WO2018092915A1 - Nucleic acid molecule, and use thereof - Google Patents

Abstract

Provided is a novel nucleic acid molecule which binds to α-casein. This nucleic acid molecule, which binds to milk-derived α-casein, includes any polynucleotide set forth in (a) or (b) below: (a) a polynucleotide which comprises the base sequence of SEQ ID NO: 1, 2, or 4, or a partial sequence of the base sequence of SEQ ID NO: 1, 2, or 4; or (b) a polynucleotide which comprises a base sequence having at least 90% identity with any of the base sequences in (a), and which binds to milk-derived α-casein.

Description

Nucleic acid molecules and uses thereof

The present invention relates to nucleic acid molecules that bind to milk-derived α-casein and uses thereof.

Milk is a food that is frequently consumed on a daily basis, but in recent years, the number of patients with milk allergies has increased and has been regarded as a problem. Many processed foods such as cheese, butter, and yogurt use milk, so it is extremely difficult to analyze whether processed milk or its production line contains milk as a raw material. is important.

Allergic allergens are generally proteins and their degradation products (peptides), and analysis methods using antibodies using these as antigens are the mainstream. Regarding milk, for example, α-casein, which is a milk protein, is known as an allergen. As an analysis method for α-casein, a method using an ELISA method has been reported (Non-patent Document 1).

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 milk-derived α-casein.

The nucleic acid molecule of the present invention is a nucleic acid molecule that binds to milk-derived α-casein, which comprises any of the following polynucleotides (a) or (b).
(A) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1, 2 or 4 or a partial sequence of the nucleotide sequence of SEQ ID NO: 1, 2 or 4 (b) 90% or more identical to the nucleotide sequence of (a) A polynucleotide comprising a base sequence having sex and binding to milk-derived α-casein

The milk-derived α-casein detection reagent of the present invention includes the nucleic acid molecule of the present invention.

In the method for detecting milk-derived α-casein 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 the milk-derived α-casein in the sample is mixed with the nucleic acid molecule or the detection. Forming a complex with the reagent, and
A step of detecting the complex.

The nucleic acid molecule of the present invention can bind to milk-derived α-casein. For this reason, according to the nucleic acid molecule of the present invention, milk-derived α-casein 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 milk, 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 milk-derived α-casein in Example 1 of the present invention. FIG. 3 is a graph showing aptamer 1 binding to a milk sample in Example 1 of the present invention. FIG. 4 is a graph showing aptamer 1 binding to α-casein in Example 1 of the present invention. FIG. 5 is a graph showing the measurement result of the light emission amount in Example 2 of the present invention. FIG. 6 is a graph showing the measurement result of the light emission amount in Example 2 of the present invention. FIG. 7 is a graph showing aptamer 1 binding to a heated milk sample in Example 3 of the present invention. FIG. 8 shows the presumed secondary structures of aptamers 2 to 5 in Example 4 of the present invention. FIG. 9 is a graph showing the binding properties of aptamers 2 and 3 to α-casein in Example 4 of the present invention. FIG. 10 is a graph showing the binding properties of aptamers 4 and 5 to α-casein in Example 4 of the present invention. FIG. 11 is a graph showing the binding properties of aptamers 2 and 3 to heated α-casein in Example 5 of the present invention. FIG. 12 is a graph showing the binding properties of aptamers 4 and 5 to heated α-casein in Example 5 of the present invention. FIG. 13 is a graph showing the binding properties of aptamers 2 to 5 to milk samples in Example 6 of the present invention. FIG. 14 is a putative secondary structure of aptamers 6 and 7 in Example 7 of the present invention. FIG. 15 is a graph showing the binding properties of aptamers 6 and 7 to α-casein in Example 7 of the present invention. FIG. 16 is a putative secondary structure of aptamers 8 and 9 in Example 8 of the present invention. FIG. 17 is a graph showing the binding properties of aptamers 8 and 9 to α-casein in Example 8 of the present invention.

(1) Nucleic acid molecule As described above, the nucleic acid molecule of the present invention is a nucleic acid molecule that binds to milk-derived α-casein, 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, 2 or 4 or a partial sequence of the nucleotide sequence of SEQ ID NO: 1, 2 or 4 (b) 90% or more identical to the nucleotide sequence of (a) A polynucleotide comprising a base sequence having sex and binding to milk-derived α-casein

In the present invention, the target is α-casein derived from milk. The origin of the milk is, for example, cows and goats. Milk-derived α-casein is, for example, milk-derived α-casein. Regarding the nucleic acid of the present invention, for example, commercially available α-casein can be used as α-casein for confirming the binding ability, and specific examples include α-casein derived from milk (C6780-19, manufactured by SIGMA). Alpha casein is, for example, a native allergen. In the present invention, hereinafter, milk-derived α-casein is also simply referred to as α-casein.

As described above, the nucleic acid molecule of the present invention can bind to α-casein. In the present invention, “binding to α-casein” means, for example, having binding property to α-casein or having binding activity to α-casein. The binding between the nucleic acid molecule of the present invention and α-casein 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 α-casein, it can be used, for example, for detection of α-casein.

