JP5846623B2 - RNA molecules that bind to rabbit-derived IgG and uses thereof - Google Patents

Description

  The present invention relates to RNA molecules that bind to rabbit-derived IgG and uses thereof.

  Since the antigen-antibody reaction is a highly specific reaction, it is widely used in the detection of targets such as proteins. For example, ELISA (Enzyme-Linked Immunosorbent Assay) is performed as follows using an antigen-antibody reaction. First, an antibody whose target is an antigen is immobilized on a solid phase. Then, the sample is brought into contact with the solid phase, and the target in the sample and the immobilized antibody are bound by an antigen-antibody reaction. Next, a primary antibody that binds to the target is brought into contact with the solid phase. As the primary antibody, an antibody that recognizes an epitope different from that of the immobilized antibody is used. As a result, a complex of the immobilized antibody, the target, and the primary antibody is formed on the solid phase. Next, after removing an unreacted sample and the primary antibody from the solid phase, a secondary antibody that binds to the primary antibody is brought into contact with the solid phase. As the secondary antibody, for example, a labeled secondary antibody labeled with a labeling substance such as an enzyme is used. Thereby, the labeled secondary antibody further binds to the complex. Then, after removing the unreacted labeled secondary antibody from the solid phase, the labeled substance of the labeled secondary antibody bound to the complex is detected. When the labeling substance is an enzyme, for example, the labeling substance can be detected by adding a coloring reagent or the like and detecting the product of the enzyme reaction, and indirectly the target in the complex can be detected.

  However, the following problems have been pointed out with antibodies. That is, for example, since an antibody is prepared by injecting an antigen into an immunized animal such as a rabbit and separating a fraction having binding properties from serum or the like, the operation is complicated and expensive. In addition, the antibody binds non-specifically to, for example, a polymer container such as polypropylene in addition to an antigen such as a protein, so that there is a problem of detection accuracy. In preparation of the labeled secondary antibody, it is necessary to form a conjugate of a labeling substance such as an enzyme and the antibody, but the operation is complicated.

  Because of these problems, attention has been focused on nucleic acid molecules that specifically bind to antigens instead of antibodies. For example, as a nucleic acid molecule that can bind to rabbit-derived IgG that is a primary antibody, an RNA molecule that exhibits a higher binding force than an antibody has been reported (see Patent Document 1). When such an RNA molecule is used in place of the secondary antibody, for example, it can prevent binding of the antibody to IgG derived from animals other than rabbits, binding to denatured IgG, etc., and thus binding power superior to that of an antibody. And can achieve specificity.

  In the practical application of target detection using such RNA molecules, it is required to enable simple detection with even better accuracy.

JP 2008-125484 A

  Then, the objective of this invention is providing the RNA molecule couple | bonded with rabbit origin IgG which is excellent in binding property, specificity, and handleability, and its use.

前記式（Ｉ）において、前記Ｎ_１、Ｎ_２、Ｎ_３、Ｎ_４、Ｎ_５、Ｎ_６およびＮ_７は、ヌクレオチド残基を示し、各ｎ_１、ｎ_２、ｎ_３、ｎ_４、ｎ_５、ｎ_６およびｎ_７は、それぞれ前記ヌクレオチド残基Ｎ_１、Ｎ_２、Ｎ_３、Ｎ_４、Ｎ_５、Ｎ_６およびＮ_７の数を示し、
前記Ｎ_１およびＮ_７は、相互に水素結合してステム構造形成可能であり、前記Ｎ_３およびＮ_５は、相互に水素結合してステム構造を形成可能であり、前記Ｎ_２およびＮ_６は、インターナルループ構造を形成可能であり、前記Ｎ_４は、ヘアピンループ構造を形成可能である。 The RNA molecule of the present invention is an RNA molecule that binds to rabbit-derived IgG, which comprises a single-stranded nucleic acid represented by the following general formula (I).

In the formula (I), N ₁ , N ₂ , N ₃ , N ₄ , N ₅ , N ₆ and N ₇ represent nucleotide residues, and each n ₁ , n ₂ , n ₃ , n ₄ , n ₅ , n ₆ and n ₇ indicate the number of the nucleotide residues N ₁ , N ₂ , N ₃ , N ₄ , N ₅ , N ₆ and N ₇ , respectively.
The N ₁ and N ₇ can be hydrogen bonded to each other to form a stem structure, the N ₃ and N ₅ can be hydrogen bonded to each other to form a stem structure, and the N ₂ and N ₆ can be , An internal loop structure can be formed, and the N ₄ can form a hairpin loop structure.

  The binding agent of the present invention is a binding agent for rabbit-derived IgG, comprising the RNA molecule of the present invention.

  The detection reagent of the present invention is a rabbit-derived IgG detection reagent comprising the binding agent of the present invention.

  The detection kit of the present invention is a rabbit-derived IgG detection kit comprising the detection reagent of the present invention.

  The method for detecting rabbit IgG of the present invention comprises a reaction step of reacting rabbit-derived IgG with the binding agent of the present invention, and a detection step of detecting the binding agent bound to the rabbit-derived IgG. To do.

The target detection method of the present invention includes a first reaction step in which a target in a sample and an antibody against the target are reacted, and a complex that binds to the antibody reacts with a complex of the target and the antibody. A second reaction step, and a detection step of detecting the binding agent bound to the complex,
Using rabbit-derived IgG as an antibody against the target,
The binder of the present invention is used as the binder.

  The RNA molecule of the present invention can bind to rabbit-derived IgG with excellent specificity. Therefore, for example, the target can be detected with higher accuracy by using the RNA molecule of the present invention instead of the secondary antibody as described above.

前記式（Ｉ）において、前記Ｎ_１、Ｎ_２、Ｎ_３、Ｎ_４、Ｎ_５、Ｎ_６およびＮ_７は、ヌクレオチド残基を示し、各ｎ_１、ｎ_２、ｎ_３、ｎ_４、ｎ_５、ｎ_６およびｎ_７は、それぞれ前記ヌクレオチド残基Ｎ_１、Ｎ_２、Ｎ_３、Ｎ_４、Ｎ_５、Ｎ_６およびＮ_７の数を示し、
前記Ｎ_１およびＮ_７は、相互に水素結合してステム構造形成可能であり、前記Ｎ_３およびＮ_５は、相互に水素結合してステム構造を形成可能であり、前記Ｎ_２およびＮ_６は、インターナルループ構造を形成可能であり、前記Ｎ_４は、ヘアピンループ構造を形成可能である。 <RNA molecule>
As described above, the RNA molecule of the present invention is an RNA molecule that binds to rabbit-derived IgG, which comprises a single-stranded nucleic acid represented by the following general formula (I).

In the formula (I), N ₁ , N ₂ , N ₃ , N ₄ , N ₅ , N ₆ and N ₇ represent nucleotide residues, and each n ₁ , n ₂ , n ₃ , n ₄ , n ₅ , n ₆ and n ₇ indicate the number of the nucleotide residues N ₁ , N ₂ , N ₃ , N ₄ , N ₅ , N ₆ and N ₇ , respectively.
The N ₁ and N ₇ can be hydrogen bonded to each other to form a stem structure, the N ₃ and N ₅ can be hydrogen bonded to each other to form a stem structure, and the N ₂ and N ₆ can be , An internal loop structure can be formed, and the N ₄ can form a hairpin loop structure.

