authors of the present invention have shown that the aptamers of the present invention are unable to bind and detect rice proteins, which implies the advantage that they will not give false positive results in gluten detection assays.

In a preferred embodiment of the first aspect of the invention, the single stranded DNA molecule comprises the nucleotide sequence X-GTCT-Y, where X comprises between 3 and 29 nucleotides and Y comprises between 7 and 33 nucleotides. Preferably, the single stranded DNA molecule comprises a nucleotide sequence that is selected from SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10. Preferably, said molecule binds to the amino acid sequence SEQ ID NO: 1 with a dissociation constant K _D equal to or less than 55 nM .

In the present description, dissociation constant K _D is understood as a constant that measures the affinity with which a ligand, in the present invention the single-stranded DNA molecule, binds to a protein, in the present invention gluten. The ligand-protein affinity depends on non-covalent intermolecular interactions between two molecules such as hydrogen bonds, electrostatic interactions, Van der Waals and hydrophobic forces. The lower the KD, the greater the affinity with which the aptamer binds to the immunotoxic peptide contained in gluten. In a preferred embodiment of the first aspect of the invention, the single stranded DNA molecule comprises the nucleotide sequence SEQ ID NO: 2.

In another preferred embodiment of the first aspect of the invention, the single stranded DNA molecule comprises the nucleotide sequence SEQ ID NO: 7.

In a preferred embodiment of the first aspect of the invention, the single stranded DNA molecule further comprises a marker molecule. Preferably, the marker molecule is selected from a fluorophore, an enzyme, a peptide, a nanoparticle, an electroactive molecule, a digoxigenin and a biotin.

In the present description, a marker molecule is understood as a molecule easily detectable by different methods, which can for example have specific receptors or it can be an enzyme that catalyzes a detectable reaction directly or indirectly after the addition of the corresponding substrates for the detection of the single chain DNA molecule of interest.

The term "fluorophore", as used herein, refers to a fluorescent chemical compound that emits light upon excitation. A fluorophore bound to the single stranded DNA molecule of interest therefore allows its detection. In a preferred embodiment of the first aspect of the invention, the fluorophore is selected from fluorescein, boron-dipyrromethene, cyanine, naphthalene and rhodamine and any of its derivatives. In a preferred embodiment of the first aspect of the invention, the enzyme is selected from peroxidase, alkaline phosphatase, DNAzymes and NADH dehydrogenases. AND! The term "DNAzymes", as used herein, refers to nucleic acids with catalytic activity that can generate with said activity a detectable marking associated with the presence of said DNAzyme.

In a preferred embodiment of the first aspect of the invention, the peptide is selected from a polyhistidine, a myc epitope and a FLAG epitope. A polyhistidine is a peptide of between 2 and 12 histidines, preferably 6 histidines. A myc epitope is a 10 amino acid peptide derived from the c-myc gene and that because it is highly immunogenic it is easily detectable by antibodies, such as the monoclonal antibody called 9E10 from Developmental Studies Hybridoma Bank, A FLAG epitope is also an octapeptide that is also highly immunogenic and therefore easily detectable by antibodies. Both epitopes are well known in the state of the art.

In a preferred embodiment of the first aspect of the invention, the nanoparticle is selected from a metal nanoparticle, semiconductor nanoparticle and magnetic micro or nanoparticle. The term "metal nanoparticle", as used in the present description, refers to a nano-object with its three dimensions in the nanoscale (between 1 and 100 nm), formed by gold, silver, platinum or other metals, which together The single-stranded DNA molecule of interest allows its detection through its optical, electrochemical or catalytic properties.

The term "immobilized", as used in the present invention, refers to the fact that the 33-mer peptide, the immunogenic protein or the single stranded DNA molecule of the invention can be attached to a support without losing its activity. . The support can be the surface of a microtiter plate, surfaces of conductive materials, micro and magnetic nanoparticles, glass supports, latex particles, carbon nanotubes, among others. The term "semiconductor nanoparticle", as used herein, refers to a nanoobject with its three dimensions in the nanoscale of semiconductor material, such as sulfides, selenides and tellurides of Cd, Zn or Pb, which together The single stranded DNA molecule of interest allows its detection by its optical or electrochemical properties.

