WO2018092915A1 - Molécule d'acide nucléique, et son utilisation - Google Patents

Molécule d'acide nucléique, et son utilisation Download PDF

Info

Publication number
WO2018092915A1
WO2018092915A1 PCT/JP2017/041869 JP2017041869W WO2018092915A1 WO 2018092915 A1 WO2018092915 A1 WO 2018092915A1 JP 2017041869 W JP2017041869 W JP 2017041869W WO 2018092915 A1 WO2018092915 A1 WO 2018092915A1
Authority
WO
WIPO (PCT)
Prior art keywords
nucleic acid
casein
polynucleotide
acid molecule
milk
Prior art date
Application number
PCT/JP2017/041869
Other languages
English (en)
Japanese (ja)
Inventor
行大 白鳥
あすみ 稲熊
晃尚 清水
金子　直人
嘉仁 吉田
穣 秋冨
藤田　智子
克紀 堀井
巌 和賀
Original Assignee
Ｎｅｃソリューションイノベータ株式会社
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Ｎｅｃソリューションイノベータ株式会社 filed Critical Ｎｅｃソリューションイノベータ株式会社
Priority to JP2018551723A priority Critical patent/JPWO2018092915A1/ja
Publication of WO2018092915A1 publication Critical patent/WO2018092915A1/fr

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/115—Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith ; Nucleic acids binding to non-nucleic acids, e.g. aptamers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids

Definitions

  • the present invention relates to nucleic acid molecules that bind to milk-derived ⁇ -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.
  • milk for example, ⁇ -casein, which is a milk protein, is known as an allergen.
  • ⁇ -casein As an analysis method for ⁇ -casein, a method using an ELISA method has been reported (Non-patent Document 1).
  • 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.
  • an object of the present invention is to provide a new nucleic acid molecule that binds to milk-derived ⁇ -casein.
  • the nucleic acid molecule of the present invention is a nucleic acid molecule that binds to milk-derived ⁇ -casein, which comprises any of the following polynucleotides (a) or (b).
  • the milk-derived ⁇ -casein detection reagent of the present invention includes the nucleic acid molecule 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 ⁇ -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 ⁇ -casein. For this reason, according to the nucleic acid molecule of the present invention, milk-derived ⁇ -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 ⁇ -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 ⁇ -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 ⁇ -casein in Example 4 of the present invention.
  • FIG. 10 is a graph showing the binding properties of aptamers 4 and 5 to ⁇ -casein in Example 4 of the present invention.
  • FIG. 11 is a graph showing the binding properties of aptamers 2 and 3 to heated ⁇ -casein in Example 5 of the present invention.
  • FIG. 12 is a graph showing the binding properties of aptamers 4 and 5 to heated ⁇ -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 ⁇ -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 ⁇ -casein in Example 8 of the present invention.
  • nucleic acid molecule of the present invention is a nucleic acid molecule that binds to milk-derived ⁇ -casein, characterized in that it comprises a polynucleotide of either (a) or (b) below.
  • 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
  • 90% or more identical to the nucleotide sequence of (a) A polynucleotide comprising a base sequence having sex and binding to milk-derived ⁇ -casein
  • the target is ⁇ -casein derived from milk.
  • the origin of the milk is, for example, cows and goats.
  • Milk-derived ⁇ -casein is, for example, milk-derived ⁇ -casein.
  • commercially available ⁇ -casein can be used as ⁇ -casein for confirming the binding ability, and specific examples include ⁇ -casein derived from milk (C6780-19, manufactured by SIGMA).
  • Alpha casein is, for example, a native allergen.
  • milk-derived ⁇ -casein is also simply referred to as ⁇ -casein.
  • the nucleic acid molecule of the present invention can bind to ⁇ -casein.
  • binding to ⁇ -casein means, for example, having binding property to ⁇ -casein or having binding activity to ⁇ -casein.
  • the binding between the nucleic acid molecule of the present invention and ⁇ -casein can be determined by, for example, surface plasmon resonance molecular interaction (SPR) analysis.
  • SPR surface plasmon resonance molecular interaction
  • BIACORE 3000 (trade name, GE Healthcare UK Ltd.) can be used. Since the nucleic acid molecule of the present invention binds to ⁇ -casein, it can be used, for example, for detection of ⁇ -casein.
  • the nucleic acid molecule of the present invention has a dissociation constant indicating the binding force to ⁇ -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 aCas757BR8m4 (SEQ ID NO: 4)
  • 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 A 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
  • the “identity” is not particularly limited, and may be any range as long as the polynucleotide (b) binds to ⁇ -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.
  • 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 ⁇ -casein
  • 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.
  • 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.
  • 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 ⁇ -casein
  • “one or several” may be in the range where the polynucleotide in (d) is bound to milk-derived ⁇ -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.
  • 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 ⁇ -casein derived from milk
  • 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.
  • 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.
  • “identity” is not particularly limited, and is the same as (b), for example.
  • “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.
  • 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 ⁇ -casein
  • the polynucleotide in the nucleic acid molecule of the present invention may be, for example, the polynucleotide (h) below.
  • 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 ⁇ -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.
  • the polynucleotide in the nucleic acid molecule of the present invention may be, for example, the following polynucleotide (i).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • one single-stranded polynucleotide is any one of the polynucleotides (a) to (i), and the other single-stranded polynucleotide is not limited.
  • the other single-stranded polynucleotide include a polynucleotide having a base sequence complementary to any one of the polynucleotides (a) to (i).
  • 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.
  • 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.
  • 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.
  • nucleotide residue examples include deoxyribonucleotide residue and ribonucleotide residue.
  • nucleic acid molecule of the present invention examples 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.
  • 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.
  • examples of the purine base modification site include the 7th and 8th positions of the purine skeleton.
  • examples of the modification site of the pyrimidine base include the 5th and 6th positions of the pyrimidine skeleton.
  • modified uracil or modified thymine 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).
  • the modified base is not particularly limited.
  • 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.
  • modified adenine examples include 7'-deazaadenine and the like.
  • modified guanine examples include, for example, 7'-deazaguanine.
  • modified cytosine examples include 5'-methylcytosine.
  • modified thymine examples include 5'-benzylaminocarbonylthymine, 5'-tryptaminocarbonylthymine, 5'-isobutylaminocarbonylthymine and the like.