  In the present invention, “binding to rabbit-derived IgG” means, for example, having a binding ability to the rabbit-derived IgG or having a binding activity to the rabbit-derived IgG. The binding between the RNA molecule of the present invention and the rabbit-derived IgG can be determined by, for example, surface plasmon resonance molecular interaction (SPR) analysis. For example, Biacore X (trade name, GE Healthcare UK Ltd.) can be used for the analysis. The RNA molecule of the present invention is also referred to as an RNA aptamer.

  The RNA molecule of the present invention has a dissociation constant with the rabbit-derived IgG of, for example, 50 nmol / L or less, preferably 25 nmol / L or less, more preferably 10 nmol / L or less, and even more preferably 7 nmol / L. L or less, particularly preferably 5 nmol / L or less.

  In the present invention, “loop structure can be formed” includes, for example, that a loop structure is actually formed and that a loop structure can be formed depending on conditions even if the loop structure is not formed. “Loop structure can be formed” includes, for example, both experimental confirmation and prediction by computer simulation. In the present invention, “stem structure can be formed” includes, for example, actually forming a stem structure and being able to form a stem structure depending on conditions even if the stem structure is not formed. “Stem structure can be formed” includes, for example, both experimental confirmation and prediction by computer simulation.

  The RNA molecule of the present invention may be, for example, a single-stranded nucleic acid or a double-stranded nucleic acid. In the case of the former single-stranded nucleic acid, the single-stranded nucleic acid may be a single-stranded nucleic acid having the structure represented by the formula (I) or a single-stranded nucleic acid having the structure. In the case of the latter double-stranded nucleic acid, one single-stranded nucleic acid may be a single-stranded nucleic acid including the structure represented by the formula (I), or a single-stranded nucleic acid having the above structure, and the other one The double-stranded nucleic acid is not particularly limited. In the case of the double-stranded nucleic acid, for example, prior to use, it is preferable to make it into a single-stranded nucleic acid by denaturation or the like. Moreover, it is preferable that the structure represented by the said formula (I) forms the stem structure and the loop structure at the time of use, for example.

  The structural unit of the RNA molecule of the present invention is, for example, a nucleotide residue, and examples thereof include a ribonucleotide residue and a deoxyribonucleotide residue. The RNA molecule of the present invention may be, for example, RNA composed only of ribonucleotide residues or RNA containing 1 to several deoxyribonucleotide residues.

  The RNA molecule of the present invention may contain, for example, 1 to several modified nucleotide residues, and examples of the modified nucleotide residue include a modified ribonucleotide residue and a modified deoxyribonucleotide residue. . Examples of the modified nucleotide residue include those in which a sugar residue in the nucleotide residue is modified. Examples of the sugar residue include a ribose residue and a deoxyribose residue. The modification site in the nucleotide residue is not particularly limited, and examples thereof include the 2 'position and / or the 4' position of the sugar residue. Examples of the modification include methylation, fluorination, amination, and thiolation. The modified nucleotide residue is, for example, a modified nucleotide residue having a pyrimidine base (pyrimidine nucleus) as a base, or a modified nucleotide residue having a purine base (purine nucleus) as a base. The former is preferred. Hereinafter, a nucleotide residue having a pyrimidine base is referred to as a pyrimidine nucleotide residue, a modified pyrimidine nucleotide residue is referred to as a modified pyrimidine nucleotide residue, and a nucleotide residue having a purine base is referred to as a purine nucleotide residue. The purified purine nucleotide residue is referred to as a modified purine nucleotide residue. Examples of the pyrimidine nucleotide residues include uracil nucleotide residues having uracil, cytosine nucleotide residues having cytosine, thymine nucleotide residues having thymine, and the like. 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, a 2′-methylated-cytosine nucleotide residue, and a 2′-modified ribose residue at the 2 ′ position. Fluorinated-uracil nucleotide residue, 2′-fluorinated-cytosine nucleotide residue, 2′-aminated-uracil nucleotide residue, 2′-aminated-cytosine nucleotide residue, 2′-thiolated-uracil nucleotide residue Groups, 2'-thiolated-cytosine nucleotide residues and the like.

  The base in the nucleotide residue may be, for example, a natural base (non-artificial base) of adenine (a), cytosine (c), guanine (g), thymine (t) and uracil (u), or a non-natural base ( Artificial base). Examples of the artificial base include a modified base and a modified base, and preferably have the same function as the natural base (a, c, g, t, or u). The artificial base having the same function is, for example, an artificial base capable of binding to cytosine (c) instead of guanine (g), an artificial base capable of binding to guanine (g) instead of cytosine (c), Instead of adenine (a), an artificial base capable of binding to thymine (t) or uracil (u), instead of thymine (t), an artificial base capable of binding to adenine (a), instead of uracil (u) And an artificial base capable of binding to adenine (a). Examples of the modified base include a methylated base, a fluorinated base, an aminated base, and a thiolated base. Specific examples of the modified base include, for example, 2′-methyluracil, 2′-methylcytosine, 2′-fluorouracil, 2′-fluorocytosine, 2′-aminouracil, 2′-aminocytosine, 2-thiouracil, 2-thiocytosine and the like. In the present invention, for example, the bases represented by a, g, c, t and u include the meaning of the artificial base having the same function as each of the natural bases in addition to the natural base.

  The RNA molecule of the present invention may contain, for example, 1 to several artificial nucleic acid monomer residues. Examples of the artificial nucleic acid monomer residue include PNA (peptide nucleic acid), LNA (Locked Nucleic Acid), and ENA (2'-O, 4'-C-Ethylenebridged Nucleic Acids). The nucleic acid in the monomer residue is the same as described above, for example.

  In the formula (I), for example, the sites of the deoxyribonucleotide residue, the modified nucleotide residue, and the artificial nucleic acid monomer residue are not particularly limited.

  In the formula (I), the number of the deoxyribonucleotide residues is not particularly limited, and is, for example, in the range of 1 to 77 residues, in the range of 1 to 76 residues, preferably 1 to 25 residues. More preferably, it is the range of 1 to 5 residues. In the formula (I), the number of the modified nucleotide residues is not particularly limited, and is, for example, in the range of 1 to 77 residues, in the range of 1 to 76 residues, preferably 1 to 25 residues. It is the range of group, More preferably, it is the range of 1-5 residues. In the formula (I), the number of the artificial nucleic acid monomer residues is not particularly limited, and is, for example, in the range of 1 to 77 residues, in the range of 1 to 76 residues, preferably 1 to 25 residues. It is the range of group, More preferably, it is the range of 1-5 residues. These residues may be continuous or discontinuous in the formula (I), for example.

  The RNA molecule of the present invention is preferably resistant to RNase, for example. The RNA molecule of the present invention preferably has at least one of the deoxyribonucleotide residue, the modified nucleotide residue, and the artificial nucleic acid monomer residue, for example, for RNase resistance. In addition, the RNA molecule of the present invention may be bound to, for example, several 10 kDa PEG (polyethylene glycol) or deoxythymidine, for example, at the 5 'end or 3' end due to RNase resistance.