The term "micro or magnetic nanoparticle", as used in the present description, refers to a microscopic particle with magnetic properties, which together with the single stranded DNA molecule of interest allows its detection by changing the properties optics that take place after the union between the protein and the DNA chain so marked. In the present invention said nanoparticle is used for detection as a marker molecule and also as a solid support for the immobilization of the single-stranded DNA molecule, 33-mer peptide or immunogenic protein (gliadin or gluten).

In a preferred embodiment of the first aspect of the invention, the electroactive molecule is selected from methylene blue, ferrocene, anthraquinone and thionine, and any of its derivatives, among others.

The term "electroactive molecule", as used in the present description, refers to a molecule that can be oxidized or reduced within the potential window of an electrode material and, therefore, its presence can be detected by faradic electrochemical techniques. well known in the state of the art.

In a preferred embodiment of the first aspect of the invention, the marker molecule is biotin. Biotin is also known as vitamin H and is a molecule widely used in biotechnology to label other molecules, which is why it is known as biotinylation. The avidin and streptavidin proteins bind with a very high affinity and specificity to biotin, so that this molecule is a good marker. In a preferred embodiment of the first aspect of the invention, the marker molecule is digoxigenin. Digoxigenin is a hapten and can be conjugated to the single-stranded DNA molecules of the present invention as a marker thereof, since it can be easily detected by specific anti-digoxigenin antibodies.

In a preferred embodiment of the first aspect of the invention, the single stranded DNA molecule comprises a primer at each of its ends for detection by PCR.

In the present description, "primer" means a nucleic acid chain that serves as a starting point for DNA amplification. It is a short nucleic acid sequence that contains a free 3 'hydroxyl group that forms base pairs complementary to the template strand and acts as a starting point for the addition of nucleotides in order to copy and amplify the template strand. In the present invention the primers are attached to the ends of the single stranded DNA molecule for amplification and detection by PCR. The primers can be labeled with biotin or the fluorophore 6- carboxyfluorescein (6-FAM), or unlabeled.

In the present description, PCR is understood as the polymerase chain reaction. It is a molecular biology technique whose objective is to obtain a large number of copies of a particular DNA fragment, starting from a minimum. This technique is based on the natural property of DNA polymerases to replicate strands of DNA from primers that bind at the ends of the strands, for which it uses cycles of alternating high and low temperatures to separate newly formed strands of DNA between yes after each phase of replication and primer binding and then allow the polymerases to rejoin to duplicate them again. In the present invention the PCR is used for amplification and subsequent detection of the single-stranded DNA molecule linked to gluten, 33-mer peptide, 33-mer recombinant peptide or control peptide There are several types of PCR such as real-time PCR, PCR-ELISA or PCR-ELOSA. The main characteristic of real-time or quantitative PCR is that it allows quantifying the amount of DNA present in the original sample or identifying, with a very high probability, specific DNA samples from their melting temperature. PCR-ELISA (polymerase chain reaction - enzyme-linked immunoabsorption assay) is a hybrid technique between PCR and ELISA for the quantification of DNA products. In this assay the PCR products are labeled with a biotin and with a hapten to allow their capture on surfaces with (strept) avidin and subsequent quantification using an antibody against the hapten conjugated to an enzyme. PCR-ELOSA is a technique very similar to PCR-ELISA, in which PCR products are only labeled with the hapten, so that they hybridize with an immobilized capture probe and quantify by using an antibody against said hapten conjugated with an enzyme.

