WO2018097220A1 - Nucleic acid molecule and use thereof - Google Patents

Abstract

Provided is a novel nucleic acid molecule capable of binding to gluten and/or gliadin. A nucleic acid molecule capable of binding to gluten and/or gliadin is a nucleic acid molecule comprising a polynucleotide (a) or (b): (a) a polynucleotide which comprises a nucleotide sequence represented by SEQ ID NO: 1, 8 or 10 or a partial sequence of the nucleotide sequence represented by SEQ ID NO: 1, 8 or 10; and (b) a polynucleotide which comprises a nucleotide sequence having an identity of 90% or higher with the nucleotide sequence for the polynucleotide (a) and can bind to gluten and/or gliadin.

Description

Nucleic acid molecules and uses thereof

The present invention relates to a nucleic acid molecule that binds to at least one of gluten and gliadin and use thereof.

小麦 Wheat is a food that is frequently consumed on a daily basis, but in recent years, the number of wheat allergic patients has increased and has been regarded as a problem. Since many processed foods use wheat, it is extremely important to analyze whether or not wheat is mixed as a raw material in processed foods and production lines thereof.

Allergic allergens are generally proteins and their degradation products (peptides), and analysis methods using antibodies using these as antigens are the mainstream. Regarding cereals including wheat, for example, gluten and gliadin, which is one of proteins constituting gluten, are known as allergens. As an analysis method for gluten, a method using an ELISA method has been reported (Non-Patent Document 1, Non-Patent Document 2).

However, since an antibody is a protein and has a problem in stability, it is difficult to use the antibody for a simple test method at a low cost. Further, electrophoresis, blotting on a nitrocellulose membrane, and the like are necessary, and the operation is complicated. For this reason, in recent years, attention has been focused on nucleic acid molecules that specifically bind to antigens instead of antibodies.

Therefore, an object of the present invention is to provide a new nucleic acid molecule that binds to at least one of gluten and gliadin.

The nucleic acid molecule of the present invention is a nucleic acid molecule that binds to at least one of gluten and gliadin, characterized in that it comprises a polynucleotide of either (a) or (b) below.
(A) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1, 8, or 10 or a partial sequence of the nucleotide sequence of SEQ ID NO: 1, 8, or 10 (b) 90% or more relative to the nucleotide sequence of (a) A polynucleotide that has the same nucleotide sequence and binds to at least one of gluten and gliadin

The detection reagent for gluten or gliadin of the present invention comprises the nucleic acid molecule of the present invention.

In the method for detecting gluten or gliadin of the present invention, the nucleic acid molecule of the present invention or the detection reagent of the present invention is brought into contact with a sample, and gluten or gliadin in the sample is mixed with the nucleic acid molecule or the detection reagent. Forming a complex of:
A step of detecting the complex.

The nucleic acid molecule of the present invention can bind to gluten or gliadin. Therefore, according to the nucleic acid molecule of the present invention, gluten and gliadin can be detected based on the presence or absence of binding to the allergen in the sample. Therefore, the nucleic acid molecule of the present invention can be said to be an extremely useful tool for detecting allergens derived from grains such as wheat, for example, in the fields of food production, food management, food distribution and the like.

FIG. 1 shows a presumed secondary structure of aptamer 1 in Example 1 of the present invention. FIG. 2 is a graph showing the binding property of aptamer 1 to gliadin and gluten in Example 1 of the present invention. FIG. 3 is a graph showing the binding property of aptamer 1 to gluten in Example 1 of the present invention. FIG. 4 is a graph showing the binding property of aptamer 1 to gliadin in Example 1 of the present invention. FIG. 5 shows the predicted secondary structures of aptamers 2 to 7 in Example 2 of the present invention. FIG. 6 is a graph showing the binding property of each aptamer to gliadin in Example 2 of the present invention. FIG. 7 is a graph showing the measurement results of the light emission amount in Example 3 of the present invention. FIG. 8 shows the presumed secondary structures of aptamers 8 to 11 in Example 4 of the present invention. FIG. 9 is a graph showing the binding properties of aptamers 8 and 9 to gliadin in Example 4 of the present invention. FIG. 10 is a graph showing the binding properties of aptamers 10 and 11 to gliadin in Example 4 of the present invention. FIG. 11 is a graph showing the binding properties of aptamers 8 and 9 to heated gliadin in Example 5 of the present invention. FIG. 12 is a graph showing the binding properties of aptamers 10 and 11 to heated gliadin in Example 5 of the present invention. FIG. 13 is a graph showing the binding properties of aptamers 8 to 11 to gluten and heated gluten in Example 6 of the present invention.

(1) Nucleic acid molecule The nucleic acid molecule of the present invention contains a polynucleotide of any one of the following (a) and (b) as described above, and is a nucleic acid molecule that binds to at least one of gluten and gliadin: It is.
(A) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1, 8, or 10 or a partial sequence of the nucleotide sequence of SEQ ID NO: 1, 8, or 10 (b) 90% or more relative to the nucleotide sequence of (a) A polynucleotide that has the same nucleotide sequence and binds to at least one of gluten and gliadin

In the present invention, the origin of gluten is not particularly limited, and examples thereof include grains such as wheat, rye, barley, and oats. For the nucleic acid of the present invention, for example, commercially available gluten can be used as gluten for confirming the binding ability, and specific examples include wheat-derived gluten (073-00575, manufactured by Wako Pure Chemical Industries, Ltd.). The gluten may be, for example, an unmodified allergen that has not been modified by heating or the like, or a modified allergen that has been modified by heating or the like.

In the present invention, the origin of gliadin is not particularly limited, and is the same as, for example, the origin of gluten described above. Regarding the nucleic acid of the present invention, for example, gliadin for confirming the binding ability can be exemplified by wheat-derived gliadin protein comprising the amino acid sequence disclosed in GenBank Accession No. A27319. The gliadin may be, for example, an unmodified allergen that has not been modified by heating or the like, or a modified allergen that has been modified by heating or the like.

The nucleic acid molecule of the present invention can bind to gluten or gliadin as described above. In the present invention, “binding to gluten” means, for example, having binding property to gluten or having binding activity to gluten, and “binding to gliadin” means, for example, It is said that it has binding property to gliadin or has binding activity to gliadin. The binding between the nucleic acid molecule of the present invention and gluten or gliadin can be determined by, for example, surface plasmon resonance molecular interaction (SPR) analysis. For the analysis, for example, BIACORE 3000 (trade name, GE Healthcare UK Ltd.) can be used. Since the nucleic acid molecule of the present invention binds to gluten or gliadin, it can be used, for example, for detection of gluten or gliadin.

The nucleic acid molecule of the present invention has a dissociation constant indicating a binding force to gliadin of, for example, 581 nmol / L or less, 132 nmol / L or less, 102 nmol / L or less, 23 nmol / L or less, 20 nmol / L or less.

The nucleic acid molecule of the present invention is also called a DNA molecule or a DNA aptamer. The nucleic acid molecule of the present invention may be, for example, a molecule composed of the polynucleotide (a) or (b) or a molecule containing the polynucleotide.

The polynucleotide (a) may be, for example, a polynucleotide containing the base sequence of SEQ ID NO: 1, 8, or 10, or a polynucleotide comprising the base sequence of SEQ ID NO: 1, 8, or 10. Further, it may be a polynucleotide containing a partial sequence of the base sequence of SEQ ID NO: 1, 8, or 10 or a polynucleotide comprising the partial sequence. The polynucleotides of SEQ ID NOs: 1, 8, and 10 are shown below.

Glu392BR8m4 (SEQ ID NO: 1)
GGTATGGAGGCAAGTCCCAATTCG T G C AGAGAAA C G T G TCT G T A TTT A TT AA TC G TTTC AG CC AGA C AGGG TTT A T G
Gli95_395TR8m1 (SEQ ID NO: 8)
GGAAACGCCGCCTAGATCATTTG C G TCCTCCCT GG T GGGGA TT GG C GAAAA TT GA C A TCCTC AAG TTCCT G C GAAA T G
Gli — 395BR8m2 (SEQ ID NO: 10)
GGAAACGCCGCCTAGATCATTTGAAAA C G TT G TT A CCTC A CCT A TT A TCT A TT GA C A TCCTC AAG TTCCT G C GAAA T G

The partial sequence of SEQ ID NO: 1 is not particularly limited, and examples thereof include base sequences of SEQ ID NOs: 2 to 7.