The nucleic acid molecule of the present invention has a dissociation constant indicating the binding force to α-casein, for example, 20 nmol / L or less, 17 nmol / L or less, 13 nmol / L or less, 9 nmol / L or less, 7 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 of (a) may be, for example, a polynucleotide containing the base sequence of SEQ ID NO: 1, 2, or 4, may be a polynucleotide consisting of the base sequence of SEQ ID NO: 1, 2, or 4, The polynucleotide may include a partial sequence of the base sequence of SEQ ID NO: 1, 2, or 4 or may be a polynucleotide comprising the partial sequence. The partial sequence is not particularly limited, and may be, for example, a sequence in which at least one of the 5 'end and 3' end is deleted from the original sequence, or a sequence in which an intermediate region sequence is deleted. The polynucleotide of SEQ ID NO: 1, 2 or 4 is shown below.

aCas392BR8m2 (SEQ ID NO: 1)
GGTATGGAGGCAAGTCCCAATTC T AAGAAG T GGAG T AGG T GGG TTT AAGGA T A C G TTTC AG CC AGA C AGGG TTT A T G
aCas757BR8m3 (SEQ ID NO: 2)
GGATAGCAGCAGGGACCTCTTATACG TC GG T G CT GG T G TT G T A T AGA CCCCCTT A T A TT A T AA CC GAA T GA TTT G CCC G CT A C GA T A T G
aCas757BR8m4 (SEQ ID NO: 4)
GGATAGCAGCAGGGACCTCTTATAC CT GAG C GG CTC A TT A CCCTTCC GA CT GG TC G CCC G CTT A CC GAA T GA TTT G CCC G CT A C GA T A T G

The partial sequence of SEQ ID NO: 2 is not particularly limited, and examples thereof include the base sequences of SEQ ID NOs: 3, 6, 8, and 9. The base sequence of SEQ ID NO: 6 is a sequence obtained by further miniaturizing the base sequence of SEQ ID NO: 3.
aCas757BR8m3s69 (SEQ ID NO: 3)
GGATAGCAGCAGGGACCTCTTATACG TC GG T G CT GG T G TT G T A T AGA CCCCCTT A T A TT A T AA CC GAA T
aCas757BR8m3s63 (SEQ ID NO: 6)
GGATAGCAGCAGGGACCTCTTATACG TC GG T G CT GG T G TT G T A T AGA CCCCCTT A T A TT A T AA
aCas757BR8m3s63b (SEQ ID NO: 8)
GGATAGACCTCTTATACG TCT G TT G T A T AGA CCCCCTT A T A TT A T AA
aCas757BR8m3s63c (SEQ ID NO: 9)
GGATAGCAGCACTCTTATAC T G CT GG T G TT G T A T AGA CCCCCTT A T A TT A T AA

The partial sequence of SEQ ID NO: 4 is not particularly limited, and examples thereof include the nucleotide sequences of SEQ ID NOs: 5 and 7.
aCas757BR8m4s62 (SEQ ID NO: 5)
GGATAGCAGCAGGGACCTCTTATAC CT GAG C GG CTC A TT A CCCTTCC GA CT GG TC G CCC G CT
aCas757BR8m4s44 (SEQ ID NO: 7)
TTATAC CT GAG C GG CTC A TT A CCCTTCC GA CT GG TC G CCC G CTC

In (b), the “identity” is not particularly limited, and may be any range as long as the polynucleotide (b) binds to α-casein. 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 milk-derived α-casein

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 which binds to milk-derived α-casein

In the above (d), “one or several” may be in the range where the polynucleotide in (d) is bound to milk-derived α-casein, for example. 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 SEQ ID NO: 2 or 4, wherein either one of SEQ ID NOs: 3 and 6, or any one of 5 and 7, respectively A polynucleotide comprising a base sequence and binding to α-casein derived from milk

In (e), “identity” is not particularly limited, and is the same as (b), for example.

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 80% or more identity to at least one base sequence selected from the group consisting of SEQ ID NOs: 1 to 9, and represented by formulas (I) to (IX), respectively. A polynucleotide that binds to milk-derived α-casein

In (f), “identity” is not particularly limited, and is the same as (b), for example. In (f) above, “can form a secondary structure” means, for example, that the polynucleotide of (f) can form a stem structure and a loop structure in the above formula. 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: 2 and 4, respectively represented by formulas (III) and (VI), and formula A polynucleotide capable of forming a secondary structure represented by (V) and formula (VII) and binding to milk-derived α-casein

The polynucleotide in the nucleic acid molecule of the present invention may be, for example, the polynucleotide (h) below. In this case, the nucleic acid molecule of the present invention may be, for example, a molecule composed of the polynucleotide (h) or a molecule containing the polynucleotide.
(H) Binds to milk-derived α-casein comprising a base sequence having 80% or more identity to the base sequence of SEQ ID NO: 2, 3, or 4 and comprising the base sequence of SEQ ID NO: 6 or 7, respectively. Polynucleotide