In a preferred embodiment of the first aspect of the invention, at least one phosphodiester bond of the single stranded DNA molecule contains at least one oxygen (O) substituted by a sulfur (S). The term "phosphodiester bond", as used herein, refers to a covalent bond that occurs between a hydroxyl group (OH) in the 3 'carbon and a phosphate group (PO ₄ ^{3 ~} ) in the 5 'carbon of the ribose sugar of the nucleotides of the single stranded DNA molecule, thus forming a double ester bond. To improve the resistance of aptamers against degradation by nucleases, modified aptamers are included in the present invention, in which at least one of the oxygen atoms participating in the phosphodiester bond has been replaced by a sulfur atom. In a second aspect, the present invention relates to a kit for the detection of gluten characterized in that it comprises at least one single stranded DNA molecule of the first aspect of the invention. Preferably, said kit It also includes a solid support. More preferably, the single chain DNA molecule is anchored to the solid support. In a preferred embodiment, the kit comprises at least two aptamers, one for capture and one for detection. In a preferred embodiment, the kit further comprises the peptide with SEQ ID NO: 1 or gliadin or gluten, anchored to the solid support. Such a kit would allow a competitive ELISA to be carried out, which would consist of the competition between the gluten present in the corresponding sample and the SEQ ID NO: 1 peptide immobilized on solid supports for a fixed and limited quantity of aptamer in solution previously marked and / or modified as described above.

Preferably, said kit further comprises an antibody that specifically recognizes and binds gluten. Gluten has several repetitions of SEQ ID NO: 1, that is, it has several binding sites whereby both the aptamers of the invention and a specific antibody could bind at the same time. Therefore, more than one aptamer or one or several aptamers and one or more antibodies could be used in combination for the separation and / or detection of gluten.

The kit of the invention may further comprise, without any limitation, conjugated or unconjugated primary antibodies, peptides, labeled or unlabeled primers, buffers, conjugated secondary antibodies, proteins or standard peptides, agents to prevent contamination, marker compounds, although not limited, fluorochromes, etc. On the other hand, the kit of the invention can include all the supports and containers necessary for its implementation and optimization. The kit of the invention may also contain other proteins or peptides that serve as positive and negative controls. Preferably, this kit further comprises instructions for carrying out the detection and / or quantification of the single stranded DNA molecule of the invention.

AND! term "antibody", as used herein, refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, that is, molecules that contain an antigen binding site that specifically binds (immunoreacts) with the gluten protein, with any of its fragments or with other indicator molecules used in the detection process.

In a third aspect, the present invention relates to a use of the single stranded DNA molecule of the first aspect of the invention or of the kit! second aspect of the invention, for the detection of gluten. As demonstrated in the examples and figures of the present description, the aptamers of the invention have proven useful in the specific detection of gluten.

In a fourth aspect, the present invention relates to a gluten detection method comprising the following steps:

 a) contacting a single stranded DNA molecule of the first aspect of the invention with a test sample; Y

 b) detect the presence of the complex formed by said molecule and the gluten of the test sample in step (a).

"Problem sample" means any sample, be it a food or other sample, capable of containing gluten or gluten-derived fragments that may cause intolerance in a celiac individual.

In a preferred embodiment of the fourth aspect of the invention, step (a) is carried out in solution.

In a preferred embodiment of the fourth aspect of the invention, step (a) is carried out on a solid support.

In a preferred embodiment of the fourth aspect of the invention, step (b) is carried out by PCR. In a preferred embodiment of the fourth aspect of the invention, step (b) is carried out by means of an aptaassay. The term "aptaassay", as used herein, refers to binding assays where the receptor molecule that binds to gluten is a single DNA chain or aptamer completely analogous to immunoassays where the receptor molecule is an antibody By analogy with immunoassays, this term includes all the variety of known formats for immunoassays, with no more than substituting antibody for aptamer. In a preferred embodiment of the fourth aspect of the invention, step (b) is carried out by an immunoassay. Preferably, said immunoassay is selected from westem blot, dot blot or ELISA. More preferably, said immunoassay is an ELISA. The aptamers of the present invention can be used to capture, concentrate and / or detect gluten in solution or anchored on solid surfaces. For example, to capture and / or preconcentrate gluten, biotinylated aptamers conjugated to a solid support containing streptavidin or any of its variants (magnetic microparticles, microtiter plates, etc.) could be used to allow separation of the matrix containing gluten. .