Glu392BR8m4_s59A (SEQ ID NO: 2)
GGTATGGAGGCAAGTCCCAATTCG T G C AGAGAAA C G T G TCT G T A TTT A TT AA TC G TTTC
Glu392BR8m4_s51 (SEQ ID NO: 3)
GGCAAGTCCCAATTCG T G C AGAGAAA C G T G TCT G T A TTT A TT AA TC G TTTC
Glu392BR8m4_s38 (SEQ ID NO: 4)
TC G T G C AGAGAAA C G T G TCT G T A TTT A TT AA TC G TTTC
Glu392BR8m4_s67 (SEQ ID NO: 5)
GGTATGGAGGCAAGTCCCAATTCG T G C AGAGAAA C G T G TCT G T A TTT A TT AA TC G TTTC AG CC AGA C
Glu392BR8m4_s59B (SEQ ID NO: 6)
GGCAAGTCCCAATTCG T G C AGAGAAA C G T G TCT G T A TTT A TT AA TC G TTTC AG CC AGA C
Glu392BR8m4_s46 (SEQ ID NO: 7)
TCGTGCAGAGAAACG T G TCT G T A TTT A TT AA TC G TTTC AG CC AGA C

The partial sequence of SEQ ID NO: 8 is not particularly limited, and examples thereof include the base sequence of SEQ ID NO: 9.
Gli95_395TR8m1s50 (SEQ ID NO: 9)
CGCCTAGATCATTTG C G TCCTCCCT GG T GGGGA TT GG C GAAAA TT GA C A T

The partial sequence of SEQ ID NO: 10 is not particularly limited, and examples thereof include the base sequence of SEQ ID NO: 11.
Gli — 395BR8m2s51 (SEQ ID NO: 11)
GGAAACGCCGCCTAGATCATTTGAAAA C G TT G TT A CCTC A CCT A TT A TCT A

In the above (b), “identity” is not particularly limited, and may be any range as long as the polynucleotide of (b) binds to at least one of gluten and gliadin. The identity is, for example, 80% or more, preferably 85% or more, more preferably 90% or more, further preferably 95% or more, 96% or more, 97% or more, particularly preferably 98% or more, and most preferably 99%. % Or more. The identity can be calculated with default parameters using analysis software such as BLAST and FASTA (hereinafter the same).

The polynucleotide in the nucleic acid molecule of the present invention may be, for example, the polynucleotide (c) below. In this case, the nucleic acid molecule of the present invention may be, for example, a molecule composed of the polynucleotide (c) or a molecule containing the polynucleotide.
(C) a polynucleotide comprising a base sequence complementary to a polynucleotide that hybridizes under stringent conditions to the polynucleotide comprising the base sequence of (a) and binding to at least one of gluten and gliadin

In (c), the “hybridizing polynucleotide” is not particularly limited, and is, for example, a polynucleotide that is completely or partially complementary to the base sequence of (a). The hybridization can be detected by, for example, various hybridization assays. The hybridization assay is not particularly limited, for example, Zanburuku (Sambrook) et al., Eds., "Molecular Cloning: A Laboratory Manual 2nd Edition (Molecular Cloning:. A Laboratory Manual 2 nd Ed) ," [Cold Spring Harbor Laboratory Press (1989)] and the like can also be employed.

In the above (c), the “stringent conditions” may be, for example, any of low stringent conditions, medium stringent conditions, and high stringent conditions. “Low stringent conditions” are, for example, conditions of 5 × SSC, 5 × Denhardt's solution, 0.5% SDS, 50% formamide, and 32 ° C. “Medium stringent conditions” are, for example, 5 × SSC, 5 × Denhardt's solution, 0.5% SDS, 50% formamide, 42 ° C. “High stringent conditions” are, for example, conditions of 5 × SSC, 5 × Denhardt's solution, 0.5% SDS, 50% formamide, 50 ° C. The degree of stringency can be set by those skilled in the art by appropriately selecting conditions such as temperature, salt concentration, probe concentration and length, ionic strength, time, and the like. "Stringent conditions" are, for example, Zanburuku previously described (Sambrook) et al., Eds., "Molecular Cloning: A Laboratory Manual 2nd Edition (Molecular Cloning:. A Laboratory Manual 2 nd Ed) ," [Cold Spring Harbor Laboratory Press ( 1989)]] and the like.

The polynucleotide in the nucleic acid molecule of the present invention may be, for example, the polynucleotide (d) below. In this case, the nucleic acid molecule of the present invention may be, for example, a molecule composed of the polynucleotide (d) or a molecule containing the polynucleotide.
(D) a polynucleotide comprising a base sequence in which one or several bases are deleted, substituted, inserted and / or added in the base sequence of (a), and binding to at least one of gluten and gliadin

In the above (d), “one or several” may be in the range where the polynucleotide in (d) binds to at least one of gluten and gliadin. The “one or several” in the base sequence of (a) is, for example, 1 to 10, preferably 1 to 7, more preferably 1 to 5, further preferably 1 to 3, particularly preferably. Is one or two.

The polynucleotide in the nucleic acid molecule of the present invention may be, for example, the polynucleotide (e) below. In this case, the nucleic acid molecule of the present invention may be, for example, a molecule composed of the polynucleotide (e) or a molecule containing the polynucleotide.
(E) consisting of a base sequence having 80% or more identity to the base sequence of (a), each of the base sequence of SEQ ID NO: 2 to 7, the base sequence of SEQ ID NO: 9, or the sequence A polynucleotide comprising the nucleotide sequence of No. 11 and binding to at least one of gluten and gliadin

The polynucleotide in the nucleic acid molecule of the present invention may be, for example, the polynucleotide (f) below. In this case, the nucleic acid molecule of the present invention may be, for example, a molecule composed of the polynucleotide (f) or a molecule containing the polynucleotide.
(F) a base sequence having at least 80% identity to at least one base sequence selected from the group consisting of SEQ ID NOs: 1 to 11, and represented by formulas (I) to (XI), respectively. A polynucleotide that binds to at least one of gluten and gliadin

In the above (f), “a secondary structure can be formed” means, for example, that the polynucleotide of (f) can form a stem structure and a loop structure in the above formula (the same applies hereinafter). The stem structure and loop structure will be described later.

The polynucleotide in the nucleic acid molecule of the present invention may be, for example, the polynucleotide (g) below. In this case, the nucleic acid molecule of the present invention may be, for example, a molecule composed of the polynucleotide (g) or a molecule containing the polynucleotide.
(G) consisting of a base sequence having 80% or more identity to at least one base sequence selected from the group consisting of SEQ ID NOs: 1, 8, and 10, each of formulas (II) to (VII) A polynucleotide that binds to at least one of gluten and gliadin, which is capable of forming a secondary structure represented by any one of formula (IX) and formula (XI):

The nucleic acid molecule of the present invention may include, for example, one of the polynucleotide sequences (a) to (g) or a plurality of the polynucleotide sequences. In the latter case, it is preferable that a plurality of polynucleotide sequences are linked to form a single-stranded polynucleotide. For example, the sequences of the plurality of polynucleotides may be directly linked to each other or indirectly linked via a linker. The polynucleotide sequences are preferably linked directly or indirectly at the respective ends. The sequences of the plurality of polynucleotides may be the same or different, for example, but are preferably the same. When the polynucleotide includes a plurality of sequences, the number of the sequences is not particularly limited, and is, for example, 2 or more, preferably 2 to 12, more preferably 2 to 6, and further preferably 2. is there.

The linker is, for example, a polynucleotide, and the structural unit is, for example, a nucleotide residue. Examples of the nucleotide residue include a ribonucleotide residue and a deoxyribonucleotide residue. The length of the linker is not particularly limited, and is, for example, 1 to 24 bases long, preferably 12 to 24 bases long, more preferably 16 to 24 bases long, and further preferably 20 to 24 bases long. It is long.

In the nucleic acid molecule of the present invention, the polynucleotide is preferably a single-stranded polynucleotide. The single-stranded polynucleotide is preferably capable of forming a stem structure and a loop structure by, for example, self-annealing. The polynucleotide is preferably capable of forming a stem loop structure, an internal loop structure, and / or a bulge structure, for example.

The nucleic acid molecule of the present invention may be, for example, double stranded. In the case of a double strand, for example, one single-stranded polynucleotide is any one of the polynucleotides (a) to (g), and the other single-stranded polynucleotide is not limited. Examples of the other single-stranded polynucleotide include a polynucleotide having a base sequence complementary to any one of the polynucleotides (a) to (g). When the nucleic acid molecule of the present invention is double-stranded, it is preferably dissociated into a single-stranded polynucleotide by denaturation or the like prior to use. In addition, the dissociated single-stranded polynucleotide of any one of (a) to (g) preferably has a stem structure and a loop structure as described above, for example.