The number of nucleotide residues in the entire single-stranded nucleic acid represented by the formula (I) is not particularly limited, and is, for example, in the range of 39 to 77 residues, preferably in the range of 39 to 76 residues, preferably It is the range of 39-70 residues, More preferably, it is the range of 39-60 residues, Most preferably, it is the range of 39-50 residues.
The nucleotide residue number _{n 1} of said _{N 1} is not particularly limited, for example, in the range of 4 to 15 residues, preferably in the range of 4 to 10 residues, more preferably 4-7 residues The range is particularly preferably 4 to 5 residues.
The number of N ₂ nucleotide residues n ₂ is not particularly limited, and is, for example, in the range of 4 to 15 residues, preferably in the range of 4 to 10 residues, and more preferably in the range of 4 to 7 residues. The range is particularly preferably 4 to 5 residues.
The nucleotide residue number _{n 3} of said _{N 3} is not particularly limited, for example, in the range of 7 to 15 residues, preferably in the range of from 7 to 12 residues, more preferably 7-10 residues The range is particularly preferably 7 to 8 residues.
The number of N ₄ nucleotide residues n ₄ is not particularly limited, and is, for example, in the range of 8 to 15 residues, in the range of 9 to 15 residues, and preferably in the range of 9 to 13 residues. More preferably, it is the range of 9-11 residues, Most preferably, it is the range of 9-10 residues.
The number of N ₅ nucleotide residues n ₅ is not particularly limited, and is, for example, in the range of 7 to 15 residues, preferably in the range of 7 to 12 residues, and more preferably in the range of 7 to 10 residues. The range is particularly preferably 7 to 8 residues.
The number of N ₆ nucleotide residues n ₆ is not particularly limited, and is, for example, in the range of 4 to 15 residues, preferably in the range of 4 to 10 residues, and more preferably in the range of 4 to 7 residues. The range is particularly preferably 4 to 5 residues.
The number of N ₇ nucleotide residues n ₇ is not particularly limited, and is, for example, in the range of 4 to 15 residues, preferably in the range of 4 to 10 residues, and more preferably in the range of 4 to 5 residues. The range is particularly preferably 4 residues.
The number of nucleotide residues n ₁ , n ₂ , n ₃ , n ₄ , n ₅ , n ₆ and n ₇ preferably satisfy, for example, the following equations and inequalities:
n ₁ = n ₇ , n ₃ = n ₅ , (n ₂ + n ₆ )> n ₄

  When the total length of the single-stranded nucleic acid is in the above-described range, it can be said that the size of the single-stranded nucleic acid is smaller than that of a conventional RNA molecule that binds to rabbit IgG. The RNA molecule having a single-stranded nucleic acid thus miniaturized is easy to synthesize, for example, and it is easy to reduce costs and to modify the binding of a labeling substance and a linker. Therefore, it can be said that the RNA molecule of the present invention is suitable for use as a detection reagent, application to a detection device, and the like.

Specific examples of the formula (I), for example, the number of single-stranded nucleic acid entire nucleotide residues of the formula (I) is a 43 residue, nucleotide residue number n ₁ of said N ₁ is a 5 residue, nucleotide residue number _{n 2} of said _{n 2} is 5 residues, nucleotides residues _{n 3} of said _{n 3} is 8 residue, nucleotide residues of the _{n 4} The number n ₄ is 8 residues, the N ₅ nucleotide residue number n ₅ is 8 residues, the N ₆ nucleotide residue number n ₆ is 4 residues, and the N ₇ nucleotide residues _{n 7} is 5 residues. Examples of such RNA molecules containing single-stranded nucleic acids include RNA molecules comprising the following sequences or RNA molecules comprising the following sequences.
mini3 (SEQ ID NO: 1)
GGGACCAGAAGUUUUUAAACCGCGCCUUGGAAGCGUACGUCCC
mini3m (SEQ ID NO: 2)
GCCACCAGAAGUUUUUAAACCGCGCCUUGGAAGCGUACGUGGC
rd06-unmodified (SEQ ID NO: 18)
GCCACCAGAAGUUUUUAAACCGCGCCUUGGAAGCG A ACGUGGC
rmd06-unmodified (SEQ ID NO: 19)
GCCACCAGAAGUUUUUAAACC C CGCCUUGGAAGCGUACGUGGC

As a specific example of the formula (I), the secondary structure of the mini3 (SEQ ID NO: 1) is shown in the following formula (II).

The sequences of these SEQ ID NOs may be substituted with, for example, the deoxyribonucleotide residue, the modified nucleotide residue, and the artificial nucleic acid monomer residue. Specific examples of the substituted sequence are shown below. In the following sequences, “dN” such as dA, dG, dC and dT represents a deoxyribonucleotide residue, “lN” such as 1G and 1C represents an LNA residue, and “mN” such as mC and mU represents , Represents a methylated ribonucleotide residue, and “fN” such as fC and fU represents a fluorinated ribonucleotide (hereinafter the same).
R10_M3-fC (SEQ ID NO: 3)
GGGA [fC] [fC] AGAAGUUUUUAAA [fC] [fC] G [fC] G [fC] [fC] UUGGAAG [fC] GUA [fC] GU [fC] [fC] [fC]
R10_M3-fU (SEQ ID NO: 4)
GGGACCAGAAG [fU] [fU] [fU] [fU] [fU] AAACCGCGCC [fU] [fU] GGAAGCG [fU] ACG [fU] CCC
rm01 (SEQ ID NO: 5)
G [mC] [mC] A [mC] [mC] AGAAGUUUUUAAA [mC] [mC] G [mC] G [mC] [mC] UUGGAAGCGUA [mC] GUGG [mC]
rm02 (SEQ ID NO: 6)
G [mC] [mC] A [mC] [mC] AGAAGUUUUUAAA [mC] [mC] G [mC] GCCUUGGAAG [mC] GUA [mC] GUGG [mC]
rm03 (SEQ ID NO: 7)
GCCAC [mC] AGAAGUUUUUAAA [mC] [mC] G [mC] G [mC] [mC] UUGGAAGCGUA [mC] GUGGC
rm04 (SEQ ID NO: 8)
GCCACCAGAAGUUUUUAAACCGCGCCUUGGAAGCG [mU] ACGUGGC
rd03 (SEQ ID NO: 9)
GCCAC [dC] AGAAGUUUUUAAA [dC] [dC] G [dC] G [dC] [dC] UUGGAAGCGUA [dC] GUGGC
rd06 (SEQ ID NO: 10)
GCCAC [dC] AGAAGUUUU [dU] AAAC [dC] GCGCCUUGGAAGCGAACGUGGC
rmd02 (SEQ ID NO: 11)
GCCAC [dC] AGAAGUUUU [mU] AAAC [dC] GCGCCUUGGAAGCG [mU] ACGUGGC
rmd03 (SEQ ID NO: 12)
GCCAC [dC] AGAAGUUUU [dU] AAAC [dC] GCGCCUUGGAAGCG [mU] ACGUGGC
rmd04 (SEQ ID NO: 13)
GCCAC [dC] AGAAGUUUU [dU] AAAC [dC] GCGCCUUGGAAGCG [dU] ACGUGGC
rmd05 (SEQ ID NO: 14)
GCCAC [dC] AGAAGUUUU [dU] AAAC [dC] ACGCCUUGGAAGCG [mU] ACGUGGC
rmd06 (SEQ ID NO: 15)
GCCAC [dC] AGAAGUUUU [dU] AAAC [dC] CCGCCUUGGAAGCG [mU] ACGUGGC
rmde01 (SEQ ID NO: 16)
[lG] [lC] CAC [dC] AGAAGUUUU [dU] AAAC [dC] GCGCCUUGGAAGCG [mU] ACGUG [lG] [lC]