Throughout the description and the claims the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and features of the invention will be derived partly from the description and partly from the practice of the invention. The following examples and figures are provided by way of illustration, and are not intended to be limiting of the present invention. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. A) Representation of the concentration of aptamers of each selection cycle that do not bind (dark gray) and that bind (light gray) to the peptide with SEQ ID NO: 1 after interaction with it for 10 min in BS (binding buffer) obtained by fluorescence. B) Evolution of the enrichment percentage in DNA sequences of affinity towards the peptide with SEQ ID NO: 1 with the successive rounds of selection. FIG. 2. More stable secondary structure of aptamers with A) SEQ ID NO: 2, B) SEQ ID NO: 5, C) SEQ ID NO: 7, D) SEQ ID NO: 8, E) SEQ ID NO: 9 and F) SEQ ID NO: 10, obtained through the open-access network server (mfold), hosted by "The RNA Institute, College of Arts and Sciences, State University of New York at Albany".

FIG. 3. Calorimetric evaluation of 33.4 μΜ of the aptamer with SEQ ID NO: 2 with 0.338 mM of the peptide with SEQ ID NO: 1 in BS (50 mM TRIS pH 7.4 + 0.25 M NaCI + 5 mM MgCI ₂ ) at 15 ° C (A) and 35 ° C (B). Superior graphics: Power as a function of time obtained after each peptide addition. Lower graphs: Integrated values of heat variation in each addition after correction with dilution heat.

FIG. 4. Link curves of aptamers with SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10 to modified magnetic particles A) with the peptide with SEQ ID NO: 1 B) with the gliadin standard PWG (Prolamin Working Group).

FIG. 5. Bonding curves of aptamers with SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 7 and SEQ ID NO: 8 to magnetic particles modified with ethanol extracts of A) rye flour (as a percentage of fraction linked) and B) oatmeal (current intensity). EXAMPLES

The invention will now be illustrated by tests carried out by the inventors, which demonstrates the efficacy of the aptamers of the invention and the gluten detection method of the invention.

Example 1: Obtaining aptamers for the peptide with SEQ ID NO: 1 of a-2- gliadin.

materials

The selected target, the peptide with SEQ ID NO: 1, was obtained by recombinant DNA techniques. From a synthetic oligonucleotide (SEQ ID NO: 20) obtained in Sigma-Genosys with the sequence coding for the 33 amino acids ranging from position 57 to 89 of gliadin (Gen Bank, accession number AJ133612.1) and the direct (SEQ ID NO: 11) and reverse (SEQ ID NO: 12) primers, multiple copies were obtained by PCR. The recombinant peptide was obtained after cloning this PCR product and expressing it in the pETBIue-2 system, following the manufacturer's instructions (Novagen), whereby a histidine tail is added. For the purification of the peptide with the histidine tail (SEQ! D NO: 13) a 1 mL HisTrap FF column was used in an AKTA FPLC equipment (GE Healthcare). For the counter-selection the histidine tail (control peptide, SEQ ID NO: 14) obtained with the pETBIue-2 system was used. The 80 nucleotide DNA sequence collection was designed so that its general sequence corresponds to SEQ ID NO: 15. The collection was synthesized and purified by PAGE by Sigma-Genosys. The sequences of the primers used in the PCR amplification step correspond to SEQ ID NO: 16 (direct primer) and SEQ ID NO: 17 (biotinylated reverse primer). Magnetic microparticles (MagneHis ™ Ni particles, from Promega, USA) modified with the target peptide or with the control were used for each round. The protocol Modification consisted of balancing the particles in modification buffer (B, 100 mM HEPES pH 7.5 + 10 mM imidazole + 0.5 M NaCI). They were then incubated with peptide with SEQ ID NO: 1 or with control peptide with SEQ ID NO. 14 in concentration between 0.25 and 1 pg / pL on BM for 15 min with rotation at room temperature. After decanting and washing twice with BM + 0.01% Tween-20 and 1 time with binding buffer (BS: 50 mM TRIS pH 7.4 + 0.25 M NaCI + 5 mM MgCk), the modified magnetic particles were diluted to 5 pmol of target / pL in BS. Process