In the present invention, “the stem structure and the loop structure can be formed” means, for example, that the stem structure and the loop structure are actually formed, and even if the stem structure and the loop structure are not formed, the stem structure depending on the conditions. And the ability to form a loop structure. “A stem structure and a loop structure can be formed” includes, for example, both experimental confirmation and prediction by a computer simulation.

The structural unit of the nucleic acid molecule of the present invention is, for example, a nucleotide residue. The length of the nucleic acid molecule is not particularly limited, and the lower limit thereof is, for example, 15 base length, preferably 75 base length or 80 base length, and the upper limit thereof is, for example, 1000 base length, preferably Is 200 bases, 100 bases or 90 bases long.

Examples of the nucleotide residue include deoxyribonucleotide residue and ribonucleotide residue. Examples of the nucleic acid molecule of the present invention include DNA composed only of deoxyribonucleotide residues, DNA containing one or several ribonucleotide residues, and the like. In the latter case, “1 or several” is not particularly limited, and is, for example, 1 to 3 in the polynucleotide. In the present invention, the numerical range of numbers such as the number of bases and the number of sequences, for example, discloses all positive integers belonging to the range. That is, for example, the description “1 to 3 bases” means all disclosures of “1, 2, 3 bases” (hereinafter the same).

The polynucleotide includes, for example, at least one modified base. The modified base is not particularly limited, and examples thereof include a base modified with a natural base (non-artificial base), and preferably has the same function as the natural base. The natural base is not particularly limited, and examples thereof include a purine base having a purine skeleton and a pyrimidine base having a pyrimidine skeleton. The purine base is not particularly limited, and examples thereof include adenine (a) and guanine (g). The pyrimidine base is not particularly limited, and examples thereof include cytosine (c), thymine (t), uracil (u) and the like. The base modification site is not particularly limited. When the base is a purine base, examples of the purine base modification site include the 7th and 8th positions of the purine skeleton. When the base is a pyrimidine base, examples of the modification site of the pyrimidine base include the 5th and 6th positions of the pyrimidine skeleton. In the pyrimidine skeleton, when “═O” is bonded to carbon at position 4 and a group other than “—CH ₃ ” or “—H” is bonded to carbon at position 5, it is called modified uracil or modified thymine. Can do.

The modifying group of the modifying base is not particularly limited, and examples thereof include a methyl group, a fluoro group, an amino group, a thio group, a benzylaminocarbonyl group represented by the following formula (1), and a tryptaminocarbonyl represented by the following formula (2). Group (tryptaminocarbonyl), isobutylaminocarbonyl group (isobutylaminocarbonyl) and the like.

The modified base is not particularly limited. For example, modified adenine modified with adenine, modified thymine modified with thymine, modified guanine modified with guanine, modified cytosine modified with cytosine and modified modified with uracil Examples include uracil and the like, and the modified thymine, the modified uracil and the modified cytosine are preferable.

Specific examples of the modified adenine include 7'-deazaadenine and the like.

Specific examples of the modified guanine include, for example, 7'-deazaguanine.

Specific examples of the modified cytosine include 5'-methylcytosine.

Specific examples of the modified thymine include 5'-benzylaminocarbonylthymine, 5'-tryptaminocarbonylthymine, 5'-isobutylaminocarbonylthymine and the like.

Specific examples of the modified uracil include 5'-benzylaminocarbonyluracil (BndU), 5'-tryptaminocarbonyluracil (TrpdU), 5'-isobutylaminocarbonyluracil and the like.

The polynucleotide is not particularly limited, and may include, for example, only one type of the modified base, or may include two or more types of the modified base.

The number of the modified base is not particularly limited. In the polynucleotide, the number of the modified base is, for example, one or more. In the polynucleotide, the modified base is, for example, 1 to 80, preferably 1 to 70, more preferably 1 to 50, still more preferably 1 to 40, particularly preferably 1 to 30, and most preferably. 1 to 20 and all the bases may be the modified bases. The number of the modified bases may be, for example, the number of any one of the modified bases or the total number of the two or more modified bases. In addition, the modified base in the entire length of the nucleic acid molecule containing the polynucleotide is not particularly limited, and is, for example, 1 to 80, 1 to 50, or 1 to 20, preferably in the same range as described above. It is.

In the polynucleotide, the ratio of the modified base is not particularly limited. The ratio of the modified base is, for example, 1/100 or more, preferably 1/40 or more, more preferably 1/20 or more, still more preferably 1/10 or more, particularly preferably, of the total number of bases of the polynucleotide. 1/4 or more, most preferably 1/3 or more. Further, the ratio of the modified base in the entire length of the nucleic acid molecule containing the polynucleotide is not particularly limited, and is the same as the above range. Here, the total number of bases is, for example, the total number of natural bases and modified bases in the polynucleotide. The ratio of the modified base is expressed as a fraction, and the total number of bases and the number of modified bases that satisfy this are positive integers.

When the modified base in the polynucleotide is the modified thymine, the number of the modified thymine is not particularly limited. In the polynucleotide, for example, natural thymine can be substituted for the modified thymine. In the polynucleotide, the number of the modified thymine is, for example, one or more. In the polynucleotide, the modified thymine is, for example, 1 to 80, preferably 1 to 70, more preferably 1 to 50, still more preferably 1 to 40, particularly preferably 1 to 30, and most preferably. 1 to 21 and all the thymines may be the modified thymines.

In the polynucleotide, the ratio of the modified thymine is not particularly limited. The ratio of the modified thymine is, for example, 1/100 or more, preferably 1/40 or more, more preferably 1/20 or more, further preferably 1 out of the total number of the natural thymine and the modified thymine. / 10 or more, particularly preferably 1/4 or more, and most preferably 1/3 or more.

When the modified base in the polynucleotide is the modified uracil, the number of the modified uracil is not particularly limited. In the polynucleotide, for example, natural thymine can be substituted for the modified uracil. In the polynucleotide, the number of the modified uracil is, for example, one or more. In the polynucleotide, the modified uracil is, for example, 1 to 80, preferably 1 to 70, more preferably 1 to 50, still more preferably 1 to 40, particularly preferably 1 to 30, and most preferably. 1 to 21 and all the uracils may be the modified uracils.

In the polynucleotide, the ratio of the modified uracil is not particularly limited. The ratio of the modified uracil is, for example, 1/100 or more, preferably 1/40 or more, more preferably 1/20 or more, and further preferably 1 out of the total number of the natural thymines and the number of the modified uracils. / 10 or more, particularly preferably 1/4 or more, and most preferably 1/3 or more.

Examples of the number of the modified thymine and the modified uracil may be, for example, the total number of both.

In the polynucleotide, for example, the thymine indicated by the underline in each base sequence may be at least one of the modified thymine and the modified uracil. Specifically, for example, 5'-benzylaminocarbonyluracil (BndU) may be used as the thymine indicated by the underline in any one of the nucleotide sequences of SEQ ID NOS: 1 to 7, 10 and 11. Further, for example, the underlined thymine in any one of the nucleotide sequences of SEQ ID NOs: 8 and 9 may be 5'-tryptaminocarbonyluracil (TrpdU). In the polynucleotide, thymine other than thymine indicated by the underlined portion in each base sequence is, for example, a natural base. However, the present invention is not limited to this, and thymine other than thymine shown by the underlined portion in each base sequence may be the modified base.

When the modified base in the polynucleotide is the modified cytosine, the number of the modified cytosines is not particularly limited. In the polynucleotide, for example, natural cytosine can be substituted for the modified cytosine. In the polynucleotide, the number of the modified cytosines is, for example, one or more. In the polynucleotide, the modified cytosine is, for example, 1 to 80, preferably 1 to 70, more preferably 1 to 50, still more preferably 1 to 40, particularly preferably 1 to 30, and most preferably. 1 to 21 and all cytosines may be the modified cytosine.

In the polynucleotide, the ratio of the modified cytosine is not particularly limited. The ratio of the modified cytosine is, for example, 1/100 or more, preferably 1/40 or more, more preferably 1/20 or more, further preferably 1 out of the total number of the natural cytosine and the modified cytosine. / 10 or more, particularly preferably 1/4 or more, and most preferably 1/3 or more.

In the polynucleotide, for example, the cytosine indicated by the underline in each base sequence may be the modified cytosine. Specifically, for example, the cytosine indicated by the underline in any of the nucleotide sequences of SEQ ID NOS: 1 to 11 may be 5'-methylcytosine. In the polynucleotide, a cytosine other than the cytosine indicated by the underlined portion in each base sequence is, for example, a natural base. However, the present invention is not limited to this, and cytosine other than cytosine indicated by the underlined portion in each base sequence may be the modified base.