The polynucleotide in the nucleic acid molecule of the present invention may be, for example, the following polynucleotide (i). In this case, the nucleic acid molecule of the present invention may be, for example, a molecule comprising the polynucleotide (i) or a molecule containing the polynucleotide.
(I) It consists of a base sequence having 80% or more identity to the base sequence of SEQ ID NO: 2, 3, or 4, and has a secondary structure represented by formula (VI) or formula (VII), respectively. Polynucleotide that binds to milk-derived α-casein that can be formed

The nucleic acid molecule of the present invention may contain, for example, one of the polynucleotide sequences (a) to (i) 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 (i), 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 (i). 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. Further, the dissociated single-stranded polynucleotide of any one of (a) to (i) 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 of the nucleotide sequences of SEQ ID NOS: 1 to 9.

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 one of the nucleotide sequences of SEQ ID NOS: 1 to 9 may be 5'-methylcytosine.

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.

As described above, the nucleic acid molecule of the present invention exhibits binding to α-casein. 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 to α-casein. The nucleic acid molecule of the present invention can be used in various methods in place of, for example, an antibody against α-casein.

According to the nucleic acid molecule of the present invention, α-casein can be detected. The method for detecting α-casein is not particularly limited, and can be performed by detecting the binding between α-casein 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 milk-derived α-casein, 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. When the detection reagent of the present invention is used, for example, milk-derived α-casein can be detected as described above. The detection reagent of the present invention can be said to be a binder to milk-derived α-casein, 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 includes A kit containing a nucleic acid sensor and an allergen-labeled carrier (α-casein-labeled carrier) can be obtained.

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 milk-derived α-casein of the present invention is a method in which the nucleic acid molecule of the present invention or the detection reagent of the present invention is brought into contact with the sample, and the milk in the sample is derived. The method includes a step of forming a complex of α-casein 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 α-casein, for example, by detecting the binding between α-casein and the nucleic acid molecule or the detection reagent, α-casein can be specifically detected. Specifically, for example, since the amount of α-casein 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 with reference to an example of a method for detecting α-casein using the nucleic acid sensor of the present invention labeled with a labeling substance as the nucleic acid molecule of the present invention. 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 amount of α-casein 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 α-casein in the sample and the nucleic acid molecule as described above. By detecting the presence or absence of binding between the two, for example, the presence or absence of α-casein in the sample can be analyzed (qualitative), and by detecting the degree of binding (binding amount) between the two, for example, The amount of α-casein in the sample can be analyzed (quantified).

The method for detecting the binding between α-casein 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 α-casein and the nucleic acid molecule cannot be detected, it can be determined that α-casein is not present in the sample, and when the binding is detected, α-casein is present in the sample. I can judge. In addition, the correlation between the concentration of α-casein and the binding amount can be obtained in advance, and the concentration of α-casein in the sample can be analyzed from the binding amount based on the correlation.

As an example of detection of the binding between α-casein 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 milk-derived α-casein labeling carrier will be described below.

First, the nucleic acid sensor and the sample are mixed. As a result, when milk-derived α-casein is present in the sample, the nucleic acid molecule in the nucleic acid sensor binds to the target milk-derived α-casein. On the other hand, when milk-derived α-casein is not present in the sample, the nucleic acid molecules in the nucleic acid sensor are not bound to the target.

Next, the mixture is brought into contact with the milk-derived α-casein labeled carrier, and then the α-casein labeled carrier is removed. Examples of the carrier include beads. In the mixture, when the nucleic acid sensor is bound to milk-derived α-casein, the nucleic acid molecule in the nucleic acid sensor cannot bind to milk-derived α-casein in the α-casein labeled carrier. For this reason, when a luciferase substrate is added to the fraction from which the α-casein labeled carrier has been removed to perform a luminescence reaction, luminescence is generated by the catalytic reaction of luciferase in the nucleic acid sensor. On the other hand, in the mixture, when the nucleic acid sensor is not bound to milk-derived α-casein, the nucleic acid molecule in the nucleic acid sensor binds to milk-derived α-casein in the α-casein labeled carrier. For this reason, by removing the α-casein labeled carrier, the nucleic acid sensor is also removed while bound to the α-casein labeled carrier. For this reason, when the luciferase substrate is added to the fraction from which the α-casein labeled carrier has been removed and the luminescence reaction is performed, the luminescence due to the luciferase catalytic reaction does not occur because the nucleic acid sensor does not exist. Does not occur. For this reason, the presence or absence of milk-derived α-casein in the sample can be analyzed (qualitative analysis) based on the presence or absence of luminescence. In addition, since the amount of milk-derived α-casein in the sample and the amount of the nucleic acid sensor remaining in the fraction after removing the α-casein-labeled carrier have a correlation, depending on the intensity of light emission, The amount of α-casein derived from milk can also be analyzed (quantitative analysis).

According to the present invention, as described above, milk-derived α-casein, which is an allergen, can be detected. Further, according to the present invention, the presence or absence of milk can also be detected indirectly, for example, by detecting milk-derived α-casein, which is the allergen.