Further, as specific examples of the formula (I), in addition to these, for example, the number of nucleotide residues in the entire single-stranded nucleic acid represented by the formula (I) is 39 residues, and the N ₁ Nucleotide residue number n ₁ is 3 residues, N ₂ nucleotide residue number n ₂ is 5 residues, N ₃ nucleotide residue number n ₃ is 8 residues, The N ₄ nucleotide residue number n ₄ is 8 residues, the N ₅ nucleotide residue number n ₅ is 8 residues, and the N ₆ nucleotide residue number n ₆ is 4 residues. a group, the nucleotide residue number _{n 7} of the _{n 7} is 3 residues. Examples of such RNA molecules containing single-stranded nucleic acids include RNA molecules comprising the following sequences or RNA molecules comprising the following sequences.
rme01-unmodified (SEQ ID NO: 20)
[lG] [lC] C [dC] AGAAGUUUU [dU] AAAC [dC] GCGCCUUGGAAGCG [mU] ACG [lG] [lC]

The sequence of SEQ ID NO may be substituted with, for example, the deoxyribonucleotide residue, the modified nucleotide residue, and the artificial nucleic acid monomer residue. Specific examples of the substituted sequence are shown below. In the following sequences, “dN” such as dA, dG, dC and dT represents a deoxyribonucleotide residue, “lN” such as 1G and 1C represents an LNA residue, and “mN” such as mC and mU represents Indicates methylated ribonucleotide residues.
rme01 (SEQ ID NO: 17)
[lG] [lC] C [dC] AGAAGUUUU [dU] AAAC [dC] GCGCCUUGGAAGCG [mU] ACG [lG] [lC]

The RNA molecule of the present invention preferably has a motif represented by SEQ ID NO: 21 from (N ₄ ) _n4 to (N ₅ ) _n5 in the formula (I). The following motif preferably has the following lower case sequence at the 3 ′ end of (N ₄ ) _{n4 and} the following upper case sequence at the 5 ′ end of (N ₅ ) _n5 . Examples of RNA molecules having such a motif include RNA molecules containing the base sequences of SEQ ID NOs: 1 to 20.
Motif (SEQ ID NO: 21)
ccUUGGAA

In the RNA molecule of the present invention, for example, an underlined base in the following sequence is preferably conserved.
mini3 (SEQ ID NO: 1)
GGGACCAGAAGUUUUUAA A CCGCG CCUUG GA AG CGUACGUCCC
mini3m (SEQ ID NO: 2)
GCCACCAGAAGUUUUUAA A CCGCG CCUUG GA AG CGUACGUGGC
rd06-unmodified (SEQ ID NO: 18)
GCCACCAGAAGUUUUUAA A CCGCG CCUUG GA AG CG A ACGUGGC
rmd06-unmodified (SEQ ID NO: 19)
GCCACCAGAAGUUUUUAA A CC C CG CCUUG GA AG CGUACGUGGC

In the RNA molecule of the present invention, in the formula (I), the stem structure of (N ₃ ) _n3 and (N ₅ ) _n5 and the phosphate group of the hairpin loop structure of (N ₄ ) _n4 are retained. preferable.

Examples of the RNA molecule of the present invention include the following RNA molecules (A1) to (A3).
(A1) RNA molecule comprising any one of the nucleotide sequences of SEQ ID NOS: 1 to 20 (A2) In the nucleotide sequence of any of SEQ ID NOS: 1 to 20, one or more bases are substituted, deleted, added or inserted An RNA molecule that can bind to the rabbit IgG (A3) and a nucleotide sequence having 90% or more homology with the nucleotide sequence represented by any one of SEQ ID NOs: 1 to 20, and RNA molecules capable of binding to the rabbit IgG

  In the above (A2), “one or more” is not particularly limited. “1 or more” is, for example, 1 to 5, preferably 1 to 4, more preferably 1 to 3, more preferably 1 in the nucleotide sequences of SEQ ID NOs: 1 to 20. One or two, particularly preferably one. The “one or more” is, for example, 1 to 5, preferably 1 to 4, more preferably 1 to 3, in the full length sequence of the rabbit IgG-binding nucleic acid molecule (A1). More preferably, it is one or two, and particularly preferably one. The base used for the substitution, addition or insertion is not particularly limited, and may be, for example, the natural base or the non-natural base. For the substitution, addition or insertion of the base, for example, the nucleotide residue may be used, or the artificial nucleic acid monomer residue may be used.

  The homology is, for example, 90% or more, preferably 95% or more, more preferably 99% or more. The homology can be calculated, for example, by calculating under default conditions using BLAST or the like.

  In the RNA molecule of the present invention, the rabbit IgG may be any of subtype 1, subtype 2a and subtype 3, for example.

  In the present invention, the subtype of the mouse-derived IgG antibody is not particularly limited. For example, the subtype 1, subtype 2a, subtype 3, and a mixture of IgG antibodies having different subtypes optionally having these are mixed. can give.

  The RNA molecule of the present invention may further have a labeling substance, for example. The labeling substance is preferably bound to, for example, at least one of the 5 ′ end and the 3 ′ end of the RNA molecule, and specifically, at least one of the 5 ′ end and the 3 ′ end of the formula (I). It is preferable that it is couple | bonded with, More preferably, it is 5 'terminal.

The labeling substance is not particularly limited, and examples thereof include biotin and derivatives thereof; avidin and derivatives thereof; a fluorophore; an enzyme; a radioisotope; a functional group such as a thiol group and an amino group; an electron acceptor such as methylene blue; Electron donors such as NADPH; organic compounds, inorganic compounds, electrochemiluminescent materials such as ruthenium and the like, and biotin is preferable. The fluorophore is not particularly limited, and examples thereof include fluorescein and its derivatives, rhodamine and its derivatives, dansyl chloride and its derivatives, umbelliferone and the like. The enzyme is not particularly limited, and examples thereof include peroxidase such as horseradish peroxidase, alkaline phosphatase, β-galactosidase, urease, catalase, glucose oxidase, lactate dehydrogenase, and amylase. The radioisotope is not particularly limited. For example, iodine ( ¹³¹ I, ¹²⁵ I, ¹²³ I, ¹²¹ I), phosphorus ( ³² P), sulfur ( ³⁵ S), metals (for example, ⁶⁸ Ga, ⁶⁷ Ga, ⁶⁸ Ge, ⁵⁴ Mn, ⁹⁹ Mo, ⁹⁹ Tc, ¹³³ Xe, etc.), tritium and the like. The labeling substance, the other, for example, NADH, FMNH ₂ such electron donors, acridinium esters, luminescent substances luminol such as luciferase, bioluminescent materials luciferin, and bio-fluorescence protein such as GFP, and the like .