When the modified base is the modified adenine or the modified guanine, in the description of the number and ratio of the modified cytosine, “cytosine” and “modified cytosine” are referred to as “adenine” and “modified adenine” or “guanine” and It can be read as “modified guanine”. In the polynucleotide, for example, natural adenine can be substituted with the modified adenine, and for example, natural guanine can be substituted with the modified guanine.

The nucleic acid molecule of the present invention may contain a modified nucleotide. The modified nucleotide may be a nucleotide having the modified base described above, a nucleotide having a modified sugar in which a sugar residue is modified, or a nucleotide having the modified base and the modified sugar.

The sugar residue is not particularly limited, and examples thereof include deoxyribose residue or ribose residue. The modification site in the sugar residue is not particularly limited, and examples thereof include the 2'-position and the 4'-position of the sugar residue, and both of them may be modified. Examples of the modifying group of the modified sugar include a methyl group, a fluoro group, an amino group, and a thio group.

In the modified nucleotide residue, when the base is a pyrimidine base, for example, the 2'-position and / or the 4'-position of the sugar residue is preferably modified. Specific examples of the modified nucleotide residue include, for example, a 2′-methylated-uracil nucleotide residue and a 2′-methylated-cytosine nucleotide residue in which the deoxyribose residue or the 2 ′ position of the ribose residue is modified. 2'-fluorinated-uracil nucleotide residues, 2'-fluorinated-cytosine nucleotide residues, 2'-aminated-uracil nucleotide residues, 2'-aminated-cytosine nucleotide residues, 2'-thiolated -Uracil nucleotide residue, 2'-thiolated-cytosine nucleotide residue and the like.

The number of the modified nucleotides is not particularly limited. For example, in the polynucleotide, for example, 1 to 80, preferably 1 to 70, more preferably 1 to 50, still more preferably 1 to 40, particularly The number is preferably 1 to 30, and most preferably 1 to 21. Further, the modified nucleotides in the entire length of the nucleic acid molecule including the polynucleotide are not particularly limited, and are, for example, 1 to 80, 1 to 50, and 1 to 20, preferably in the same range as described above. It is.

The nucleic acid molecule of the present invention may contain, for example, one or several artificial nucleic acid monomer residues. The “one or several” is not particularly limited, and for example, in the polynucleotide, for example, 1 to 80, preferably 1 to 70, more preferably 1 to 50, still more preferably 1 to 40 Particularly preferred is 1 to 30, most preferably 1 to 21. Examples of the artificial nucleic acid monomer residue include PNA (peptide nucleic acid), LNA (Locked Nucleic Acid), ENA (2'-O, 4'-C-Ethylenebridged Nucleic Acids) and the like. The nucleic acid in the monomer residue is the same as described above, for example. In addition, the artificial nucleic acid monomer residue in the entire length of the nucleic acid molecule containing the polynucleotide is not particularly limited, and is, for example, 1 to 80, 1 to 50, or 1 to 20, preferably the above-mentioned Similar to range.

The nucleic acid molecule of the present invention is preferably nuclease resistant, for example. The nucleic acid molecule of the present invention preferably has, for example, the modified nucleotide residue and / or the artificial nucleic acid monomer residue for nuclease resistance. Since the nucleic acid molecule of the present invention is nuclease resistant, for example, tens of kDa PEG (polyethylene glycol) or deoxythymidine may be bound to the 5 'end or 3' end.

The nucleic acid molecule of the present invention may further have an additional sequence, for example. The additional sequence is preferably bound to, for example, at least one of the 5 'end and the 3' end of the nucleic acid molecule, and more preferably the 3 'end. The additional sequence is not particularly limited. The length of the additional sequence is not particularly limited, and is, for example, 1 to 200 bases long, preferably 1 to 50 bases long, more preferably 1 to 25 bases long, and further preferably 18 to 24 bases long. It is. The structural unit of the additional sequence is, for example, a nucleotide residue, and examples thereof include a deoxyribonucleotide residue and a ribonucleotide residue. The additional sequence is not particularly limited, and examples thereof include polynucleotides such as DNA consisting of deoxyribonucleotide residues and DNA containing ribonucleotide residues. Specific examples of the additional sequence include poly dT and poly dA.

The nucleic acid molecule of the present invention can be used, for example, immobilized on a carrier. In the nucleic acid molecule of the present invention, for example, either the 5 'end or the 3' end can be immobilized. When immobilizing the nucleic acid molecule of the present invention, for example, the nucleic acid molecule may be immobilized directly or indirectly on the carrier. In the latter case, the nucleic acid molecule of the present invention is immobilized on the carrier, for example, via the additional sequence. Examples of the carrier include beads, plates, filters, columns, substrates, containers and the like.

For example, the nucleic acid molecule of the present invention may further have a labeling substance, and specifically, the labeling substance may be bound to the nucleic acid molecule. The nucleic acid molecule to which the labeling substance is bound can also be referred to as a nucleic acid sensor of the present invention, for example. The labeling substance may be bound to, for example, at least one of the 5 'end and the 3' end of the nucleic acid molecule. The labeling with the labeling substance may be, for example, binding or chemical modification. The labeling substance is not particularly limited, and examples thereof include enzymes, fluorescent substances, dyes, isotopes, drugs, toxins, and antibiotics. Examples of the enzyme include luciferase and NanoLuc luciferase. Examples of the fluorescent substance include pyrene, TAMRA, fluorescein, Cy3 dye, Cy5 dye, FAM dye, rhodamine dye, Texas red dye, JOE, MAX, HEX, TYE and the like, and the dye includes, for example, And Alexa dyes such as Alexa 488 and Alexa 647. The labeling substance may be linked directly to the nucleic acid molecule or indirectly via a linker, for example. The linker is not particularly limited and is, for example, a polynucleotide linker.

The method for producing the nucleic acid molecule of the present invention is not particularly limited, and can be synthesized by, for example, a known method such as a nucleic acid synthesis method using chemical synthesis or a genetic engineering technique.

The nucleic acid molecule of the present invention exhibits binding property to gluten as described above. For this reason, the use of the nucleic acid molecule of the present invention is not particularly limited as long as it uses the binding property to gluten. The nucleic acid molecule of the present invention can be used in various methods in place of, for example, an antibody against gluten.

According to the nucleic acid molecule of the present invention, gluten can be detected. The method for detecting gluten is not particularly limited, and can be performed by detecting the binding between gluten and the nucleic acid molecule.

Further, as described above, the nucleic acid molecule of the present invention exhibits binding property to gliadin. For this reason, the nucleic acid molecule of the present invention can be used, for example, in applications utilizing the binding property to gliadin. The nucleic acid molecule of the present invention can be used in various methods in place of, for example, an antibody against gliadin.

According to the nucleic acid molecule of the present invention, for example, gliadin can be detected. The method for detecting gliadin is not particularly limited, and can be performed by detecting the binding between gliadin and the nucleic acid molecule.

(2) Detection reagent and kit As described above, the detection reagent of the present invention is a detection reagent for gluten or gliadin, and includes the nucleic acid molecule of the present invention. The detection reagent of this invention should just contain the nucleic acid molecule of the said this invention, and another structure is not restrict | limited at all. If the detection reagent of the present invention is used, for example, detection of gluten or gliadin can be performed as described above. The detection reagent of the present invention can be said to be a binder to gluten or a binder to gliadin, for example.

The detection reagent of the present invention may further have a labeling substance, for example, and the labeling substance may be bound to the nucleic acid molecule. For the labeling substance, for example, the description in the nucleic acid molecule of the present invention can be used. The detection reagent of the present invention may have, for example, a carrier, and the nucleic acid molecule may be immobilized on the carrier. For the carrier, for example, the description in the nucleic acid molecule of the present invention can be incorporated.

The detection kit of the present invention includes the nucleic acid molecule of the present invention or the detection reagent of the present invention. The detection kit of the present invention may further include other components, for example. Examples of the constituent element include a buffer solution for preparing the sample, an instruction manual, and the like. In addition, in the case of a method using a nucleic acid sensor in which a luciferase that is a labeling substance is bound to the nucleic acid molecule and an allergen-labeled carrier shown in the detection method of the present invention described later, the detection kit of the present invention is, for example, A kit comprising a nucleic acid sensor and an allergen-labeled carrier (gluten-labeled carrier or gliadin-labeled carrier) can be provided.

For the detection reagent and detection kit of the present invention, for example, the description of the nucleic acid molecule of the present invention can be used, and the nucleic acid molecule of the present invention and the detection method of the present invention described later can also be used for the method of use. .