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 milk origin (alpha) casein was confirmed by SPR analysis.

(1) Aptamer Aptamer 1 of the following polynucleotide was synthesized as an aptamer of Examples. 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 1: aCas392BR8m2 (SEQ ID NO: 1)
GGTATGGAGGCAAGTCCCAATTC T AAGAAG T GGAG T AGG T GGG TTT AAGGA T A C 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 Milk-derived α-casein (C6780-19, manufactured by SIGMA) 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 α-casein. This was used as an α-casein sample. In addition, milk (manufactured by Ashigara Dairy Co., Ltd.) was diluted with SB1T buffer and dissolved overnight, then centrifuged (3000 g, 20 minutes, room temperature) and separated, and the separated supernatant was filtered with a 0.8 mm filter. The obtained extract was used as a milk sample. 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 preparation of the gliadin sample, the gluten sample, and the lysozyme sample was performed in the same manner as the preparation of the α-casein sample. Egg samples, raw peanut samples, and roasted peanut samples were separated from the materials shown below after being crushed with a food processor, suspended in SB1T buffer, dissolved overnight, and centrifuged (3000 g, 20 minutes, room temperature). The separated supernatant was filtered with a 0.8 mm filter, and the resulting extract was used as a sample.
Gliadin (101778, manufactured by MP Biomedicals)
Wheat-derived gluten (073-00575, manufactured by Wako Pure Chemical Industries, Ltd.)
Lysozyme derived from egg white of chicken egg (120-02674, manufactured by Wako Pure Chemical Industries, Ltd.)
Whole egg peanuts from chicken eggs (made by Indian tea curry shop R-Tea)
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 protein 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. It was. After the injection of the sample, the signal intensity (RU) was measured, and the average value of RU (RU _295-315 ) at 295 to 315 seconds was determined with the start of injection of the sample as 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 milk-derived α-casein. The horizontal axis represents each sample, and the vertical axis represents signal intensity (RU). On the horizontal axis, an α-casein sample, a milk sample, a gliadin sample, a gluten sample, a lysozyme sample, an egg sample, a raw peanut sample, and a roasted peanut sample are shown in order from the left. Concentration (ppm) in each sample indicates the concentration of each protein for α-casein sample, gliadin sample, gluten sample, and lysozyme sample, and each for milk sample, egg sample, raw peanut sample, and roasted peanut sample The concentration of total protein contained in the sample is shown. As shown in FIG. 2, aptamer 1 showed binding properties to α-casein samples and milk samples. Since aptamer 1 selectively binds to milk-derived α-casein, it can be said that the ability to bind to a milk sample showed binding to milk-derived α-casein contained in the milk sample. Since about 80% of the protein contained in milk is α casein, it can be said that most of the milk-derived α casein contained in the milk sample was detected by the aptamer 1. On the other hand, the aptamer 1 showed no signal intensity to the gliadin sample, gluten sample, lysozyme sample, egg sample, raw peanut sample, and roasted peanut sample, and showed no binding.

Next, the binding analysis was performed in the same manner except that the milk sample was used and the protein concentration in the sample was changed to 0.37, 1.1, 3.3, 10 and 30 ppm.

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

Next, using the α-casein sample, the binding analysis was performed in the same manner except that the α-casein concentration in the sample was 12.5, 25, 50, 100, and 200 nmol / 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 α-casein, 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 aptamer 1 increased as the α-casein concentration increased.

Further, kinetic parameters were calculated from the results of the SPR analysis in FIG. As a result, the aptamer 1 has a dissociation constant (KD) in the α-casein sample of 8.95 × 10 ⁻⁹ M, which indicates that the aptamer 1 has excellent binding properties.

[Example 2]
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 milk-derived α-casein was confirmed. The confirmation of the binding was performed using target solid-phased beads on which milk-derived α-casein as a target 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 α-casein 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 binds to α-casein 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, milk-derived α-casein can be detected by detecting luminescence by luciferase using the nucleic acid sensor and the target solid-phased beads.

The nucleic acid sensor was prepared by labeling the 5 ′ end of the aptamer 1 of Example 1 (aCas392BR8m2) using the fluorescent substance NanoLuc ™ luciferase (manufactured by Promega) according to the instruction manual. .

As the sample, the α casein sample of Example 1 was used.

The target-immobilized beads were prepared using the α-casein sample as a target and NHS-activated Sepharose 4 Fast Flow Lab Packs (manufactured by GE Healthcare) according to the instructions for use.

Using the nucleic acid sensor and the target-immobilized beads, the binding property of the nucleic acid sensor to α-casein 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. . After removing the buffer by centrifugation, 50 μL of the α-casein sample (final concentration 0, 0.03, 0.12, 0.47, 1.9, 7.5, 30 ppm) and 50 μL of the α-casein sample were added to each well. A nucleic acid sensor (4 × 10 ⁵ fold dilution) was added and mixed at room temperature for 5 minutes to react the α-casein sample, the target solid-phased 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.