For each round of selection 250 pmoles of the DNA collection selected and amplified in the previous round (1 nmol of the initial collection in the first round were used to ensure a variety of sequences of 5x10 ¹⁴ -1x10 ¹⁵ molecules) diluted in BS. They were heated at 98 ° C for 4 min and immediately cooled on ice another 4 min. The selection medium consisted of BS to which 1 pg / mL of BSA (bovine serum albumin) was added to avoid nonspecific binding and a competing transfer RNA at a concentration 10 times lower than that of the DNA collection. In this medium the DNA molecules were contacted with a concentration of peptide (peptide with SEQ ID NO: 1 or control peptide with SEQ ID NO: 14 in the counterselections) 10 times lower than that of DNA to limit the amount of target and favor the competition. After an incubation time that was progressively decreasing in successive rounds according to Table 1, several washes were done in BS + 0.01 Tween-20 (see table 1).

Finally, the DNA-peptide complex was eluted by incubating the particles in 100 mM HEPES pH 7.5 + 500 mM imidazole (BME) for 10 min under stirring. The eluate was transferred to PCR tubes for amplification. Each 100 μ! _ Of PCR contained: 2 μΙ_ of template (eluate), 1 μΜ of each primer (SEQ ID NO: 16 and SEQ ID NO: 17), 0.2 mM dNTP, 3 mM Mg ²⁺ , 1x PCR buffer and 2.8 U of immolase DNA polymerase (Disbiotec). The PCR conditions were: initial incubation at 37 ° C for 10 min, 20 min at 95 ° C to activate the enzyme and 15 cycles of 94 ° C- 57 ° C- 72 ° C for 45 s each. The final extension was performed at 72 ° C for 10 min. The amplification was confirmed by 2% agarose gel electrophoresis. Quantification of the amplified double-stranded DNA was fluorometrically performed with a "MiniFluorimeter" model TBS-380 from the Turner Biosystems house. Obtaining the single strand DNA strand was achieved using streptavidin modified magnetic particles (Dynabeads® MyOne ™ Streptavidin C1, Life Technologies, Madrid). These particles were contacted with the DNA for 15 min at room temperature and under rotation after which, the strands were separated by incubation in 100 mM NaOH for 10 min under stirring. The resulting supernatant contains the single stranded DNA that is used in the next round.

Table 1. Variable experimental conditions used in the successive rounds of the selection.

Before rounds 3, 6 and 9, a counter-selection was made by incubating the DNA of the previous round with magnetic particles modified with the control peptide with SEQ ID NO: 14 under the same conditions as with the peptide with SEQ ID NO: 1, but in this case the particles were discarded and the supernatant was used in the corresponding selection round.

After 10 rounds of selection, 12.8% of the selected DNA was able to bind to the recombinant peptide, while no sequences that bound to the control peptide were detected. The percentage of binding was determined with aliquots of each cycle amplified by PCR under the same experimental conditions as in each cycle except that a direct primer with SEQ ID NO: 16 labeled with 6-FAM (SEQ ID NO: 18) was used. The 6-FAM-labeled strand was separated from the biotin-labeled strand identically to the strand separation prior to each round and its concentration was determined fluorimetrically (Biotek Instrument flx800) versus a calibration performed with random DNA-6- sequences. FAM of known concentration using an excitation λ of 480 nm and an emission λ of 528 nm.

The binding assay was carried out by contacting equal amounts of DNA-6-FAM of each cycle and recombinant peptide (SEQ ID NO: 13) immobilized on magnetic particles (15 pmoles) for 10 minutes in BS. The unbound fraction was collected and after 2 washes in BS + 0.01% Tween-20, the bound aptamers were eluted in B E for 10 min under stirring, thus obtaining the bound fraction. Both fractions were analyzed fluorimetrically (Figure 1A), observing a progressive decrease in the fraction not linked with the cycles simultaneously to an increase in the fraction linked (Figure 1B). A binding assay was performed with the control peptide and the DNA sequences obtained in cycle 10. The fluorescence emitted by the unbound fraction was lower than the detection limit, indicating that no affinity sequences were amplified by the tail of histidines or solid support.