(3) Detection method As described above, the method for detecting gluten or gliadin of the present invention comprises contacting the nucleic acid molecule of the present invention or the detection reagent of the present invention with a sample, and then gluten or gliadin in the sample. And a step of forming a complex with the nucleic acid molecule or the detection reagent, and a step of detecting the complex. The detection method of the present invention is characterized by using the nucleic acid molecule of the present invention or the detection reagent, and the other steps and conditions are not particularly limited. Hereinafter, the use of the nucleic acid molecule of the present invention will be described as an example, but the nucleic acid molecule of the present invention can be read as the detection reagent of the present invention.

According to the present invention, since the nucleic acid molecule of the present invention specifically binds to gluten or gliadin, for example, by detecting the binding between gluten or gliadin and the nucleic acid molecule or the detection reagent, Gluten or gliadin can be specifically detected. Specifically, for example, since the amount of gluten or gliadin in a sample can be analyzed, it can be said that qualitative analysis or quantitative analysis is also possible.

In the present invention, the sample is not particularly limited. Examples of the sample include foods, food materials, food additives, and the like. Examples of the sample include a deposit in a food processing shop or a cooking place, a cleaning liquid after cleaning, and the like.

The sample may be, for example, a liquid sample or a solid sample. For example, the sample is preferably a liquid sample because it is easy to contact with the nucleic acid molecule and is easy to handle. In the case of the solid sample, for example, a mixed solution, an extract, a dissolved solution, and the like may be prepared using a solvent and used. The solvent is not particularly limited, and examples thereof include water, physiological saline, and buffer solution.

The detection method of the present invention will be described by taking as an example a method for detecting gluten using the nucleic acid sensor of the present invention labeled with a labeling substance as the nucleic acid molecule of the present invention. Since gliadin is a constituent of gluten, detection of gluten can be read as detection of gliadin. In addition, this invention is not restrict | limited to these illustrations.

The detection step further includes, for example, a step of analyzing the presence or absence or amount of gluten in the sample based on the detection result of the complex.

In the complex formation step, the method for contacting the sample and the nucleic acid molecule is not particularly limited. The contact between the sample and the nucleic acid molecule is preferably performed in a liquid, for example. The liquid is not particularly limited, and examples thereof include water, physiological saline, and buffer solution.

In the complex formation step, the contact condition between the sample and the nucleic acid molecule is not particularly limited. The contact temperature is, for example, 4 to 37 ° C., preferably 18 to 25 ° C., and the contact time is, for example, 10 to 120 minutes, preferably 30 to 60 minutes.

In the complex formation step, the nucleic acid molecule may be, for example, an immobilized nucleic acid molecule (solid phase carrier) immobilized on a carrier or an unfixed free nucleic acid molecule. In the latter case, for example, the sample is contacted in a container. In the former case, the carrier is not particularly limited, and examples thereof include a plate, a filter, a column, a substrate, a bead, and a container. Examples of the container include a microplate and a tube. The nucleic acid molecule is immobilized as described above, for example.

The detection step is a step of detecting the binding between gluten in the sample and the nucleic acid molecule as described above. By detecting the presence / absence of binding between the two, for example, the presence / absence of gluten in the sample can be analyzed (qualitative), and by detecting the degree of binding (binding amount) between the two, for example, the sample The amount of gluten can be analyzed (quantified).

The method for detecting the binding between gluten and the nucleic acid molecule is not particularly limited. As the method, for example, a conventionally known method for detecting binding between substances can be adopted, and specific examples thereof include the SPR described above.

Then, when the binding between gluten and the nucleic acid molecule cannot be detected, it can be determined that gluten is not present in the sample, and when the binding is detected, it can be determined that gluten is present in the sample. In addition, a correlation between the concentration of gluten and the binding amount can be obtained in advance, and the concentration of gluten in the sample can be analyzed from the binding amount based on the correlation.

As an example of detection of the binding between gluten and the nucleic acid molecule, a method using a nucleic acid sensor in which a luciferase as a labeling substance is bound to the nucleic acid molecule and a gluten-labeled carrier will be described below.

First, the nucleic acid sensor and the sample are mixed. Thereby, when gluten is present in the sample, the nucleic acid molecule in the nucleic acid sensor binds to gluten as a target. On the other hand, when gluten is not present in the sample, the nucleic acid molecule in the nucleic acid sensor is not bound to the target.

Next, after contacting the mixture with a gluten-labeled carrier, the gluten-labeled carrier is removed. Examples of the carrier include beads. In the mixture, when the nucleic acid sensor is bound to gluten, the nucleic acid molecule in the nucleic acid sensor cannot bind to gluten in the gluten-labeled carrier. Therefore, when a luciferase substrate is added to the fraction from which the gluten-labeled carrier has been removed to perform a luminescence reaction, luminescence is generated by the luciferase catalytic reaction in the nucleic acid sensor. On the other hand, in the mixture, when the nucleic acid sensor is not bound to gluten, the nucleic acid molecule in the nucleic acid sensor is bound to gluten in the gluten-labeled carrier. For this reason, by removing the gluten-labeled carrier, the nucleic acid sensor is also removed while bound to the gluten-labeled carrier. For this reason, when the luciferase substrate is added to the fraction from which the gluten-labeled carrier has been removed and the luminescence reaction is performed, the nucleic acid sensor does not exist, and thus luminescence due to the luciferase catalytic reaction occurs. Absent. Therefore, the presence or absence of gluten in the sample can be analyzed (qualitative analysis) based on the presence or absence of luminescence. In addition, the amount of gluten in the sample and the amount of the nucleic acid sensor remaining in the fraction after removing the gluten-labeled carrier have a correlation, so that the amount of gluten in the sample depends on the intensity of luminescence. The amount can also be analyzed (quantitative analysis).

According to the present invention, as described above, gluten or gliadin can be detected. According to the present invention, it is also possible to detect the presence or absence of wheat indirectly, for example, by detecting gluten or gliadin.

Next, examples of the present invention will be described. However, the present invention is not limited by the following examples. Commercial reagents were used based on those protocols unless otherwise indicated.

[Example 1]
About the aptamer of this invention, the binding property with respect to gluten and gliadin was confirmed by SPR analysis.

(1) Aptamer Aptamer 1 of the following polynucleotide was synthesized as an aptamer of Examples. In the polynucleotide of SEQ ID NO: 1, the underlined “T” is a deoxyribonucleotide having 5′-benzylaminocarbonyluracil (BndU) substituted at the 5-position of thymine in place of natural thymine (T) The underlined “C” is a deoxyribonucleotide residue having 5′-methylcytosine substituted at the 5-position of cytosine in place of natural cytosine (C).

Aptamer 1: Glu392BR8m4 (SEQ ID NO: 1)
GGTATGGAGGCAAGTCCCAATTCG T G C AGAGAAA C G T G TCT G T A TTT A TT AA TC G TTTC AG CC AGA C AGGG TTT A T G

The estimated secondary structure of aptamer 1 is shown in FIG. However, it is not limited to this.

The aptamer was added with polydeoxyadenine (poly dA) having a length of 20 bases at the 3 'end and used as a poly dA added aptamer in SPR described later. The poly dA-added aptamer used was heat-denatured at 95 ° C. for 5 minutes.

(2) Sample Wheat-derived gluten (073-00575, manufactured by Wako Pure Chemical Industries, Ltd.) was suspended in SB1T buffer, dissolved overnight, and then centrifuged (12,000 rpm, 15 minutes, room temperature) for separation. The separated supernatant was obtained as an extract containing native gluten. This was used as a gluten sample. In addition, using gliadin derived from wheat (cat # 101778, manufactured by MP Biomedicals), an extract containing unmodified gliadin was prepared in the same manner. The composition of the SB1T buffer was 40 mmol / L HEPES, 125 mmol / L NaCl, 5 mmol / L KCl, 1 mmol / L MgCl ₂ and 0.01% Tween (registered trademark) 20, and the pH was 7.5.

In the following binding test, each sample was prepared from the materials shown below for confirmation of the cross-reaction of the aptamer. The α-casein sample was prepared in the same manner as the gluten sample. Milk, eggs, and peanuts were crushed with a food processor, suspended in SB1T buffer, dissolved overnight, centrifuged (3000 g, 20 minutes, room temperature) and separated. The obtained extract was filtered as a sample.
Lysozyme derived from egg white of chicken egg (120-02674, manufactured by Wako Pure Chemical Industries, Ltd.)
Α-casein derived from whole egg milk of chicken eggs (C6780-19, manufactured by SIGMA)
Milk (made by Ashigara Dairy Co., Ltd.)
Raw peanuts (made by Indian curry shop Earl Tee)
Roasted peanut (KFV Fruit)

(3) Analysis of binding by SPR For analysis of binding, ProteON XPR36 (BioRad) was used according to the instruction manual.

