JP2004229582A - Method for detecting target base sequence, reagent for enhancing detection sensitivity, and kit for detection - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for detecting a target base sequence by which the bonding affinity between a nucleic acid aptamer and a ligand is heightened, and the target base sequence in a specimen can be detected even if the concentration is low; and to provide a reagent for enhancing detection sensitivity, and a kit for the detection. <P>SOLUTION: The method for detecting the target base sequence in the test specimen comprises (a) a step for forming a nucleic acid aptamer obtained by hybridizing the target base sequence in the test specimen with a probe by mixing the test specimen with the probe hybridizable with the target base sequence, (b) a step for reacting the nucleic acid aptamer with the ligand in the presence of a compound having ≥1.8 dipole moment in the reaction solution, and (c) a step for detecting the target base sequence by using the bonding affinity of the nucleic acid aptamer with the ligand as an index. <P>COPYRIGHT: (C)2004,JPO&NCIPI

Description

【００６２】
ポジティブ標的塩基配列又はネガティブ標的塩基配列により形成されるアプタマーをそれぞれ図１Ａ及びＢに示す。ポジティブ標的塩基配列の場合にはＳＮＰの塩基においてプローブとの結合が起こり、このスリーウェイジャンクション構造にリガンドは結合することができる。一方、ネガティブ標的塩基配列の場合にはＳＮＰの塩基においてプローブとの結合がおこらず、リガンドは結合することはできない。従って、ポジティブ標的塩基配列とネガティブ標的塩基配列とではリガンドの結合親和性が異なるはずである。表１から明らかなように、ポジティブ配列又はネガティブ配列を用いた場合に、核酸アプタマーとリガンドとの結合親和性の変化が、リガンドを標識した蛍光の偏光度の差により証明された。すなわち、一塩基の違い（ポジティブ配列とネガティブ配列の差）により、核酸アプタマーとリガンドとの結合親和性が変化することが示された。また、偏光度の差異は極性物質の添加量に依存することが確認され、極性物質の存在下では核酸アプタマーとリガンドとの結合親和性の検出感度が向上することが示された。極性物質としてＴＭＡＦを用いた場合の添加量の最適領域は、表１に示すように、約１〜３％と評価される。表１のデータから逆算すると、極性物質を添加剤として試薬に加えることにより感度が２桁以上向上することが示された。
【００６３】
【発明の効果】
本発明により、アプタマー法において、標的塩基配列を高感度に特異的に検出するための方法、並びに検出感度増強剤及び検出用キットが提供される。本発明では、微量の標的塩基配列に対しても高感度で検出を行うことができるため、あらゆる試料中の標的塩基配列の検出に有用である。
【００６４】
【配列表】