Example 2: Identification of the DNA aptamers obtained. To identify aptamers with affinity for the SEQ ID NO: 1 peptide, an aliquot of cycle 10 was amplified using the unmodified primers (SEQ ID NO: 16 and SEQ ID NO: 19) using the same conditions as in previous stages of amplification, and was cloned using the pETBIue-1 Acceptor Vector Kit system from Novagen. After cloning, plates of the host strain (Nova Competent cells, Novagen) were seeded and 25 clones were sequenced using the BigDye Terminator v3.1 Cycle Sequencing kit (Applied Biosystems) and an Abi PRISM 3130x1 Genetic Analyzer. The sequences obtained were analyzed using the Chromas Lite 2.01 software.

The secondary structure of the six aptamers was determined SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10 using the network server " mfoid "under the binding conditions (25 ° C, 250 mM NaCI and 5 mM MgCl ₂ ). Figure 2 and Table 2 show the most stable structure and Gibbs free energies for each of the aptamers, respectively.

Table 2. Thermodynamic parameters of aptamers.

Example 3: Characterization of the SEQ ID NO: 2 aptamer. The aptamer with SEQ ID NO: 2 was selected for further characterization by isothermal calorimetric titration. The SEQ ID NO: 1 peptide chemically synthesized by Biomedal (Seville, Spain) was used as the target molecule. The aptamer binding with SEQ ID NO: 2 to the peptide SEQ ID NO: 1 was studied by isothermal calorimetric titration. The concentration of the aptamer in the calorimetric cell was 33.4 μΜ, while the concentration of peptide in the syringe was 0.338 mM. The following series of peptide injections was performed: a first 4 L injection followed by 18 injections of 15 μί. Figure 3 shows the valuations at both temperatures 15 ⁰ C and 35 ⁰ C. The analysis of the measurements have provided the thermodynamic values summarized in table 3.

Table 3. Thermodynamic parameters found by ITC.

The K _D was determined by isothermal calorimetric titration (ITC), a usual technique for this type of measurement. The aptamer was evaluated by successive additions of peptide SEQ ID NO: 1 chemically synthesized, at 15 and 35 ° C, obtaining a KD of 45 ± 10 nM and 17 ± 10 nM, respectively. These data confirm the binding between the aptamer with SEQ ID NO: 2 object of the invention and the peptide SEQ ID NO: 1. In addition, the aptamer with SEQ ID NO: 2 has a high affinity for the target, as follows from a constant of nM dissociation for a hydrophobic molecule, which does not contain the functional groups that favor interaction with nucleic acids.

Example 4 Binding curves of the aptamers selected to the peptide with SEQ! D NO: 1.

The affinity evaluation of the aptamers by the peptide with SEQ ID NO: 1 was carried out by binding assays on streptavidin coated magnetic particles, on which the peptide with biotinylated SEQ ID NO: 1 was immobilized at its C-terminus. The fraction of each of the six biotin modified aptamers that binds to said particles was measured chronoamperometrically after addition of! streptavidin-HRP and enzyme substrate conjugate (tetramethylbenzidine + H ₂ 0 ₂ ). All selected aptamers had affinity for the target.

For this, streptavidin-coated magnetic particles (Dynabeads® MyOne ™ Streptavidin C1, Life Technologies, Madrid) were modified with SEQ ID NO: 1 biotinylated peptide at its C-terminal end (Biomedal, Sevilla) 2 μΜ in PBS + 0.01% Tween -20 throughout the night. After two washes with PBS + 0.01% Tween-20, they were blocked with 500 μΜ of biotin in PBS + 0.01% Tween-20 for 30 min. After 2 new washes they were reconstituted in BS. Increasing concentrations of each of the biotinylated aptamers were contacted (SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10) in BS for 30 min. After 2 washes with BS + 0.01% Tween-20, 2.5 g mL streptavidin-HRP from Sigma-Aldrich was added for 30 min. After 3 washes with BS + 0.01% Tween-20, an aliquot was placed on a screen-printed carbon electrode (Dropsens, Oviedo) and the substrate was added. After 1 minute of enzymatic reaction, the amount of product generated was measured chronoamperometrically at 0 V using a μ-AutoLab potentiostat type II PGstat-12 equipped with GPES 4.9 software (EcoChemie, The Netherlands). Figure 4A shows that all aptamers have affinity for the target (peptide SEQ ID NO: 1). Example 5 Link curves of the aptamers selected to PWG.