  • modified uracil examples 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.
  • the number of the modified base is, for example, one or more.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the number of the modified thymine is not particularly limited.
  • natural thymine can be substituted for the modified thymine.
  • the number of the modified thymine is, for example, one or more.
  • 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.
  • 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.
  • the number of the modified uracil is not particularly limited.
  • natural thymine can be substituted for the modified uracil.
  • the number of the modified uracil is, for example, one or more.
  • 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.
  • 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.
  • the thymine indicated by the underline in each base sequence may be at least one of the modified thymine and the modified uracil.
  • the modified thymine and the modified uracil may be used as the thymine indicated by the underline in any of the nucleotide sequences of SEQ ID NOS: 1 to 9.
  • the number of the modified cytosines is not particularly limited.
  • natural cytosine can be substituted for the modified cytosine.
  • the number of the modified cytosines is, for example, one or more.
  • 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.
  • 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.
  • the cytosine indicated by the underline in each base sequence may be the modified cytosine.
  • the cytosine indicated by the underline in any one of the nucleotide sequences of SEQ ID NOS: 1 to 9 may be 5'-methylcytosine.
  • the modified base is the modified adenine or the modified guanine
  • cytosine and modified cytosine are referred to as “adenine” and “modified adenine” or “guanine” and It can be read as “modified guanine”.
  • natural adenine can be substituted with the modified adenine
  • 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.
  • the base 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.
  • the number of the modified nucleotides is not particularly limited.
  • 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.
  • 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.
  • 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.
  • 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.
  • a carrier In the nucleic acid molecule of the present invention, for example, either the 5 'end or the 3' end can be immobilized.
  • 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.
  • the carrier include beads, plates, filters, columns, substrates, containers and the like.
  • 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.
  • the fluorescent substance examples 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 to ⁇ -casein.
  • the use of the nucleic acid molecule of the present invention is not particularly limited as long as it uses the binding to ⁇ -casein.
  • the nucleic acid molecule of the present invention can be used in various methods in place of, for example, an antibody against ⁇ -casein.
  • ⁇ -casein can be detected.
  • the method for detecting ⁇ -casein is not particularly limited, and can be performed by detecting the binding between ⁇ -casein and the nucleic acid molecule.
  • the detection reagent of the present invention is a detection reagent for milk-derived ⁇ -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
  • milk-derived ⁇ -casein can be detected as described above.
  • the detection reagent of the present invention can be said to be a binder to milk-derived ⁇ -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.
  • 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.
  • 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.
  • the constituent element include a buffer solution for preparing the sample, an instruction manual, and the like.
  • the detection kit of the present invention includes A kit containing a nucleic acid sensor and an allergen-labeled carrier ( ⁇ -casein-labeled carrier) can be obtained.
  • 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. .
  • the method for detecting milk-derived ⁇ -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 ⁇ -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.
  • 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.
  • the nucleic acid molecule of the present invention specifically binds to ⁇ -casein, for example, by detecting the binding between ⁇ -casein and the nucleic acid molecule or the detection reagent, ⁇ -casein can be specifically detected. Specifically, for example, since the amount of ⁇ -casein in a sample can be analyzed, it can be said that qualitative analysis or quantitative analysis is also possible.
  • the sample is not particularly limited.
  • 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.
  • the sample is preferably a liquid sample because it is easy to contact with the nucleic acid molecule and is easy to handle.
  • 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 ⁇ -casein using the nucleic acid sensor of the present invention labeled with a labeling substance as the nucleic acid molecule of the present invention.
  • this invention is not restrict
  • the detection step further includes, for example, a step of analyzing the presence or amount of ⁇ -casein in the sample based on the detection result of the complex.
  • 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.
  • 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.
  • the contact time is, for example, 10 to 120 minutes, preferably 30 to 60 minutes.
  • 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.
  • the sample is contacted in a container.
  • 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 ⁇ -casein in the sample and the nucleic acid molecule as described above.
  • detecting the presence or absence of binding between the two for example, the presence or absence of ⁇ -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 ⁇ -casein in the sample can be analyzed (quantified).
  • the method for detecting the binding between ⁇ -casein and the nucleic acid molecule is not particularly limited.
  • a conventionally known method for detecting binding between substances can be adopted, and specific examples thereof include the SPR described above.
  • the binding between ⁇ -casein and the nucleic acid molecule cannot be detected, it can be determined that ⁇ -casein is not present in the sample, and when the binding is detected, ⁇ -casein is present in the sample. I can judge.