First, as a ProteON dedicated sensor chip, a chip (trade name: ProteOn NLC Sensor Chip, BioRad) on which streptavidin was immobilized was set in the ProteON XPR36. 1 μmol / L of biotinylated poly dT was injected into the flow cell of the sensor chip using ultrapure water (DDW) and allowed to bind until the signal intensity (RU: Resonance Unit) was about 900 RU. The biotinylated poly dT was prepared by biotinylating the 5 ′ end of 20 base deoxythymidine. Then, 1 μmol / L of the poly dA-added aptamer was injected into the flow cell of the chip at a flow rate of 25 μL / min for 80 seconds using SB1T buffer, and was bound until the signal intensity reached about 800 RU. Subsequently, the samples having a predetermined concentration (500 ppm or 100 ppm) were each injected with the buffer at a flow rate of 25 μL / min for 240 seconds, and then the buffer was flowed under the same conditions for washing. After the injection of the sample, the signal intensity was measured, and the average value (RU _295-315 ) of the signal intensity (RU) at 295 to 315 seconds was determined with the injection start of the sample being 0 second. Then, a ratio (RU _295-315 / RU _immob ) between the RU value (RU _immob ) and RU _295-315 when the poly dA-added aptamer was bound to the biotinylated poly dT was calculated.

These results are shown in FIG. FIG. 2 is a graph showing the binding property of aptamer 1 to gliadin and gluten, the horizontal axis represents each sample, and the vertical axis represents signal intensity (RU). On the horizontal axis, gliadin sample, gluten sample, lysozyme sample, egg sample, α-casein sample, milk sample, raw peanut sample, and roasted peanut sample are shown in order from the left. The concentration (ppm) in each sample indicates the concentration of each protein for the gliadin sample, gluten sample, lysozyme sample, and α-casein sample, and for each egg sample, milk sample, raw peanut sample, and roast peanut sample The concentration of total protein contained in the sample is shown. As shown in FIG. 2, aptamer 1 showed binding properties to gliadin and gluten. On the other hand, aptamer 1 has a signal intensity of 0.00 or less and exhibits no binding to lysozyme sample, egg sample, α-casein sample, milk sample, raw peanut sample, and roasted peanut sample. It was.

Next, the binding analysis was performed in the same manner except that the gluten sample was used and the gluten concentration in the sample was changed to 3.7, 11, 33, 100, and 300 ppm.

This result is shown in FIG. FIG. 3 is a graph showing the binding property of aptamer 1 to gluten, the horizontal axis indicates the gluten concentration (ppm), and the vertical axis indicates the signal intensity (RU). As shown in FIG. 3, the signal intensity of the aptamer 1 increased as the concentration of gluten increased. From this result, it was found that the gluten concentration in the sample can be quantitatively analyzed by measuring the signal intensity using the aptamer of the present invention.

Next, binding analysis was performed in the same manner except that the gliadin sample was used, and the gliadin concentration in the sample was changed to 0.25, 0.5, 1, 2, and 4 μmol / L. The signal intensity at a predetermined time after the start of sample injection was determined.

This result is shown in FIG. FIG. 4 is a graph showing the binding property of aptamer 1 to gliadin, the horizontal axis indicates the elapsed time (seconds) after the start of injection of the sample, and the vertical axis indicates the signal intensity (RU). As shown in FIG. 4, the signal intensity of the aptamer 1 increased as the concentration of gliadin increased.

Further, kinetic parameters were calculated from the results of the SPR analysis in FIG. As a result, it was found that the aptamer 1 has a dissociation constant (KD) for gliadin of 5.81 × 10 ⁻⁷ mol / L and excellent binding properties.

[Example 2]
About the downsized aptamer of this invention, the binding property with respect to a gliadin was confirmed by SPR analysis.

The following polynucleotide aptamers 2 to 7 were synthesized as miniaturized aptamers of the examples. Aptamers 2 to 7 are aptamers obtained by miniaturizing the aptamer 1 (SEQ ID NO: 1) of Example 1. In the following polynucleotide, the underlined “T” is a deoxyribonucleotide residue having 5′-benzylaminocarbonyluracil (BndU) substituted at the 5-position of thymine in place of natural thymine (T), The underlined “C” was a deoxyribonucleotide residue having 5′-methylcytosine substituted at the 5-position of cytosine in place of natural cytosine (C).

Aptamer 2: Glu392BR8m4_s59A (SEQ ID NO: 2)
GGTATGGAGGCAAGTCCCAATTCG T G C AGAGAAA C G T G TCT G T A TTT A TT AA TC G TTTC
Aptamer 3: Glu392BR8m4_s51 (SEQ ID NO: 3)
GGCAAGTCCCAATTCG T G C AGAGAAA C G T G TCT G T A TTT A TT AA TC G TTTC
Aptamer 4: Glu392BR8m4_s38 (SEQ ID NO: 4)
TC G T G C AGAGAAA C G T G TCT G T A TTT A TT AA T CG TTTC
Aptamer 5: Glu392BR8m4_s67 (SEQ ID NO: 5)
GGTATGGAGGCAAGTCCCAATTCG T G C AGAGAAA C G T G TCT G T A TTT A TT AA TC G TTTC AG CC AGA C
Aptamer 6: Glu392BR8m4_s59B (SEQ ID NO: 6)
GGCAAGTCCCAATTCG T G C AGAGAAA C G T G TCT G T A TTT A TT AA TC G TTTC AG CC AGA C
Aptamer 7: Glu392BR8m4_s46 (SEQ ID NO: 7)
TCGTGCAGAGAAACG T G TCT G T A TTT A TT AA TC G TTTC AG CC AGA C

The estimated secondary structure of aptamers 2 to 7 is shown in FIG. However, it is not limited to this.

The aptamer 1 of Example 1 and the miniaturized aptamers 2 to 7 were used as aptamers, and the gliadin sample (4 μmol / L) and lysozyme sample (25 nmol / L or 100 nmol) prepared in Example 1 were used as samples. / L), and α casein sample (100 nmol / L or 400 nmol / L) were used in the same manner as in Example 1 to analyze binding by SPR.

This result is shown in FIG. FIG. 6 is a graph showing the binding of each aptamer to gliadin (4 μmol / L), lysozyme (25 nmol / L or 100 nmol / L), and α-casein (100 nmol / L or 400 nmol / L). Each sample is shown, and the vertical axis shows the signal intensity (RU). Each graph shows the downsized aptamer 2, the downsized aptamer 3, the downsized aptamer 4, the downsized aptamer 5, the downsized aptamer 6, the downsized aptamer 7, and the aptamer 1 in order from the left.

As shown in FIG. 6, all of the miniaturized aptamers 2 to 7 in which the aptamer 1 was miniaturized showed higher binding to gliadin than the aptamer 1. Aptamers 1 to 7 showed no signal binding to lysozyme and α-casein, and the signal intensity was 0.00 or less. From these results, it was found that the miniaturized aptamers 2 to 7 bind to gliadin with excellent specificity.

[Example 3]
A nucleic acid sensor in which a labeling substance luciferase was bound to the aptamer of the present invention was prepared, and the binding property of the nucleic acid sensor to gliadin and gluten was confirmed. The confirmation of the binding was performed using the target solid-phased beads on which the target gliadin or gluten was solid-phased and the nucleic acid sensor.

As described above, in the reaction solution, when the nucleic acid sensor is bound to the target, the nucleic acid molecule in the nucleic acid sensor cannot bind to gliadin or gluten immobilized on the target immobilized beads. . For this reason, when a luminescence reaction is performed on the fraction from which the target solid-phased beads have been removed, luminescence is generated by the catalytic reaction of luciferase in the nucleic acid sensor. On the other hand, in the reaction solution, when the nucleic acid sensor is not bound to the target, the nucleic acid molecule in the nucleic acid sensor is bound to gliadin or gluten immobilized on the target immobilized beads. For this reason, by removing the target-immobilized beads, the nucleic acid sensor is also removed while bound to the target-immobilized beads. For this reason, when the luciferase substrate is added to the fraction from which the target solid-phased beads have been removed and the luminescence reaction is performed, the luminescence due to the luciferase catalytic reaction is not caused by the absence of the nucleic acid sensor. Does not occur. Therefore, gliadin or gluten can be detected by detecting luminescence by luciferase using the nucleic acid sensor and the target solid-phased beads.