The affinity of aptamers was evaluated by the gliadin pattern commonly used in commercial immunoassays obtained from a mixture of wheat flour from different sources, called PWG. Binding assays were performed on magnetic particles activated with surface tosyl groups, on which the PWG was immobilized. The fraction of each of the six biotin modified aptamers that binds to said particles was measured chronoamperometrically after the addition of the streptavidin-HRP conjugate and the enzyme substrate (tetramethylbenzidine + H2O2).

To this end, magnetic particles were modified with superficial tosyl groups (Dynabeads® MyOne ™ Tosylactivated, Life Technologies, Madrid) with PWG (Prolamin Working Group gliadin standard) at 0.32 mg / mL in Borate 0.058 M pH 9.5 + ( NH ₄ ) ₂ SO ₄ 1 M for 24 h at 37 ° C. After two washes in PBS + 0.01% Tween-20, they are left in the same buffer overnight at 37 ° C. After 2 new washes, they were reconstituted in PBS + 0.01% Tween-20 and used to obtain the binding curves. For this, identical aliquots of the PWG modified microparticles were contacted with increasing concentrations of each of the biotinylated aptamers (SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9) in BS + 0.01% Tween-20 for 30 min. After 2 washes with BS + 0.01% Tween-20, 2.5 pg / mL streptavidin-HRP from Sigma-Aldrich was added and incubated for 30 min. After 2 washes with BS + 0.01% Tween-20 and one in BS, they were resuspended in BS. An aliquot was placed on a screen-printed carbon electrode (Dropsens, Oviedo) and the substrate was added. After 1 minute of enzymatic reaction, the amount of product generated was measured chronoamperometrically at 0 V using a PG-typeLamp-12 potentiostat type II equipped with GPES 4.9 software (EcoChemie, The Netherlands). Figure 4B shows that all aptamers have affinity for the complete gliadin protein, as expected after the demanding selection made by SELEX. Example 6: Specificity of the interaction.

The specificity of the affinity of aptamers towards other proteins susceptible to trigger celiac disease was evaluated and their cross-reactivity was evaluated with other proteins present in cereals that do not trigger it. Binding tests were carried out on magnetic particles activated with surface tosyl groups, on which a diluted ethanolic extract of rye, oatmeal and rice flour was immobilized. The fraction of each of the biotin-modified aptamers that bind to said particles was measured chronoamperometrically after the addition of the streptavidin-HRP conjugate and the enzyme substrate (tetramethylbenzidine + H ₂ O ₂ ) (Figure 5).

For this, link curves of the aptamers with sequences SEQ ID NO: 7, SEQ ID NO: 2 and SEQ ID NO: 8, were made to the prolamines of other cereals that cause celiac disease (rye and possibly oats) and others that do not cause it (rice). The extraction of 1 g of each flour was performed with 10 mL of 60% ethanol for 30 mtn under stirring, after which it was centrifuged at 2500 g for 45 min. The extract was centrifuged again and the resulting supernatant collected for use. Tosylated magnetic particles were modified in the manner indicated in example 5 and the binding curves were performed in a completely analogous manner. Figure 5 shows that all aptamers tested have an affinity for rye, although less than for wheat. As shown in Figure 5B, the SEQ ID NO: 7 aptamer shows affinity for oats, although at higher concentrations than for wheat and rye, so it is able to recognize avenins, whose role in triggering the Celiac disease is not yet fully clarified. The SEQ ID NO: 2 aptamer showed no affinity for rice proteins since the amperometric signals obtained are indistinct from the target.

Claims

1. - A single-stranded DNA molecule, where said sequence recognizes and specifically binds to a peptide that comprises the amino acid sequence SEQ ID NO:1.

2. - The DNA molecule according to claim 1, characterized in that said molecule recognizes and specifically binds to gluten.