Fig. 5 shows the measurement results of the luminescence amount. FIG. 5 is a graph showing the measurement results of the luminescence amount for the α-casein sample. In FIG. 5, the horizontal axis indicates the concentration of the α casein sample, and the vertical axis indicates the light emission amount (RLU). As shown in FIG. 5, light emission due to the catalytic reaction of luciferase was observed, and the light emission amount increased as the protein concentration of the α-casein sample increased.

Furthermore, the detection limit for the milk-derived α-casein of the nucleic acid sensor was calculated from the measurement result of FIG. 5 by the 3σ method (n = 3). As a result, the detection limit (LOD) for the milk-derived α-casein of the nucleic acid sensor was 0.12 ppm. From this, it was found that the nucleic acid sensor can detect a small amount of milk-derived α-casein.

Next, instead of the α-casein sample, the milk sample prepared in Example 1 and the egg sample, gluten sample, and raw peanut sample (final concentration) prepared in Example 1 were used for confirmation of the cross reaction. 0, 0.032, 0.16, 0.8, 4 ppm), and the same measurement was performed.

FIG. 6 is a graph showing the measurement results of the amount of luminescence when milk, eggs, gluten, and raw peanuts are used as samples. In FIG. 6, the horizontal axis indicates the concentration of each sample, and the vertical axis indicates the light emission amount (RLU). As shown in FIG. 6, in the case of the milk sample, luminescence was observed due to the catalytic reaction of luciferase, and the amount of luminescence increased with an increase in the protein concentration of the sample. On the other hand, in the case of the egg sample, gluten sample, and raw peanut sample, the amount of luminescence did not change. From this, it was found that the nucleic acid sensor can specifically detect milk-derived α-casein in the milk sample, specifically, the milk sample.

Further, the detection limit of the nucleic acid sensor in the milk sample was calculated from the measurement result of FIG. 6 by the 3σ method (n = 3). As a result, the detection limit (LOD) of the nucleic acid sensor in the milk sample was 0.16 ppm. From this, it was found that the nucleic acid sensor can sufficiently detect the milk-derived α-casein in the milk sample.

From the above results, the aptamer of the present invention specifically binds to milk-derived α casein and can be detected by measurement, and according to the aptamer of the present invention, the milk-derived α It was found that the amount of casein can be analyzed.

[Example 3]
Using the nucleic acid sensor, the binding property to the heated milk sample was confirmed.

The binding property of the nucleic acid sensor to the sample was confirmed in the same manner as in Example 2 except that the sample shown below was used as the sample.

The milk sample of Example 1 was processed at 95 ° C. for 10 minutes to prepare a heated milk sample. The heated milk sample was further centrifuged at 12000 rpm for 10 minutes at room temperature, and the resulting supernatant was used as a heated supernatant sample. As a control, the milk sample of Example 1 was used.

The result is shown in FIG. FIG. 7 is a graph showing the measurement results of the amount of luminescence regarding the milk sample, the heated milk sample, and the heated supernatant sample. In FIG. 7, the horizontal axis indicates each sample, and the vertical axis indicates the light emission amount (RLU). Each graph shows the total protein concentration (ppm) in each sample, and shows 0 ppm and 100 ppm in order from the left. As shown in FIG. 4, compared to 0 ppm, at 100 ppm, both the heated milk sample and the heated supernatant sample showed an increase in the amount of luminescence comparable to that of the milk sample.

From the above results, it was found that the aptamer of the present invention also binds to heated milk-derived α-casein and can be detected by measurement.

[Example 4]
About the aptamer of this invention, the binding property with respect to milk origin (alpha) casein was confirmed by SPR analysis.

The following polynucleotide aptamers 2 and 3 were synthesized in the same manner as in Example 1. Aptamer 3 is a miniaturized sequence of aptamer 2. In the polynucleotides of SEQ ID NOs: 2 and 3, the underlined “T” has 5′-benzylaminocarbonyluracil (BndU) substituted at the 5-position of thymine in place 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 2: aCas757BR8m3 (SEQ ID NO: 2)
GGATAGCAGCAGGGACCTCTTATACG TC GG T G CT GG T G TT G T A T AGA CCCCCTT A T A TT A T AA CC GAA T GA TTT G CCC G CT A C GA T A T G
Aptamer 3: aCas757BR8m3s69 (SEQ ID NO: 3)
GGATAGCAGCAGGGACCTCTTATACG TC GG T G CT GG T G TT G T A T AGA CCCCCTT A T A TT A T AA CC GAA T

The presumed secondary structure of aptamers 2 and 3 is shown in FIG. However, it is not limited to this.

As in Example 1 except that aptamers 2 and 3 were used, the α-casein sample was used as a sample, and the α-casein concentration in the sample was 50, 100, and 200 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 2 and 3 to α-casein, respectively, and the horizontal axis represents the elapsed time (seconds) after the start of injection of the sample, and the vertical axis Indicates signal intensity (RU). As shown in FIGS. 9A and 9B, aptamer 2 and aptamer 3, which is a miniaturized sequence thereof, increased in signal intensity as the α-casein concentration increased.