The nucleic acid sensor is prepared by using the fluorescent substance NanoLuc ™ luciferase (manufactured by Promega) and labeling the 5 ′ end of the aptamer 1 (Glu392BR8m4) of Example 1 according to the instruction manual. did.

As the sample, the gliadin sample or the gluten sample of Example 1 was used.

The target solid-phased beads were prepared using NHS-activated Sepharose 4 Fast Flow Lab Packs (manufactured by GE Healthcare) using the gliadin sample or the gluten sample as a target.

Then, using the nucleic acid sensor and the target-immobilized beads, the binding property of the nucleic acid sensor to gliadin and gluten was confirmed as follows. First, a filter plate (manufactured by millipore, cat # MSGVN2250) was set on a 96-well U-bottom plate, and the target solid-phased beads were added to each well of the U-bottom plate so as to be 50 μL / well. . In each well, 90 μL of the gliadin sample or the gluten sample (final concentrations 0, 0.4, 1.235, 3.7, 11.1, 33.3, 100 ppm) and 10 μL of the nucleic acid sensor (2 5 × 10 ⁶ dilution), and mixing at 1200 rpm for 5 minutes at room temperature to react the gliadin sample or the gluten sample, the target immobilized beads, and the nucleic acid sensor. Thereafter, the U bottom plate was centrifuged at 3000 g for 2 minutes at room temperature, and the target solid-phased beads were removed by centrifugation. By the centrifugation, the reaction solution that passed through the filter plate was collected from each well and subjected to measurement of the amount of luminescence. For the measurement of the light emission amount, Infinite M1000 Pro (TECAN) was used according to the instruction manual. In the measurement of the luminescence amount, NanoGlo (trademark, manufactured by Promega, cat # N2012) was used as a substrate. In addition, in order to confirm the cross-reaction, the same measurement was performed using the above-described egg, milk, and raw peanut instead of the gliadin sample and the gluten sample.

Fig. 7 shows the measurement results of light emission. FIG. 7 is a graph showing the measurement results of the amount of luminescence when gliadin, gluten, eggs, milk, and raw peanuts are used as samples. In FIG. 7, the horizontal axis indicates the concentration of each sample, and the vertical axis indicates the light emission amount (RLU). As shown in FIG. 7, in the case of the gliadin sample and the gluten sample, the amount of luminescence increased as the protein concentration of the gliadin sample and the gluten sample in which luminescence was observed due to the catalytic reaction of luciferase. On the other hand, in the case of the egg sample, the milk sample, and the raw peanut sample, the luminescence amount did not change.

Furthermore, from the measurement results of FIG. 7, the detection limit for gliadin and gluten of the nucleic acid sensor was calculated by the 3σ method (n = 3). As a result, the detection limit (LOD) for gliadin and gluten of the nucleic acid sensor was 1.2 ppm, respectively. From this, it was found that the nucleic acid sensor can detect a small amount of gliadin and gluten.

[Example 4]
Regarding the aptamer of the present invention, the binding property to gliadin was confirmed by SPR analysis.

The following polynucleotide aptamers 8 and 9 were synthesized in the same manner as in Example 1. Aptamer 9 is a miniaturized sequence of aptamer 8. In the polynucleotides of SEQ ID NOs: 8 and 9, the underlined “T” has 5′-tryptaminocarbonyluracil (TrpdU) substituted at position 5 of thymine instead of natural thymine (T). A deoxyribonucleotide residue having a 5′-methylcytosine substituted at the 5-position of cytosine in place of natural cytosine (C) was designated as a deoxyribonucleotide residue.

Aptamer 8: Gli95_395TR8m1 (SEQ ID NO: 8)
GGAAACGCCGCCTAGATCATTTG C G TCCTCCCT GG T GGGGA TT GG C GAAAA TT GA C A TCCTC AAG TTCCT G C GAAA T G
Aptamer 9: Gli95_395TR8m1s50 (SEQ ID NO: 9)
CGCCTAGATCATTTG C G TCCTCCCT GG T GGGGA TT GG C GAAAA TT GA C A T

The presumed secondary structure of aptamers 8 and 9 is shown in FIG. However, it is not limited to this.

As in Example 1 except that aptamers 8 and 9 were used, the gliadin sample was used as a sample, and the gliadin concentration in the sample was 50, 100, 200, and 400 nmol / L. The signal intensity at a predetermined time after the start of injection of the sample was determined.

The result is shown in FIG. FIGS. 9A and 9B are graphs showing the binding properties of aptamers 8 and 9 to gliadin, respectively. The horizontal axis represents the elapsed time (seconds) after the start of injection of the sample, and the vertical axis represents Shows the signal intensity (RU). As shown in FIGS. 9A and 9B, aptamer 8 and aptamer 9, which is a miniaturized sequence thereof, increased in signal intensity as the concentration of gliadin increased.

Furthermore, kinetic parameters were calculated from the results of the SPR analysis in FIG. As a result, the aptamers 8 and 9 have dissociation constants (KD) with respect to gliadin of 19.4 × 10 ⁻⁹ mol / L and 22.7 × 10 ⁻⁹ mol / L, respectively. I found out.

Next, aptamers 10 and 11 of the following polynucleotides were synthesized in the same manner as in Example 1. Aptamer 11 is a miniaturized sequence of aptamer 10. In the polynucleotides of SEQ ID NOS: 10 and 11 below, “T” indicated by underlining has 5′-benzylaminocarbonyluracil (BndU) substituted at the 5-position of thymine instead of natural thymine (T). A deoxyribonucleotide residue having a 5′-methylcytosine substituted at the 5-position of cytosine in place of natural cytosine (C) was designated as a deoxyribonucleotide residue.

Aptamer 10: Gli_395BR8m2 (SEQ ID NO: 10)
GGAAACGCCGCCTAGATCATTTGAAAA C G TT G TT A CCTC A CCT A TT A TCT A TT GA C A TCCTC AAG TTCCT G C GAAA T G
Aptamer 11: Gli_395BR8m2s51 (SEQ ID NO: 11)
GGAAACGCCGCCTAGATCATTTGAAAA C G TT G TT A CCTC A CCT A TT A TCT A

The estimated secondary structure of the aptamers 10 and 11 is shown in FIG. However, it is not limited to this.

The binding analysis was performed in the same manner except that aptamers 10 and 11 were used, the gliadin sample was used as a sample, and the gliadin concentration in the sample was 100, 200, 400, and 800 nmol / L. The signal intensity at a predetermined time after the start of injection of the sample was determined.

The result is shown in FIG. FIGS. 10A and 10B are graphs showing the binding properties of aptamers 10 and 11 to gliadin, respectively. The horizontal axis represents the elapsed time (seconds) after the start of injection of the sample, and the vertical axis represents Shows the signal intensity (RU). As shown in FIGS. 10A and 10B, the aptamer 10 and the aptamer 11, which is a miniaturized sequence thereof, increased in signal intensity as the concentration of gliadin increased.

Furthermore, kinetic parameters were calculated from the results of the SPR analysis in FIG. As a result, the aptamers 10 and 11 have excellent dissociation properties with dissociation constants (KD) for gliadin of 132 × 10 ⁻⁹ mol / L and 102 × 10 ⁻⁹ mol / L, respectively. It was.

[Example 5]
About the aptamer of this invention, the binding property with respect to the heated gliadin was confirmed by SPR analysis.

The binding analysis was performed in the same manner as in Example 4 except that a heated gliadin sample was used as a sample, and the signal intensity at a predetermined time after the start of injection of the sample was obtained. The heated gliadin sample was prepared by heat-treating the gliadin sample at 95 ° C. for 10 minutes, centrifuging at 12000 × g for 10 minutes, and then collecting the supernatant.

The results are shown in FIGS. FIGS. 11A and 11B are graphs showing the binding properties of aptamers 8 and 9 to heated gliadin, the horizontal axis indicates the elapsed time (seconds) after the start of injection of the sample, and the vertical axis is Signal intensity (RU) is shown. As shown in FIGS. 11A and 11B, aptamer 8 and aptamer 9, which is a miniaturized sequence thereof, increased in signal intensity as the concentration of heated gliadin increased.

12 (A) and (B) are graphs showing the binding properties of aptamers 10 and 11 to heated gliadin, the horizontal axis indicates the elapsed time (seconds) after the start of injection of the sample, and the vertical axis is Signal intensity (RU) is shown. As shown in FIGS. 12A and 12B, the aptamer 10 and the aptamer 11, which is a miniaturized sequence thereof, increased in signal intensity as the concentration of heated gliadin increased.