3.- The DNA molecule according to claim 2, wherein said gluten is from wheat, barley, rye or oats.

4.- The DNA molecule according to claim 3, wherein said gluten is wheat.

5.- The DNA molecule according to any of claims 2 or 3, wherein said molecule recognizes and specifically binds to gliadin, hordein, secalin or avenin.

6. - The DNA molecule according to any of claims 1 to 5, wherein said molecule recognizes and specifically binds to gliadin.

7. - The DNA sequence according to any of claims 1 to 6, wherein said sequence is incapable of specifically recognizing and binding corn, soy or rice proteins.

8. - The DNA sequence according to any of claims 1 to 7, where said sequence comprises the nucleotide sequence X-GTCT-Y, where X comprises between 3 and 29 nucleotides and Y comprises between 7 and 33 nucleotides.

9.- The DNA sequence according to any of claims 1 to 8, where said sequence comprises a nucleotide sequence that is selected from between SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10.

10. - The DNA sequence according to claim 9, wherein said sequence joins the amino acid sequence SEQ ID NO: 1 with a dissociation constant

K _D equal to or less than 55 nM.

11. - The DNA sequence according to any of claims 1 to 10, where said sequence comprises the nucleotide sequence SEQ ID NO: 2.

12. The DNA sequence according to any of claims 1 to 10 wherein said sequence comprises the nucleotide sequence SEQ ID NO: 7.

13. - The DNA sequence according to any of claims 1 to 12, wherein said sequence also comprises a marker molecule.

14. - The DNA sequence according to claim 13, wherein said marker molecule is selected from among a fluorophore, an enzyme, a peptide, a nanoparticle, an electroactive molecule, a digoxigenin and a biotin.

15. - The DNA sequence according to claim 14, wherein said electroactive molecule is selected from methylene blue, ferrocene, anthraquinone and thionine.

16.- The DNA sequence according to claim 14, wherein said fluorophore is selected from fluorescein, boron-dipyrromethene, cyanine, naphthalene and rhodamine.

17.- The DNA sequence according to claim 14, wherein said enzyme is selected from peroxidase, alkaline phosphatase, DNAzymes and NADH dehydrogenases.

18. - The DNA sequence according to claim 14, wherein the peptide is selected from a polyhistidine, a myc epitope and a flag epitope.

19. - The DNA sequence according to claim 14, wherein said nanoparticle is selected from a metallic nanoparticle, semiconductor nanoparticle and magnetic micro or nanoparticle.

20. - The DNA sequence according to claim 14, where the marker molecule is biotin.

21. - The DNA sequence according to claim 14, where the marker molecule is digoxigenin.

22. - The DNA sequence according to any of claims 1 to 21, where at least one phosphodiester bond of said sequence contains at least one O substituted by an S.

23. - A kit for the detection of gluten characterized in that it comprises at least one single-stranded DNA sequence according to any of claims 1 to 22.

24. - The kit according to claim 23, characterized in that it also comprises a solid support.

25.- The kit according to any of claims 23 or 24, wherein the DNA sequence is anchored to the solid support.

26.- The kit according to any of claims 23 or 24, which also comprises the peptide with SEQ ID NO: 1 or gliadin or gluten, anchored to the solid support.

27. - The kit according to any of claims 23 to 26, where it also comprises an antibody that recognizes and specifically binds to gluten.

28. - Use of the single-stranded DNA sequence according to any of claims 1 to 22 or the kit according to any of claims 23 to 27, for the detection of gluten.

29 - A gluten detection method comprising the following steps: a) contacting a single-stranded DNA sequence according to any of claims 1 to 22 with a test sample; and b) detect the presence of the complex formed by said sequence and the gluten of the test sample in step (a).

30 - The method according to claim 29, wherein step (a) is carried out in solution.

31.- The method according to claim 29, wherein step (a) is carried out on a solid support.

32.- E! method according to any of claims 29 to 31, where step (b) is carried out by PCR.

33. - The method according to any of claims 29 to 31, where step (b) is carried out by means of an apta-assay.

34. - The method according to claim 33, wherein the aptaassay is an ELOSA.