Furthermore, kinetic parameters were calculated from the results of the SPR analysis in FIG. As a result, aptamers 2 and 3 have dissociation constants (KD) for α-casein of 19.1 × 10 ⁻⁹ mol / L and 6.2 × 10 ⁻⁹ mol / L, respectively, and excellent binding properties I found out that

Next, aptamers 4 and 5 of the following polynucleotides were synthesized in the same manner as in Example 1. Aptamer 5 is a miniaturized sequence of aptamer 4. In the polynucleotides of SEQ ID NOs: 4 and 5, the underlined “T” has 5′-benzylaminocarbonyluracil (BndU) substituted at the 5-position of thymine in place 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 4: aCas757BR8m4 (SEQ ID NO: 4)
GGATAGCAGCAGGGACCTCTTATAC CT GAG C GG CTC A TT A CCCTTCC GA CT GG TC G CCC G CTT A CC GAA T GA TTT G CCC G CT A C GA T A T G
Aptamer 5: aCas757BR8m4s62 (SEQ ID NO: 5)
GGATAGCAGCAGGGACCTCTTATAC CT GAG C GG CTC A TT A CCCTTCC GA CT GG TC G CCC G CT

The estimated secondary structure of aptamers 4 and 5 is shown in FIG. However, it is not limited to this.

Analysis of binding properties in the same manner except that aptamers 4 and 5 were used, the α casein sample was used as a sample, and the α casein 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. 10 (A) and (B) are graphs showing the binding properties of aptamers 4 and 5 to α-casein, respectively. The horizontal axis represents the elapsed time (seconds) after the start of injection of the sample, and the vertical axis Indicates signal intensity (RU). As shown in FIGS. 10A and 10B, aptamer 4 and aptamer 5, which is a miniaturized sequence thereof, increased in signal intensity as the α-casein concentration increased.

Furthermore, kinetic parameters were calculated from the results of the SPR analysis in FIG. As a result, aptamers 4 and 5 have dissociation constants (KD) for α-casein of 12.3 × 10 ⁻⁹ mol / L and 16.7 × 10 ⁻⁹ mol / L, respectively, and excellent binding properties I found out that

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

The binding analysis was performed in the same manner as in Example 4 except that a heated α-casein 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 α-casein sample was prepared by heat-treating the α-casein sample at 95 ° C. for 10 minutes.

The results are shown in FIGS. FIGS. 11 (A) and (B) are graphs showing the binding properties of aptamers 2 and 3 to heated α-casein, the horizontal axis indicates the elapsed time (seconds) after the start of injection of the sample, and the vertical axis is Shows the signal intensity (RU). As shown in FIGS. 11A and 11B, aptamer 2 and aptamer 3, which is a miniaturized sequence thereof, increased in signal intensity as the concentration of heated α-casein increased.

12 (A) and (B) are graphs showing the binding properties of aptamers 4 and 5 to heated α-casein, the horizontal axis indicates the elapsed time (seconds) after the start of injection of the sample, and the vertical axis is Shows the signal intensity (RU). As shown in FIGS. 12A and 12B, aptamer 4 and aptamer 5, which is a miniaturized sequence thereof, increased in signal intensity as the concentration of heated α-casein increased.

Furthermore, kinetic parameters were calculated from the results of SPR analysis in FIGS. As a result, aptamers 2 to 5 have dissociation constants (KD) for heated α-casein of 21.3 × 10 ⁻⁹ mol / L, 8.29 × 10 ⁻⁹ mol / L, and 14.7 × 10 ^{−, respectively. It was 9} mol / L, and 22.2 × 10 ⁻⁹ mol / L, which was found to be excellent binding properties.

[Example 6]
About the aptamer of this invention, the binding property with respect to a milk sample was confirmed by SPR analysis.

Bindability analysis was performed in the same manner as in Example 1 except that aptamers 2 to 5 were used as aptamers, and 100 ppm of the milk sample and the heated milk sample were used as samples. In addition, in order to confirm the cross-reaction of the aptamer, binding analysis was performed in the same manner except that the gluten sample, the egg 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 2 to 5 to a heated milk sample. The horizontal axis indicates the type of aptamer, and the vertical axis indicates signal intensity (RU). In the horizontal axis, aptamer 2, aptamer 4, aptamer 3, and aptamer 5 are shown in order from the left. Each graph shows a milk sample, a heated milk sample, a gluten sample, an egg sample, and a raw peanut sample in order from the left. As shown in FIG. 13, aptamers 2 to 5 showed binding properties to milk samples and heated milk samples. On the other hand, aptamers 2 to 5 had a signal intensity of 0 or less and showed no binding to gluten samples, egg samples, and raw peanut samples.

From the above results, it was found that the aptamer of the present invention specifically binds to heated milk-derived α-casein and can be detected by measurement.

[Example 7]
About the aptamer of this invention, the binding property with respect to milk origin (alpha) casein was confirmed by SPR analysis.