  • the correlation between the concentration of ⁇ -casein and the binding amount can be obtained in advance, and the concentration of ⁇ -casein in the sample can be analyzed from the binding amount based on the correlation.
  • 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 ⁇ -casein labeling carrier will be described below.
  • the nucleic acid sensor and the sample are mixed.
  • the nucleic acid molecule in the nucleic acid sensor binds to the target milk-derived ⁇ -casein.
  • the nucleic acid molecules in the nucleic acid sensor are not bound to the target.
  • the mixture is brought into contact with the milk-derived ⁇ -casein labeled carrier, and then the ⁇ -casein labeled carrier is removed.
  • the carrier include beads.
  • the nucleic acid molecule in the nucleic acid sensor cannot bind to milk-derived ⁇ -casein in the ⁇ -casein labeled carrier.
  • a luciferase substrate is added to the fraction from which the ⁇ -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.
  • the nucleic acid molecule in the nucleic acid sensor binds to milk-derived ⁇ -casein in the ⁇ -casein labeled carrier. For this reason, by removing the ⁇ -casein labeled carrier, the nucleic acid sensor is also removed while bound to the ⁇ -casein labeled carrier. For this reason, when the luciferase substrate is added to the fraction from which the ⁇ -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.
  • the presence or absence of milk-derived ⁇ -casein in the sample can be analyzed (qualitative analysis) based on the presence or absence of luminescence.
  • the amount of milk-derived ⁇ -casein in the sample and the amount of the nucleic acid sensor remaining in the fraction after removing the ⁇ -casein-labeled carrier have a correlation, depending on the intensity of light emission,
  • the amount of ⁇ -casein derived from milk can also be analyzed (quantitative analysis).
  • milk-derived ⁇ -casein which is an allergen
  • the presence or absence of milk can also be detected indirectly, for example, by detecting milk-derived ⁇ -casein, which is the allergen.
  • Example 1 About the aptamer of this invention, the binding property with respect to milk origin (alpha) casein was confirmed by SPR analysis.
  • Aptamer Aptamer 1 of the following polynucleotide was synthesized as an aptamer of Examples.
  • 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.
  • poly dA polydeoxyadenine
  • the poly dA-added aptamer used was heat-denatured at 95 ° C. for 5 minutes.
  • 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.
  • 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 ⁇ -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)
  • 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 ⁇ mol / 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.
  • FIG. 2 is a graph showing the binding property of aptamer 1 to milk-derived ⁇ -casein.
  • the horizontal axis represents each sample, and the vertical axis represents signal intensity (RU).
  • RU signal intensity
  • an ⁇ -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 ⁇ -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.
  • aptamer 1 showed binding properties to ⁇ -casein samples and milk samples. Since aptamer 1 selectively binds to milk-derived ⁇ -casein, it can be said that the ability to bind to a milk sample showed binding to milk-derived ⁇ -casein contained in the milk sample.
  • the aptamer 1 Since about 80% of the protein contained in milk is ⁇ casein, it can be said that most of the milk-derived ⁇ 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.
  • 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.
  • 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).
  • 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 ⁇ -casein concentration in the milk sample can be quantitatively analyzed by measuring the signal intensity using the aptamer of the present invention.
  • the binding analysis was performed in the same manner except that the ⁇ -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.
  • FIG. 4 is a graph showing the binding property of aptamer 1 to ⁇ -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 ⁇ -casein concentration increased.
  • the aptamer 1 has a dissociation constant (KD) in the ⁇ -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 ⁇ -casein was confirmed. The confirmation of the binding was performed using target solid-phased beads on which milk-derived ⁇ -casein as a target was solid-phased and the nucleic acid sensor.
  • the nucleic acid molecule in the nucleic acid sensor cannot bind to ⁇ -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 ⁇ -casein immobilized on the target immobilized beads.
  • the nucleic acid sensor is also removed while bound to the target-immobilized beads.
  • 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 ⁇ -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 TM luciferase (manufactured by Promega) according to the instruction manual. .
  • Example 1 As the sample, the ⁇ casein sample of Example 1 was used.
  • the target-immobilized beads were prepared using the ⁇ -casein sample as a target and NHS-activated Sepharose 4 Fast Flow Lab Packs (manufactured by GE Healthcare) according to the instructions for use.
  • the binding property of the nucleic acid sensor to ⁇ -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 ⁇ L / well. . After removing the buffer by centrifugation, 50 ⁇ L of the ⁇ -casein sample (final concentration 0, 0.03, 0.12, 0.47, 1.9, 7.5, 30 ppm) and 50 ⁇ L of the ⁇ -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 ⁇ -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 ⁇ -casein sample.
  • the horizontal axis indicates the concentration of the ⁇ casein sample
  • the vertical axis indicates the light emission amount (RLU).
  • RLU light emission amount
  • the detection limit (LOD) for the milk-derived ⁇ -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 ⁇ -casein.
  • Example 1 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.
  • the horizontal axis indicates the concentration of each sample, and the vertical axis indicates the light emission amount (RLU).
  • RLU light emission amount
  • 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 ⁇ -casein in the milk sample.
  • the aptamer of the present invention specifically binds to milk-derived ⁇ casein and can be detected by measurement, and according to the aptamer of the present invention, the milk-derived ⁇ 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.
  • Example 1 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.
  • 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.