  The method for producing the RNA molecule of the present invention is not limited at all, and can be synthesized by a genetic engineering technique such as a nucleic acid synthesis method using chemical synthesis, or a known method. The RNA molecule of the present invention can also be obtained, for example, by the SELEX method (Systematic Evolution of Ligands by exponential enrichment) using an RNA pool and rabbit-derived IgG.

  As described above, the RNA molecule of the present invention exhibits binding to the rabbit IgG. For this reason, the use of the RNA molecule of the present invention is not particularly limited as long as it uses the binding property to the rabbit IgG. The RNA molecule of the present invention can be used in various methods, for example, instead of the antibody against the rabbit IgG.

  According to the RNA molecule of the present invention, for example, the rabbit IgG can be detected. For example, the reaction can be performed by reacting the RNA molecule with the rabbit IgG, forming a complex of the RNA molecule and the rabbit IgG, and detecting the RNA molecule in the complex. The target can also be detected by using it together with the rabbit IgG that binds to the target. For example, the target and the rabbit IgG are reacted, and the target and the rabbit IgG are bound to form a complex. Then, the target can be detected indirectly by binding the RNA molecule to the complex via the rabbit IgG and detecting the bound RNA molecule. The method for detecting the rabbit IgG and the target is not particularly limited. A specific example will be described later.

  According to the RNA molecule of the present invention, for example, the rabbit IgG can be purified. For purification of the rabbit IgG, for example, the RNA molecule and a non-purified fraction of the rabbit IgG are contacted to form a complex of the RNA molecule and the rabbit IgG, and a non-binding substance to the RNA molecule is removed. And it can carry out by collect | recovering the said composite_body | complex.

  When the RNA molecule of the present invention is used for purification of rabbit-derived IgG, for example, the RNA molecule may be used by binding to a solid phase. Examples of the shape of the solid phase include beads, and examples of the material of the solid phase include agarose and synthetic resin. The method for binding the RNA molecule to the solid phase is not particularly limited, and examples thereof include a method in which one of the RNA molecule and the solid phase is biotinylated, the other is avidin, and bound by an avidin-biotin bond. It is done. For this reason, the RNA molecule of the present invention may be subjected to various modifications such as biotinylation or avidinization.

<Binders>
As described above, the binding agent of the present invention is a binding agent for rabbit-derived IgG characterized by containing the RNA molecule of the present invention. The binding agent of the present invention is not limited as long as it contains the RNA molecule of the present invention. When the binding agent of the present invention is used, for example, the detection of the rabbit IgG and the target and the purification of the rabbit IgG can be performed as described above.

  As described above, the detection reagent of the present invention is a rabbit-derived IgG detection reagent comprising the binding agent of the present invention. The detection reagent of this invention should just contain the binder of the said this invention, and another structure is not restrict | limited at all. If the detection reagent of the present invention is used, for example, the rabbit IgG and the target can be detected as described above.

  As described above, the detection kit of the present invention is a rabbit-derived IgG detection kit comprising the detection reagent of the present invention. The detection kit of the present invention only needs to contain the detection reagent of the present invention, and other configurations are not limited at all. The detection kit may further include other reagents that can be used for detection of the rabbit-derived IgG. The other reagents are not particularly limited, and examples thereof include reagents that can be used in the detection method of the present invention described later. The detection reagent of the present invention and the other reagent may be housed in the same container or in separate containers, for example. The detection kit may further include instructions for use, for example.

<Rabbit-derived IgG detection method>
The method for detecting rabbit-derived IgG of the present invention comprises a reaction step of reacting rabbit-derived IgG with the binding agent of the present invention, and a detection step of detecting the binding agent bound to the rabbit-derived IgG. And The present invention is characterized in that the binding agent of the present invention, that is, the RNA molecule of the present invention is bound to the rabbit-derived IgG, and other steps and conditions are not particularly limited.

  The method for detecting the binding agent bound to the rabbit-derived IgG is not particularly limited. For example, the binding agent is detected by binding a labeling substance to the RNA molecule in the binding agent and detecting the labeling substance. it can. The labeling substance is not particularly limited, and examples thereof include the aforementioned substances. For the method for detecting the binding agent bound to the rabbit-derived IgG, the description in the target detection method of the present invention described later can be cited.

<Target detection method>
In the target detection method of the present invention, as described above, the first reaction step in which the target in the sample and the antibody against the target are reacted, and the complex of the target and the antibody binds to the antibody. A second reaction step of reacting with a binding agent, and a detection step of detecting the binding agent bound to the complex,
Rabbit-derived IgG is used as an antibody against the target, and the binding agent of the present invention is used as the binding agent.

  The detection method of the present invention is characterized in that the binding agent of the present invention is used for detecting the complex of the target and the antibody, and other steps and conditions are not particularly limited. The binding agent of the present invention can be used in place of, for example, a known secondary antibody.

  In the detection method of the present invention, for example, the antibody unbound to the target is further removed from the reaction solution of the first reaction between the first reaction step and the second reaction step. It is preferable to include a removal step. By removing the antibody unbound to the target, for example, the binding of the binding agent to the unbound antibody can be removed, so that the detection accuracy can be further improved. The removing step is preferably performed, for example, by immobilizing the target on a solid phase, contacting the antibody, and then washing the solid phase. By immobilizing the target, for example, in addition to the unreacted antibody, other substances contained in the sample can be removed. The immobilization of the target is not particularly limited, and examples thereof include immobilization on a membrane. Immobilization to the membrane can be performed, for example, by electrophoresis of the sample using an electrophoresis gel and blotting from the electrophoresis gel to the membrane. The electrophoresis is not particularly limited, and examples thereof include PAGE and SDS-PAGE, and preferably SDS-PAGE.

  The detection method of the present invention preferably includes, for example, a separation step of separating the target in the sample and other components in the sample prior to the first reaction step. By separating the target from the other components, for example, the detection accuracy can be further improved. The separation step can be performed, for example, by electrophoresis of the sample. The electrophoresis of the sample is not particularly limited, for example, and is the same as described above. By electrophoresis of the sample, for example, the target of the sample and the other components can be separated, and further, for example, the unreacted antibody can be removed by immobilization on the membrane. . In the separation step, the target and the other components are preferably denatured, and specifically, the sample is preferably subjected to SDS-PAGE.

  The detection method of the present invention is, for example, between the second reaction step and the detection step, and further from the reaction solution of the second reaction, the binding unbound to the rabbit-derived IgG in the complex. It is preferable to include a removing step for removing the body. For example, the detection accuracy can be further improved by removing the unbound complex.

  The detection method of the present invention can be performed, for example, according to the Northwestern method. For example, the first reaction step, the second reaction step, and the detection step are preferably Western blot methods.