The aptamers 6 and 7 of the following polynucleotides were synthesized in the same manner as in Example 1. The aptamer 6 is a sequence obtained by further miniaturizing the aptamer 3 which is a miniaturized sequence of the aptamer 2, and the aptamer 7 is a miniaturized sequence of the aptamer 4. In the polynucleotides of SEQ ID NOs: 6 and 7, the underlined “T” has 5′-benzylaminocarbonyluracil (BndU) substituted at the 5-position of thymine in place 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 6: aCas757BR8m3s63 (SEQ ID NO: 6)
GGATAGCAGCAGGGACCTCTTATACG TC GG T G CT GG T G TT G T A T AGA CCCCCTT A T A TT A T AA
Aptamer 7: aCas757BR8m4s44 (SEQ ID NO: 7)
TTATAC CT GAG C GG CTC A TT A CCCTTCC GA CT GG TC G CCC G CTC

The estimated secondary structure of aptamers 6 and 7 is shown in FIG. However, it is not limited to this.

The aptamer was biotin-modified at the 5 'end and used for SPR described later. The biotin-modified aptamer used was thermally denatured at 95 ° C. for 5 minutes.

The sample was separated by suspending milk-derived α-casein (C6780-19, manufactured by SIGMA) in SB1T buffer and dissolving overnight, followed by centrifugation (12,000 rpm, 15 minutes, room temperature). The separated supernatant was obtained as an extract containing native α-casein. This was diluted with SB1T (+) buffer to obtain an α-casein sample. The composition of the SB1T (+) buffer was 40 mmol / L HEPES, 125 mmol / L NaCl, 5 mmol / L KCl, 1 mmol / L MgCl _2, 0.01% Tween® 20 and 0.1 mmol / L Sodium Dextran Sulfate. The pH was 5000 and the pH was 7.5.

For the binding analysis, ProteON XPR36 (BioRad) was used according to the instruction manual.

First, as a ProteON dedicated sensor chip, a chip (product name: ProteOn NLC Sensor Chip, BioRad) on which streptavidin was immobilized was set in the ProteON XPR36. 200 nmol / L of the biotin-modified aptamer was injected into the flow cell of the sensor chip at a flow rate of 25 μL / min for 80 seconds using the SB1T buffer, and was bound until the signal intensity reached about 800 RU. Then, the flow cell of the chip was blocked using ultrapure water (DDW) containing 10 μmol / L biotin. Subsequently, the α casein samples having predetermined protein concentrations (100, 200, 400, and 800 nmol / L) were each injected with the SB1T (+) buffer at a flow rate of 50 μL / min for 120 seconds, and the same. Under the conditions, the SB1T (+) buffer was flowed, and washing was performed at a flow rate of 50 μL / min for 300 seconds. After the injection of the sample was started, the signal intensity (RU) at a predetermined time was obtained.

The results are shown in FIG. FIGS. 15A and 15B are graphs showing the binding properties of aptamers 6 and 7 to α-casein, respectively. The horizontal axis represents the elapsed time (seconds) after the start of injection of the sample, and the vertical axis Indicates signal intensity (RU). As shown in FIGS. 15 (A) and (B), aptamers 6 and 7 increased in signal intensity as the α-casein concentration increased.

Furthermore, kinetic parameters were calculated from the results of the SPR analysis in FIG. As a result, aptamers 6 and 7 have excellent dissociation properties with dissociation constants (KD) for α-casein of 18 × 10 ⁻⁹ mol / L and 17.2 × 10 ⁻⁹ mol / L, respectively. I understood it.

[Example 8]
About the aptamer of this invention, the binding property with respect to milk origin (alpha) casein was confirmed by SPR analysis.

The following polynucleotide aptamers 8 and 9 were synthesized in the same manner as in Example 1. Aptamers 8 and 9 are partial sequences of aptamer 2. In the polynucleotides of SEQ ID NOs: 8 and 9, the underlined “T” has 5′-benzylaminocarbonyluracil (BndU) substituted at the 5-position of thymine in place 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: aCas757BR8m3s63b (SEQ ID NO: 8)
GGATAGACCTCTTATACG TCT G TT G T A T AGA CCCCCTT A T A TT A T AA
Aptamer 9: aCas757BR8m3s63c (SEQ ID NO: 9)
GGATAGCAGCACTCTTATAC T G CT GG T G TT G T A T AGA CCCCCTT A T A TT A T AA

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

The binding analysis was performed in the same manner as in Example 7 except that the aptamers 8 and 9 were used, and the signal intensity at a predetermined time after starting the injection of the sample was determined.

The result is shown in FIG. FIGS. 17A and 17B are graphs showing the binding properties of aptamers 8 and 9 to α-casein, respectively. The horizontal axis represents the elapsed time (seconds) after the start of injection of the sample, and the vertical axis Indicates signal intensity (RU). As shown in FIGS. 17 (A) and (B), aptamers 8 and 9 increased in signal intensity as the α-casein concentration increased.