  • 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.
  • ppm total protein concentration
  • 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.
  • the aptamer of the present invention also binds to heated milk-derived ⁇ -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.
  • aptamers 2 and 3 were synthesized in the same manner as in Example 1.
  • Aptamer 3 is a miniaturized sequence of aptamer 2.
  • the underlined “T” has 5′-benzylaminocarbonyluracil (BndU) substituted at the 5-position of thymine in place of natural thymine (T)
  • BndU 5′-benzylaminocarbonyluracil
  • 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.
  • the ⁇ -casein sample was used as a sample, and the ⁇ -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.
  • FIGS. 9A and 9B are graphs showing the binding properties of aptamers 2 and 3 to ⁇ -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).
  • aptamer 2 and aptamer 3 which is a miniaturized sequence thereof, increased in signal intensity as the ⁇ -casein concentration increased.
  • aptamers 2 and 3 have dissociation constants (KD) for ⁇ -casein of 19.1 ⁇ 10 ⁇ 9 mol / L and 6.2 ⁇ 10 ⁇ 9 mol / L, respectively, and excellent binding properties I found out that
  • 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.
  • the underlined “T” has 5′-benzylaminocarbonyluracil (BndU) substituted at the 5-position of thymine in place of natural thymine (T).
  • BndU 5′-benzylaminocarbonyluracil
  • 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.
  • the ⁇ casein sample was used as a sample, and the ⁇ 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.
  • FIGS. 10 (A) and (B) are graphs showing the binding properties of aptamers 4 and 5 to ⁇ -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).
  • aptamer 4 and aptamer 5 which is a miniaturized sequence thereof, increased in signal intensity as the ⁇ -casein concentration increased.
  • aptamers 4 and 5 have dissociation constants (KD) for ⁇ -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 ⁇ -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 ⁇ -casein sample was prepared by heat-treating the ⁇ -casein sample at 95 ° C. for 10 minutes.
  • FIGS. 11 (A) and (B) are graphs showing the binding properties of aptamers 2 and 3 to heated ⁇ -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).
  • aptamer 2 and aptamer 3 which is a miniaturized sequence thereof, increased in signal intensity as the concentration of heated ⁇ -casein increased.
  • FIGS. 12A and 12B are graphs showing the binding properties of aptamers 4 and 5 to heated ⁇ -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).
  • aptamer 4 and aptamer 5 which is a miniaturized sequence thereof, increased in signal intensity as the concentration of heated ⁇ -casein increased.
  • aptamers 2 to 5 have dissociation constants (KD) for heated ⁇ -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.
  • 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).
  • 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.
  • aptamers 2 to 5 showed binding properties to milk samples and heated milk samples.
  • 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.
  • the aptamer of the present invention specifically binds to heated milk-derived ⁇ -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.
  • the underlined “T” has 5′-benzylaminocarbonyluracil (BndU) substituted at the 5-position of thymine in place of natural thymine (T).
  • BndU 5′-benzylaminocarbonyluracil
  • 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 ⁇ -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 ⁇ -casein. This was diluted with SB1T (+) buffer to obtain an ⁇ -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.
  • 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 ⁇ L / 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 ⁇ mol / L biotin.
  • DDW ultrapure water
  • the ⁇ 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 ⁇ L / min for 120 seconds, and the same.
  • the SB1T (+) buffer was flowed, and washing was performed at a flow rate of 50 ⁇ L / min for 300 seconds.
  • the signal intensity (RU) at a predetermined time was obtained.
  • FIGS. 15A and 15B are graphs showing the binding properties of aptamers 6 and 7 to ⁇ -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).
  • aptamers 6 and 7 increased in signal intensity as the ⁇ -casein concentration increased.
  • aptamers 6 and 7 have excellent dissociation properties with dissociation constants (KD) for ⁇ -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.
  • aptamers 8 and 9 were synthesized in the same manner as in Example 1.
  • Aptamers 8 and 9 are partial sequences of aptamer 2.
  • the underlined “T” has 5′-benzylaminocarbonyluracil (BndU) substituted at the 5-position of thymine in place of natural thymine (T).
  • BndU 5′-benzylaminocarbonyluracil
  • 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.
  • FIGS. 17A and 17B are graphs showing the binding properties of aptamers 8 and 9 to ⁇ -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).
  • aptamers 8 and 9 increased in signal intensity as the ⁇ -casein concentration increased.
  • aptamers 8 and 9 have dissociation constants (KD) for ⁇ -casein of 12.9 ⁇ 10 ⁇ 9 mol / L and 8.59 ⁇ 10 ⁇ 9 mol / L, respectively, and excellent binding properties I found out that
  • the nucleic acid molecule of the present invention can bind to milk-derived ⁇ -casein. For this reason, according to the nucleic acid molecule of the present invention, milk-derived ⁇ -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.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Plant Pathology (AREA)
  • Analytical Chemistry (AREA)
  • Immunology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne une nouvelle molécule d'acide nucléique qui se lie à l'α-caséine. Cette molécule d'acide nucléique, qui se lie à l'α-caséine dérivée du lait, comprend n'importe quel polynucléotide présenté en (a) ou (b) ci-dessous : (a) un polynucléotide qui comprend la séquence de bases de SEQ ID NO : 1, 2, ou 4, ou une séquence partielle de la séquence de bases de SEQ ID NO : 1, 2, ou 4 ; ou (b) un polynucléotide qui comprend une séquence de bases ayant au moins 90 % d'identité avec l'une quelconque des séquences de bases en (a), et qui se lie à l'α-caséine dérivée du lait.
PCT/JP2017/041869 2016-11-21 2017-11-21 Molécule d'acide nucléique, et son utilisation WO2018092915A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP2018551723A JPWO2018092915A1 (ja) 2016-11-21 2017-11-21 核酸分子およびその用途