  In the present invention, the sample is not particularly limited, and may be a liquid system such as a solution, suspension, or dispersion, or a solid system such as a cell or tissue.

  The detection method of the present invention will be described with an example. In the present invention, the binding between the target and the RNA molecule of the present invention may be used, and the present invention is not limited to the following examples.

  First, the sample is subjected to SDS-PAGE and blotted on a membrane. The SDS-PAGE conditions, the blotting conditions, and the membrane to be used are not particularly limited, and known methods and materials can be used.

  Next, the membrane is contacted with rabbit-derived IgG that binds to the target. Thereby, the target immobilized on the membrane and the rabbit-derived IgG are bound to form a complex. After the reaction between the target and the rabbit-derived IgG, the membrane is washed to remove the unreacted rabbit-derived IgG and other components contained in the sample.

  Subsequently, the membrane of the present invention is brought into contact with the membrane. As a result, the binding agent of the present invention binds to the rabbit-derived IgG in the complex on the membrane. After the reaction between the rabbit-derived IgG and the binding agent, the membrane is washed to remove the rabbit-derived IgG and the unreacted binding agent.

  Then, the binding agent bound to the rabbit-derived IgG in the complex is detected. The target in the complex can be detected indirectly by detecting the binding agent. The detection of the binding agent is not particularly limited, and can be appropriately determined according to the RNA molecule. The RNA molecule is preferably labeled with a labeling substance as described above, and can be appropriately determined according to the type of the labeling substance. When the labeling substance is, for example, an enzyme, a substrate for the enzyme is added to the membrane, and a product obtained by an enzyme reaction is detected to detect the labeling substance and indirectly detect a target. it can. Such detection allows qualitative and quantitative determination of the target.

  In addition, in the target detection method, for example, in the first reaction step, after the target in the sample and the antibody are reacted, the reaction solution of the first reaction is subjected to electrophoresis, and the membrane is supplied to the membrane. After blotting, the binder of the present invention may be brought into contact with the membrane.

  Next, examples of the present invention will be described. However, the present invention is not limited by the following examples. Commercially available reagents were used based on their protocol unless otherwise indicated.

[Example 1]
An RNA aptamer was prepared, and binding ability to rabbit IgG was confirmed by SPR analysis.

(1) RNA aptamer A 24 d-long poly dA was added as a tag to the 3 ′ end of the RNA aptamer of the following SEQ ID NO to prepare a tagged RNA aptamer.
mini3 (SEQ ID NO: 1)
GGGACCAGAAGUUUUUAAACCGCGCCUUGGAAGCGUACGUCCC
mini3m (SEQ ID NO: 2)
GCCACCAGAAGUUUUUAAACCGCGCCUUGGAAGCGUACGUGGC
R10_M3-fC (SEQ ID NO: 3)
GGGA [fC] [fC] AGAAGUUUUUAAA [fC] [fC] G [fC] G [fC] [fC] UUGGAAG [fC] GUA [fC] GU [fC] [fC] [fC]
R10_M3-fU (SEQ ID NO: 4)
GGGACCAGAAG [fU] [fU] [fU] [fU] [fU] AAACCGCGCC [fU] [fU] GGAAGCG [fU] ACG [fU] CCC
rm01 (SEQ ID NO: 5)
G [mC] [mC] A [mC] [mC] AGAAGUUUUUAAA [mC] [mC] G [mC] G [mC] [mC] UUGGAAGCGUA [mC] GUGG [mC]
rm02 (SEQ ID NO: 6)
G [mC] [mC] A [mC] [mC] AGAAGUUUUUAAA [mC] [mC] G [mC] GCCUUGGAAG [mC] GUA [mC] GUGG [mC]
rm03 (SEQ ID NO: 7)
GCCAC [mC] AGAAGUUUUUAAA [mC] [mC] G [mC] G [mC] [mC] UUGGAAGCGUA [mC] GUGGC
rm04 (SEQ ID NO: 8)
GCCACCAGAAGUUUUUAAACCGCGCCUUGGAAGCG [mU] ACGUGGC
rd03 (SEQ ID NO: 9)
GCCAC [dC] AGAAGUUUUUAAA [dC] [dC] G [dC] G [dC] [dC] UUGGAAGCGUA [dC] GUGGC
rd06 (SEQ ID NO: 10)
GCCAC [dC] AGAAGUUUU [dU] AAAC [dC] GCGCCUUGGAAGCGAACGUGGC
rmd02 (SEQ ID NO: 11)
GCCAC [dC] AGAAGUUUU [mU] AAAC [dC] GCGCCUUGGAAGCG [mU] ACGUGGC
rmd03 (SEQ ID NO: 12)
GCCAC [dC] AGAAGUUUU [dU] AAAC [dC] GCGCCUUGGAAGCG [mU] ACGUGGC
rmd04 (SEQ ID NO: 13)
GCCAC [dC] AGAAGUUUU [dU] AAAC [dC] GCGCCUUGGAAGCG [dU] ACGUGGC
rmd05 (SEQ ID NO: 14)
GCCAC [dC] AGAAGUUUU [dU] AAAC [dC] ACGCCUUGGAAGCG [mU] ACGUGGC
rmd06 (SEQ ID NO: 15)
GCCAC [dC] AGAAGUUUU [dU] AAAC [dC] CCGCCUUGGAAGCG [mU] ACGUGGC
rmde01 (SEQ ID NO: 16)
[lG] [lC] CAC [dC] AGAAGUUUU [dU] AAAC [dC] GCGCCUUGGAAGCG [mU] ACGUG [lG] [lC]
rme01 (SEQ ID NO: 17)
[lG] [lC] C [dC] AGAAGUUUU [dU] AAAC [dC] GCGCCUUGGAAGCG [mU] ACG [lG] [lC]

As a comparative example, a tag-added RNA aptamer was prepared by adding polyA having a length of 24 bases as a tag to the 3 ′ end of an RNA aptamer having the following SEQ ID NO.
R10 (SEQ ID NO: 22)
GGGAGAAUUCCGACCAGAAGUUUUUAAACCGCGCCUUGGAAGCGUACGUUGGCCCUUUCCUCUCUCCUCCUUCUUCU

(2) SPR analysis SPR analysis was performed using BIACORE 3000 (trade name, manufactured by BIACORE) and BIAevaluation software (version 3.1, manufactured by BIOACORE). The dissociation constant _{K D} values, the BIAevaluation software for global fitting curves: in (1 1 Langmuir binding), was calculated by fitting the data.