Further, kinetic parameters were calculated from the results of the SPR analysis in FIG. As a result, aptamers 8 and 9 have dissociation constants (KD) for α-casein of 12.9 × 10 ⁻⁹ mol / L and 8.59 × 10 ⁻⁹ mol / L, respectively, and excellent binding properties I found out that

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. 2016-226350 filed on November 21, 2016, the entire disclosure of which is incorporated herein.

The nucleic acid molecule of the present invention can bind to milk-derived α-casein. For this reason, according to the nucleic acid molecule of the present invention, milk-derived α-casein can be detected based on the presence or absence of binding to the allergen in the sample. For this reason, the nucleic acid molecule of the present invention can be said to be an extremely useful tool for detecting allergens derived from milk, for example, in the fields of food production, food management, food distribution, and the like.

Claims (18)

1. A nucleic acid molecule that binds to milk-derived α-casein, comprising the polynucleotide of any one of (a) and (b) below:
   (A) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1, 2 or 4 or a partial sequence of the nucleotide sequence of SEQ ID NO: 1, 2 or 4 (b) 90% or more identical to the nucleotide sequence of (a) Polynucleotide aCas392BR8m2 (SEQ ID NO: 1) consisting of a base sequence having a property and binding to milk-derived α-casein
   GGTATGGAGGCAAGTCCCAATTC T AAGAAG T GGAG T AGG T GGG TTT AAGGA T A C G TTTC AG CC AGA C AGGG TTT A T G
   aCas757BR8m3 (SEQ ID NO: 2)
   GGATAGCAGCAGGGACCTCTTATACG TC GG T G CT GG T G TT G T A T AGA CCCCCTT A T A TT A T AA CC GAA T GA TTT G CCC G CT A C GA T A T G
   aCas757BR8m4 (SEQ ID NO: 4)
   GGATAGCAGCAGGGACCTCTTATAC CT GAG C GG CTC A TT A CCCTTCC GA CT GG TC G CCC G CTT A CC GAA T GA TTT G CCC G CT A C GA T A T G
2. The nucleic acid molecule according to claim 1, wherein the partial sequences of the base sequence of SEQ ID NO: 2 or 4 are the base sequences of SEQ ID NO: 3, 6, 8, and 9, or 5 and 7, respectively.
   aCas757BR8m3s69 (SEQ ID NO: 3)
   GGATAGCAGCAGGGACCTCTTATACG TC GG T G CT GG T G TT G T A T AGA CCCCCTT A T A TT A T AA CC GAA T
   aCas757BR8m3s63 (SEQ ID NO: 6)
   GGATAGCAGCAGGGACCTCTTATACG TC GG T G CT GG T G TT G T A T AGA CCCCCTT A T A TT A T AA
   aCas757BR8m4s62 (SEQ ID NO: 5)
   GGATAGCAGCAGGGACCTCTTATAC CT GAG C GG CTC A TT A CCCTTCC GA CT GG TC G CCC G CT
   aCas757BR8m4s44 (SEQ ID NO: 7)
   TTATAC CT GAG C GG CTC A TT A CCCTTCC GA CT GG TC G CCC G CTC
   aCas757BR8m3s63b (SEQ ID NO: 8)
   GGATAGACCTCTTATACG TCT G TT G T A T AGA CCCCCTT A T A TT A T AA
   aCas757BR8m3s63c (SEQ ID NO: 9)
   GGATAGCAGCACTCTTATAC T G CT GG T G TT G T A T AGA CCCCCTT A T A TT A T AA
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 SEQ ID NO: 2 or 4, wherein either one of SEQ ID NOs: 3 and 6, or any one of 5 and 7, respectively A polynucleotide comprising a base sequence and binding to α-casein derived from milk
11. The nucleic acid molecule according to any one of claims 1 to 9, comprising the following polynucleotide (f):
    (F) a base sequence having 80% or more identity to at least one base sequence selected from the group consisting of SEQ ID NOs: 1 to 9, and represented by formulas (I) to (IX), respectively. A polynucleotide that binds to milk-derived α-casein
    

    

    
    

    

    
    

    

    
    

    

    
    

    

    
    

    

    
    

    

    
    

    

    
    

    
12. The nucleic acid molecule according to any one of claims 1 to 11, wherein the milk-derived α-casein is a native allergen.
13. A detection reagent for milk-derived α-casein, comprising the nucleic acid molecule according to any one of claims 1 to 12.
14. Furthermore, it has a labeling substance,
    The detection reagent according to claim 13, wherein the labeling substance is bound to the nucleic acid molecule.
15. The detection reagent according to claim 14, wherein the labeling substance is an enzyme.
16. The detection reagent according to claim 15, wherein the enzyme is luciferase.
17. A nucleic acid molecule according to any one of claims 1 to 12, or a detection reagent according to any one of claims 13 to 16, and a sample, and a milk-derived α-casein in the sample; Forming a complex with the nucleic acid molecule or the detection reagent, and
    A method for detecting milk-derived α-casein, comprising a step of detecting the complex.
18. The detection method according to claim 17, wherein the detection is qualitative analysis or quantitative analysis.

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