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2016-226350 2016-11-21
JP2016226350 2016-11-21

Publications (1)

Publication Number Publication Date
WO2018092915A1 true WO2018092915A1 (fr) 2018-05-24

Family

ID=62146430

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2017/041869 WO2018092915A1 (fr) 2016-11-21 2017-11-21 Molécule d'acide nucléique, et son utilisation

Country Status (2)

Country Link
JP (1) JPWO2018092915A1 (fr)
WO (1) WO2018092915A1 (fr)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015066027A2 (fr) * 2013-10-28 2015-05-07 Dots Devices, Inc. Détection d'allergènes
WO2015151349A1 (fr) * 2014-03-31 2015-10-08 Ｎｅｃソリューションイノベータ株式会社 Molécule d'acide nucléique se liant à un allergène du sarrasin et utilisation s'y rapportant

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015066027A2 (fr) * 2013-10-28 2015-05-07 Dots Devices, Inc. Détection d'allergènes
WO2015151349A1 (fr) * 2014-03-31 2015-10-08 Ｎｅｃソリューションイノベータ株式会社 Molécule d'acide nucléique se liant à un allergène du sarrasin et utilisation s'y rapportant

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
AMAYA-GONZALEZ, S.: "Aptamer-based analysis: a promising alternative for food safety control", SENSORS, vol. 13, no. 12, 2013, pages 16292 - 16311, XP055233452 *
CAMILLE, L. A. ET AL.: "Selection and analytical applications of aptamers", TRAC TRENDS IN ANALYTICAL CHEMISTRY, vol. 25, no. 7, July 2006 (2006-07-01), pages 681 - 691, XP025029963 *
MONACI, L. ET AL.: "Milk allergens, their characteristics and their detection in food: A review.", EUROPEAN FOOD RESEARCH AND TECHNOLOGY, vol. 223, no. 2, 15 February 2006 (2006-02-15), pages 149 - 179, XP019420433 *
MONACI, L. ET AL.: "Reliable Detection of Milk Allergens in Food Using a High-Resolution, Stand-Alone Mass Spectrometer", J. AOAC. INT., vol. 94, no. 4, 2011, pages 1034 - 1042 *
ZHANG, B. ET AL.: "A novel insight in rapid allergen detection in food systems: From threshold dose to real-world concentration, Sens", ACTUATORS B CHEM., vol. 186, 2013, pages 597 - 602 *

Also Published As

Publication number Publication date
JPWO2018092915A1 (ja) 2019-06-24

Similar Documents

Publication Publication Date Title
Pinto et al. Label-free detection of gliadin food allergen mediated by real-time apta-PCR
JP6687618B2 (ja) 組換えタンパク質調製物中の残留宿主細胞タンパク質の検出
JP6212136B2 (ja) ピーナッツに結合する核酸分子およびその用途
CN104911186A (zh) 一种特异性识别黄曲霉素b1的单链dna寡核苷酸适配子
JP6744028B2 (ja) α−アミラーゼに結合する核酸分子およびその用途
WO2018092915A1 (fr) Molécule d'acide nucléique, et son utilisation
JP6414907B2 (ja) そばアレルゲンに結合する核酸分子およびその用途
JP6347498B2 (ja) 卵アレルゲンに結合する核酸分子およびその用途
WO2018097220A1 (fr) Molécule d'acide nucléique et son utilisation
JP7343138B2 (ja) ターゲットの分析方法および分析キット
JP6399611B2 (ja) エビアレルゲンに結合する核酸分子およびその用途
JP6598315B2 (ja) 小麦アレルゲンに結合する核酸分子およびその用途
JP6687264B2 (ja) 核酸分子およびその用途
EP3406735B1 (fr) Procédé d'analyse cible et trousse d'analyse cible destinée à l'utilisation dans ledit procédé
JP2018171035A (ja) 核酸分子およびその用途
JP7405400B2 (ja) 核酸分子およびその用途
JP6963221B2 (ja) 核酸分子およびその用途
JP2018171041A (ja) ターゲット分析方法およびこれに用いるターゲット分析キット
JP2020165820A (ja) 乳アレルゲンの分析キットおよび分析方法
JP2009189310A (ja) 高親和性核酸フラグメント群の塩基配列決定方法および核酸アプタマーのスクリーニング方法
JP2018170985A (ja) 核酸分子およびその用途
JP2022106581A (ja) Ｃｒｐ結合核酸分子、ｃｒｐ検出用センサ、ｃｒｐ検出試薬、ｃｒｐの検出方法、およびｃｒｐ結合領域

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 17871970

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 2018551723

Country of ref document: JP

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 17871970

Country of ref document: EP

Kind code of ref document: A1