First, 5 μmol / L biotinylated poly dT was added to a chip (trade name Sensor chip SA, manufactured by BIACORE) for 10 minutes at a flow rate of 2 μL / min. The biotinylated poly dT was obtained by biotinylating the 5 ′ end of poly dT having a length of 24 bases. As a result, about 1,000 RU of biotinylated poly dT was immobilized on the chip. 400 nmol / L of the tagged RNA aptamer was added to the chip at a flow rate of 2 μL / min for 10 minutes. Thereby, about 2,000 RU of the tagged RNA aptamer was hybridized to the biotinylated poly dT on the chip. Next, a predetermined concentration of anti-mouse IgG rabbit IgG (polyclonal antibody, trade name anti GST rabbit IgG: manufactured by Chemicon) is added to the chip on which the tagged RNA aptamer is immobilized for 2 minutes at a flow rate of 20 μL / ml. did. The predetermined concentrations of the rabbit IgG were final concentrations of 100 nmol / L, 50 nmol / L, 25 nmol / L and 12.5 nmol / L. Then, after measuring the binding reaction rate between the tagged RNA aptamer and the rabbit IgG, Buffer (50 mmol / L HEPES-NaOH [pH 7.4], 150 mmol / L NaCl, 5 mmol / L MgCl ₂ ) was introduced for 3 minutes. The dissociation of the rabbit IgG from the tagged RNA aptamer was measured as the dissociation reaction rate. In addition, after the measurement of the dissociation reaction rate, the complex of the tagged RNA aptamer and the rabbit IgG was dissociated from the chip by adding 10 mmol / L NaOH, and the tagged RNA aptamer was again performed in the same manner as described above. Hybridization and binding and dissociation of the rabbit IgG were repeated as a series of steps. Table 1 below shows these results.

(Table 1)
RNA aptamer SEQ ID NO dissociation constant, _{K D,}
(Nmol / L)
mini3 1 22.4
mini3m 2 30.8
rm02 6 50.7
rm04 8 37
rd06 10 45.3
rmd02 11 31
rmd03 12 29
rmd04 13 40
rmd05 14 27.3
rmd06 15 26.1
rmde01 16 24.3
rme01 17 22.9
Comparative Example RNA aptamer 22 51.5

  As shown in Table 1, it was found that the RNA aptamer of the present invention showed a binding force superior to that of the RNA aptamer of the comparative example.

[Example 2]
(1) RNA aptamer A 24 d-long poly dA was added as a tag to the 3 ′ end of the RNA aptamer of the following SEQ ID NO to prepare a tagged RNA aptamer.
mini3 (SEQ ID NO: 1)
GGGACCAGAAGUUUUUAAACCGCGCCUUGGAAGCGUACGUCCC
mini3m (SEQ ID NO: 2)
GCCACCAGAAGUUUUUAAACCGCGCCUUGGAAGCGUACGUGGC
R10_M3-fC (SEQ ID NO: 3)
GGGA [fC] [fC] AGAAGUUUUUAAA [fC] [fC] G [fC] G [fC] [fC] UUGGAAG [fC] GUA [fC] GU [fC] [fC] [fC]
R10_M3-fU (SEQ ID NO: 4)
GGGACCAGAAG [fU] [fU] [fU] [fU] [fU] AAACCGCGCC [fU] [fU] GGAAGCG [fU] ACG [fU] CCC
rm01 (SEQ ID NO: 5)
G [mC] [mC] A [mC] [mC] AGAAGUUUUUAAA [mC] [mC] G [mC] G [mC] [mC] UUGGAAGCGUA [mC] GUGG [mC]

(2) SPR analysis The biotinylated poly dT on the chip is hybridized with about 3,000 RU of the tagged RNA aptamer, and the chip on which the tagged RNA aptamer is immobilized is 800 nmol / L as an analyte. Anti-mouse IgG rabbit IgG (polyclonal antibody, trade name anti mouse rabbit IgG: manufactured by Chemicon) was added, and Buffers with different compositions (20 mmol / L HEPES-NaOH [pH 7.4], 150 mmol / L NaCl, Analysis was performed in the same manner as in Example 1 except that 5 mmol / L MgCl ₂ and 0.05% Tween 20) were used.

  Table 2 below shows RU (Response Units) for 120 seconds from the start of the addition of the analyte.

(Table 2)
RNA aptamer SEQ ID NO: RU
mini3 1 1666
R10 22 283

  The miniaturized aptamer mini3 showed a very high binding amount as compared with the aptamer R10. Specifically, the binding amount increased about 5.89 times. In SPR analysis, in general, an increase in signal is seen in inverse proportion to the molecular weight of the immobilized molecule. Since the molecular weight of R10 is 1.77 times that of mini3, theoretically, the apparent signal of mini10 is estimated to increase by about 1.77 times. However, in practice, the mini3 signal increased approximately 5.89 times. This suggests that the base sequence not involved in the binding at R10 inhibited the binding between rabbit IgG and aptamer. Mini3 was thought to have increased the amount of aptamer binding to rabbit IgG by eliminating unnecessary base sequences.

[Example 3]
Serum stability was evaluated for RNA aptamers.

(1) RNA aptamer The RNA aptamer of the following sequence number was produced. These RNA aptamers were each diluted in TBS to a concentration of 0.15 mg / mL and used for the following evaluation. The composition of the TBS was 20 mmol / L Tris-HCl, 0.9% NaCl, 5 mmol / L MgCl ₂ .
mini3 (SEQ ID NO: 1)
GGGACCAGAAGUUUUUAAACCGCGCCUUGGAAGCGUACGUCCC
rmd02 (SEQ ID NO: 11)
GCCAC [dC] AGAAGUUUU [mU] AAAC [dC] GCGCCUUGGAAGCG [mU] ACGUGGC
rmd03 (SEQ ID NO: 12)
GCCAC [dC] AGAAGUUUU [dU] AAAC [dC] GCGCCUUGGAAGCG [mU] ACGUGGC
rmd04 (SEQ ID NO: 13)
GCCAC [dC] AGAAGUUUU [dU] AAAC [dC] GCGCCUUGGAAGCG [dU] ACGUGGC

(2) Serum Rabbit serum (SIGMA # 4505, manufactured by Sigma) was diluted 1/100 with TBS to prepare diluted serum. The composition of the TBS (pH 7.4) was 20 mmol / L Tris-HCl, 0.9% NaCl, 5 mmol / L MgCl ₂ .

(3) Evaluation method 0.1 mL of the RNA aptamer was mixed with 0.1 mL of the diluted serum, and the mixture was allowed to stand at room temperature. Then, after the start of standing (0 minutes), 30 minutes after the start, 1 hour, 2 hours, and 4 hours, the mixed solution was sampled and subjected to electrophoresis. The electrophoresis conditions were as follows. The gel after electrophoresis was photographed on a UV transilluminator, and the RNA aptamer full length (43 mer) band was signaled by densitometry of image analysis software Image-J (free software, manufactured by NIH) using the photographed data. The intensity was quantified. And the relative value of the signal intensity | strength when the result of 0 minute was set to 100% was calculated | required, and the time when the said relative value showed 50% was made into the half life.

(Electrophoresis conditions)
Gel composition: 15% Acrylamide, 7 mol / L Urea in 1 × TBE
Sample load: 30 ng / lane
Electrophoretic conditions: 200V constant voltage, 85min
Staining: SYBR Green II staining

  The stability results in rabbit serum are shown in Table 3 below. As a result of quantifying the full length band of the aptamer with the image analysis software, as shown in Table 3 below, mini3 had a half-life of about 15 minutes in 1/100 diluted rabbit serum. The modified aptamers rmd02, rmd03, and rmd04 had a half-life of 2-3 hours. Thus, it was suggested that the degradation resistance in serum was further improved by modifying mini3.