Furthermore, kinetic parameters were calculated from the results of SPR analysis in FIGS. As a result, aptamers 8 to 11 have dissociation constants (KD) with respect to heated gliadin of 19.7 × 10 ⁻⁹ mol / L, 21.7 × 10 ⁻⁹ mol / L, and 134 × 10 ⁻⁹ mol / L, respectively. L, and 99.5 × 10 ⁻⁹ mol / L, which was found to be excellent binding properties.

[Example 6]
Regarding the aptamer of the present invention, the binding property to gluten and heated gluten was confirmed by SPR analysis.

Bondability analysis was performed in the same manner as in Example 1 except that aptamers 8 to 11 were used as aptamers, and 100 ppm of the gluten sample and the heated gluten sample were used as samples. The heated gluten sample was prepared by heating the gluten sample at 95 ° C. for 10 minutes, centrifuging at 12,000 × g for 10 minutes, and then collecting the supernatant. In addition, in order to confirm the cross-reaction of the aptamer, binding analysis was performed in the same manner except that the egg sample, the milk sample, and the raw peanut sample were used.

The result is shown in FIG. FIG. 13 is a graph showing the binding properties of aptamers 8 to 11 to gluten and heated gluten. The horizontal axis represents the type of aptamer, and the vertical axis represents signal intensity (RU). In the horizontal axis, aptamer 8, aptamer 10, aptamer 9, and aptamer 11 are shown in order from the left. Each graph shows a gluten sample, a heated gluten sample, an egg sample, a milk sample, and a raw peanut sample in order from the left. As shown in FIG. 13, aptamers 8 to 11 showed binding properties to gluten and heated gluten. On the other hand, the aptamers 8 to 11 had a signal intensity of 0.05 or less and showed no binding to egg samples, milk samples, and raw peanut samples.

From the above results, the aptamer of the present invention specifically binds to gluten and heated gluten and can be detected by measurement, and according to the aptamer of the present invention, gluten in the sample and heated It was found that the amount of gluten could be analyzed.

As mentioned above, although this invention was demonstrated with reference to embodiment and an Example, this invention is not limited to the said embodiment and Example. Various changes that can be understood by those skilled in the art can be made to the configuration and details of the present invention within the scope of the present invention.

This application claims priority based on Japanese Patent Application No. 2016-227865 filed on November 24, 2016, the entire disclosure of which is incorporated herein.

The nucleic acid molecule of the present invention can bind to gluten or gliadin. Therefore, according to the nucleic acid molecule of the present invention, gluten and gliadin can be detected based on the presence or absence of binding to the allergen in the sample. Therefore, the nucleic acid molecule of the present invention can be said to be an extremely useful tool for detecting allergens derived from grains such as wheat, for example, in the fields of food production, food management, food distribution, and the like.

Claims (17)

1. A nucleic acid molecule that binds to at least one of gluten and gliadin, comprising the polynucleotide of any one of (a) and (b) below:
   (A) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1, 8, or 10 or a partial sequence of the nucleotide sequence of SEQ ID NO: 1, 8, or 10 (b) 90% or more relative to the nucleotide sequence of (a) A polynucleotide Glu392BR8m4 (SEQ ID NO: 1) that binds to at least one of gluten and gliadin.
   GGTATGGAGGCAAGTCCCAATTCG T G C AGAGAAA C G T G TCT G T A TTT A TT AA TC G TTTC AG CC AGA C AGGG TTT A T G
   Gli95_395TR8m1 (SEQ ID NO: 8)
   GGAAACGCCGCCTAGATCATTTG C G TCCTCCCT GG T GGGGA TT GG C GAAAA TT GA C A TCCTC AAG TTCCT G C GAAA T G
   Gli — 395BR8m2 (SEQ ID NO: 10)
   GGAAACGCCGCCTAGATCATTTGAAAA C G TT G TT A CCTC A CCT A TT A TCT A TT GA C A TCCTC AAG TTCCT G C GAAA T G
2. The partial sequence of the base sequence of SEQ ID NO: 1, 8, or 10 is each at least one base sequence selected from the group consisting of SEQ ID NOs: 2 to 7, the base sequence of SEQ ID NO: 9, or the base of SEQ ID NO: 11 2. The nucleic acid molecule of claim 1 which is a sequence.
   Glu392BR8m4_s59A (SEQ ID NO: 2)
   GGTATGGAGGCAAGTCCCAATTCG T G C AGAGAAA C G T G TCT G T A TTT A TT AA TC G TTTC
   Glu392BR8m4_s51 (SEQ ID NO: 3)
   GGCAAGTCCCAATTCG T G C AGAGAAA C G T G TCT G T A TTT A TT AA TC G TTTC
   Glu392BR8m4_s38 (SEQ ID NO: 4)
   TC G T G C AGAGAAA C G T G TCT G T A TTT A TT AA TC G TTTC
   Glu392BR8m4_s67 (SEQ ID NO: 5)
   GGTATGGAGGCAAGTCCCAATTCG T G C AGAGAAA C G T G TCT G T A TTT A TT AA TC G TTTC AG CC AGA C
   Glu392BR8m4_s59B (SEQ ID NO: 6)
   GGCAAGTCCCAATTCG T G C AGAGAAA C G T G TCT G T A TTT A TT AA TC G TTTC AG CC AGA C
   Glu392BR8m4_s46 (SEQ ID NO: 7)
   TCGTGCAGAGAAACG T G TCT G T A TTT A TT AA TC G TTTC AG CC AGA C
   Gli95_395TR8m1s50 (SEQ ID NO: 9)
   CGCCTAGATCATTTG C G TCCTCCCT GG T GGGGA TT GG C GAAAA TT GA C A T
   Gli — 395BR8m2s51 (SEQ ID NO: 11)
   GGAAACGCCGCCTAGATCATTTGAAAA C G TT G TT A CCTC A CCT A TT A TCT A
3. The nucleic acid molecule according to claim 1 or 2, wherein in the polynucleotide, at least one thymine is a modified base, and the modified base of the thymine is at least one of a modified thymine and a modified uracil.
4. 4. The polynucleotide according to claim 1, wherein, in the polynucleotide, at least one-twentieth of the total number of bases of thymine is a modified base, and the modified base of thymine is at least one of modified thymine and modified uracil. The nucleic acid molecule according to claim 1.
5. In the said polynucleotide, the thymine shown by the underline part in each said base sequence is a modified base, The modified base of the said thymine is at least one of a modified thymine and a modified uracil. The nucleic acid molecule according to Item.
6. The nucleic acid molecule according to any one of claims 1 to 5, wherein in the polynucleotide, at least one cytosine is a modified base.
7. The nucleic acid molecule according to any one of claims 1 to 6, wherein in the polynucleotide, at least one-twentieth of the total number of bases of cytosine is a modified base.
8. The nucleic acid molecule according to any one of claims 1 to 7, wherein in the polynucleotide, a cytosine indicated by an underline in each base sequence is a modified base.
9. The nucleic acid molecule according to any one of claims 1 to 8, wherein the polynucleotide is DNA.
10. The nucleic acid molecule according to any one of claims 1 to 9, which comprises the following polynucleotide (e):
    (E) consisting of a base sequence having 80% or more identity to the base sequence of (a), each of the base sequence of SEQ ID NO: 2 to 7, the base sequence of SEQ ID NO: 9, or the sequence A polynucleotide comprising the nucleotide sequence of No. 11 and binding to at least one of gluten and gliadin
11. The nucleic acid molecule according to any one of claims 1 to 9, comprising the following polynucleotide (f):
    (F) a base sequence having at least 80% identity to at least one base sequence selected from the group consisting of SEQ ID NOs: 1 to 11, and represented by formulas (I) to (XI), respectively. A polynucleotide that binds to at least one of gluten and gliadin
    

    

    
    

    

    
    

    

    
    

    

    
    

    

    
    

    

    
    

    

    
    

    

    
    

    

    
    

    

    
    

    
12. A detection reagent for gluten or gliadin, comprising the nucleic acid molecule according to any one of claims 1 to 11.
13. Furthermore, it has a labeling substance,
    The detection reagent according to claim 12, wherein the labeling substance is bound to the nucleic acid molecule.
14. The detection reagent according to claim 13, wherein the labeling substance is an enzyme.
15. The detection reagent according to claim 14, wherein the enzyme is luciferase.
16. A nucleic acid molecule according to any one of claims 1 to 11 or a detection reagent according to any one of claims 12 to 15 and a sample are brought into contact, and gluten or gliadin in the sample, Forming a complex with a nucleic acid molecule or the detection reagent, and
    A method for detecting gluten or gliadin, comprising the step of detecting the complex.
17. The detection method according to claim 16, wherein the detection is qualitative analysis or quantitative analysis.
    

Images