(Table 3)
Aptamer half-life (minutes)
mini3 15
rmd02 180
rmd03 135
rmd04 150

  Although the present invention has been described with reference to the exemplary embodiments and examples, the present invention is not limited to the above exemplary embodiments and examples. 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.

  The RNA molecule of the present invention can bind to rabbit-derived IgG with excellent specificity. Therefore, for example, the target can be detected with higher accuracy by using the RNA molecule of the present invention instead of the secondary antibody as described above. For this reason, the RNA molecule of the present invention is useful, for example, as a new research tool.

Claims (14)

Look contains a single-stranded nucleic acid represented by the following general formula (I), the formula (I) single-stranded nucleic acid represented by the, rabbit, characterized in that it consists of any of the sequences of SEQ ID NO: 1 to 17 RNA molecules that bind to IgG.

In the formula (I), N ₁ , N ₂ , N ₃ , N ₄ , N ₅ , N ₆ and N ₇ represent nucleotide residues, and each n ₁ , n ₂ , n ₃ , n ₄ , n ₅ , n ₆ and n ₇ indicate the number of the nucleotide residues N ₁ , N ₂ , N ₃ , N ₄ , N ₅ , N ₆ and N ₇ , respectively.
The N ₁ and N ₇ can be hydrogen bonded to each other to form a stem structure, the N ₃ and N ₅ can be hydrogen bonded to each other to form a stem structure, and the N ₂ and N ₆ can be , An internal loop structure can be formed, and the N ₄ can form a hairpin loop structure.
mini3 (SEQ ID NO: 1)
GGGACCAGAAGUUUUUAAACCGCGCCUUGGAAGCGUACGUCCC
mini3m (SEQ ID NO: 2)
GCCACCAGAAGUUUUUAAACCGCGCCUUGGAAGCGUACGUGGC
R10_M3-fC (SEQ ID NO: 3)
GGGA [fC] [fC] AGAAGUUUUUAAA [fC] [fC] G [fC] G [fC] [fC] UUGGAAG [fC] GUA [fC] GU [fC] [fC] [fC]
R10_M3-fU (SEQ ID NO: 4)
GGGACCAGAAG [fU] [fU] [fU] [fU] [fU] AAACCGCGCC [fU] [fU] GGAAGCG [fU] ACG [fU] CCC
rm01 (SEQ ID NO: 5)
G [mC] [mC] A [mC] [mC] AGAAGUUUUUAAA [mC] [mC] G [mC] G [mC] [mC] UUGGAAGCGUA [mC] GUGG [mC]
rm02 (SEQ ID NO: 6)
G [mC] [mC] A [mC] [mC] AGAAGUUUUUAAA [mC] [mC] G [mC] GCCUUGGAAG [mC] GUA [mC] GUGG [mC]
rm03 (SEQ ID NO: 7)
GCCAC [mC] AGAAGUUUUUAAA [mC] [mC] G [mC] G [mC] [mC] UUGGAAGCGUA [mC] GUGGC
rm04 (SEQ ID NO: 8)
GCCACCAGAAGUUUUUAAACCGCGCCUUGGAAGCG [mU] ACGUGGC
rd03 (SEQ ID NO: 9)
GCCAC [dC] AGAAGUUUUUAAA [dC] [dC] G [dC] G [dC] [dC] UUGGAAGCGUA [dC] GUGGC
rd06 (SEQ ID NO: 10)
GCCAC [dC] AGAAGUUUU [dU] AAAC [dC] GCGCCUUGGAAGCGAACGUGGC
rmd02 (SEQ ID NO: 11)
GCCAC [dC] AGAAGUUUU [mU] AAAC [dC] GCGCCUUGGAAGCG [mU] ACGUGGC
rmd03 (SEQ ID NO: 12)
GCCAC [dC] AGAAGUUUU [dU] AAAC [dC] GCGCCUUGGAAGCG [mU] ACGUGGC
rmd04 (SEQ ID NO: 13)
GCCAC [dC] AGAAGUUUU [dU] AAAC [dC] GCGCCUUGGAAGCG [dU] ACGUGGC
rmd05 (SEQ ID NO: 14)
GCCAC [dC] AGAAGUUUU [dU] AAAC [dC] ACGCCUUGGAAGCG [mU] ACGUGGC
rmd06 (SEQ ID NO: 15)
GCCAC [dC] AGAAGUUUU [dU] AAAC [dC] CCGCCUUGGAAGCG [mU] ACGUGGC
rmde01 (SEQ ID NO: 16)
[lG] [lC] CAC [dC] AGAAGUUUU [dU] AAAC [dC] GCGCCUUGGAAGCG [mU] ACGUG [lG] [lC]
rme01 (SEQ ID NO: 17)
[lG] [lC] C [dC] AGAAGUUUU [dU] AAAC [dC] GCGCCUUGGAAGCG [mU] ACG [lG] [lC]
In the sequences of SEQ ID NOs: 3 to 17, [dC] or [dU] represents a deoxyribonucleotide residue having a base of C or U, respectively, and [1G] or [1C] represents a base of G or C, respectively. [MC] or [mU] represents a methylated ribonucleotide residue whose base is C or U, respectively, and [fC] or [fU] represents a base C or Fluorinated ribonucleotide that is U is shown. In the formula (I), a portion of said nucleotide residue is a modified nucleotide residue, RNA molecule of claim 1 Symbol placement. The RNA according to claim 2 , wherein the modified nucleotide residue is at least one selected from the group consisting of a methylated nucleotide residue, a fluorinated nucleotide residue, an aminated nucleotide residue, and a thiolated nucleotide residue. molecule. The RNA molecule according to claim 1 or 2 , wherein in the formula (I), a part of the nucleotide residue is at least one of LNA and DNA. The binding agent with respect to rabbit origin IgG characterized by including the RNA molecule as described in any one of Claim 1 to 4 . A detection reagent for rabbit-derived IgG, comprising the binding agent according to claim 5 . A detection kit for rabbit-derived IgG, comprising the detection reagent according to claim 6 . A reaction step of reacting rabbit-derived IgG with the binding agent according to claim 5 ; and
A method for detecting rabbit IgG, comprising a detection step of detecting the binding agent bound to the rabbit-derived IgG. A first reaction step of reacting a target in a sample with an antibody against the target;
A second reaction step in which a complex of the target and the antibody is reacted with a binding agent that binds to the antibody; and
Detecting the binding agent bound to the complex,
Using rabbit-derived IgG as an antibody against the target,
A method for detecting a target, wherein the binding agent according to claim 5 is used as the binding agent. Between the second reaction step and the first reaction step, further, from the reaction solution of the first reaction involves the removal step of removing the antibody unbound to the target, according to claim 9, wherein Detection method. The detection method according to claim 9 or 10 , further comprising a separation step of separating the target in the sample and other components in the sample prior to the first reaction step. The detection method according to claim 11 , wherein the target is denatured in the separation step. The detection method according to claim 11 or 12 , wherein the separation step is SDS-PAGE of a sample. The detection method according to any one of claims 9 to 13 , wherein the first reaction step, the second reaction step, and the detection step are Western blotting.