【００６５】
【配列表フリーテキスト】
配列番号１〜４：合成オリゴヌクレオチド
【図面の簡単な説明】
【図１】標的塩基配列とプローブとにより形成されるアプタマーの構造を示す図である。Ａはポジティブ標的塩基配列の場合、Ｂはネガティブ標的塩基配列の場合のアプタマーの構造を示す。
【図２】双極子モーメントが大きい化合物としてテトラメチルアンモニウムフルオリド（ＴＭＡＦ）を種々の濃度（％）で添加した場合の、アプタマーとリガンドとの結合親和性の変化（偏光度差）を示すグラフである。[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a method for specifically detecting a specific base sequence (hereinafter, referred to as a target base sequence) present in a sample with high sensitivity, and a detection sensitivity enhancing reagent and a detection kit used for the method.
[0002]
[Prior art]
As a method for detecting a target base sequence, there is a method utilizing hybridization between complementary base sequences represented by the Southern hybridization method. However, a nucleic acid having a complementary base sequence causes hybridization even if both base sequences are not completely complementary, so that a single nucleotide such as a single nucleotide polymorphism (hereinafter, referred to as SNP) is generated. Cannot be directly detected by the method using hybridization. As a SNP detection method, for example, a PCR-SSCP method or the like is known, but it is not suitable for processing a large amount of a sample because electrophoresis is required.
[0003]
On the other hand, there is a small nucleic acid molecule having a specific structure called an “aptamer” that specifically binds to a specific biological substance (ligand) such as a protein based on a principle different from a complementary base sequence. Aptamer search is performed by a technique called SELEX (Systematic Evolution of Ligands by Exponential Enrichment) that selects a larger nucleic acid molecule in vitro by utilizing affinity with a ligand (Non-Patent Document 1). In the SELEX method, a library of RNA having a random base sequence is brought into contact with a ligand, the substance bound to the ligand is recovered, amplified by the RT-PCR method, and the RNA using the amplified and purified product as a template is transcribed and re-transferred. This is a method for obtaining a nucleic acid having a high binding affinity to a certain ligand by repeating the step of contacting the ligand. In addition, as a completely new approach to the detection of target nucleic acids, a method of detecting a target nucleotide sequence using the binding affinity between a nucleic acid aptamer and a ligand without using hybridization between complementary nucleotide sequences so far ( Hereinafter, referred to as the “aptamer method”) (see Patent Document 1). The aptamer method is a method of forming a nucleic acid aptamer containing a probe capable of hybridizing to a target base sequence and detecting the target base sequence using the binding affinity between the nucleic acid aptamer and a ligand as an index. The magnitude of the binding affinity between a nucleic acid aptamer and a ligand depends on the base sequence that maintains the structure of the nucleic acid aptamer, and even a single nucleotide substitution in the base sequence greatly changes the binding affinity. Then, mutation such as SNP in the target base sequence can be detected. As described above, in the aptamer method, detection is performed using the binding affinity between a nucleic acid aptamer and a ligand as an index, but in order to reduce the required amount of a sample to be detected, that is, it is possible to detect a very small amount of a sample. Therefore, it is required to further increase the binding affinity between the nucleic acid aptamer and the ligand.
[0004]
[Patent Document 1]
International Publication Number WO01 / 44509 A1
[Non-patent document 1]
Bock, L .; C. et al. , Nature (UK), 1992, 355 (6360), p. 564-566
[0005]
[Problems to be solved by the invention]
Accordingly, the present invention provides a method for detecting a target base sequence, a detection sensitivity enhancement reagent and a detection kit, which can enhance the binding affinity between a nucleic acid aptamer and a ligand and enable detection even at a low concentration of the target base sequence in a sample. The task is to provide.
[0006]
[Means for Solving the Problems]
One of the binding modes of a nucleic acid aptamer and a ligand in the aptamer method for detecting a target base sequence is based on a hydrophobic interaction, and the strength of the hydrophobic interaction is considered to depend on the surrounding environment. In particular, it strongly depends on the polarity of the solvent. The lower the polarity of the solvent, the weaker the hydrophobic interaction, and the higher the polarity, the higher the hydrophobic interaction. By adding a polar substance having a high molecular polarity (compound having a hyperbolic moment of 1.8 or more) to the reaction solution, the polarity around the binding site between the nucleic acid aptamer and the ligand is increased, and the hydrophobic interaction is increased. It has been found that the stronger the action, the higher the binding affinity and the higher the detection sensitivity. The present invention has been completed based on such findings.
[0007]
That is, the present invention is a method for detecting a target base sequence in a test sample, and the detection method includes the following steps.
(A) mixing a test sample with a probe capable of hybridizing with a target base sequence to form a nucleic acid aptamer in which the target base sequence and the probe in the test sample have hybridized;
(B) reacting the nucleic acid aptamer and the ligand in a reaction solution in the presence of a compound having a dipole moment of 1.8 or more; and
(C) detecting a target base sequence using the binding affinity between the nucleic acid aptamer and the ligand as an index.
[0008]
In the above detection method, as the compound having a dipole moment of 1.8 or more in the reaction solution, for example, tetraalkylammonium fluoride (TMAF), tetraethylammonium fluoride (TEAF) , Tetrabutylammonium fluoride (TBAF), and tetraoctylammonium fluoride (TOAF). In the above detection method, examples of the target base sequence include those containing a single nucleotide polymorphism (SNP). Further, it is preferable to use a compound having a steroid skeleton as the ligand.
[0009]
In the above detection method, the detection is performed, for example, by a change in a signal generated from a labeling substance that labels the ligand. Here, it is preferable to use a fluorescent molecule as the labeling substance, in which case the polarization of the fluorescence is detected as a change in the signal.
[0010]
The present invention is also a detection sensitivity enhancing reagent comprising a compound having a dipole moment of 1.8 or more in a solution.
[0011]
Regarding the detection sensitivity enhancing reagent, examples of the compound having a dipole moment of 1.8 or more in an aqueous solution include tetraalkylammonium fluoride, such as tetramethylammonium fluoride (TMAF) and tetraethylammonium fluoride (TEAF). ), Tetrabutylammonium fluoride (TBAF), and tetraoctylammonium fluoride (TOAF).
[0012]
Further, the present invention is a kit for detecting a target base sequence, comprising a nucleic acid probe, a ligand, and the above-mentioned detection sensitivity enhancing reagent.
[0013]
In the above detection kit, the ligand is, for example, a compound having a steroid skeleton, and is preferably labeled with a fluorescent molecule.
[0014]
BEST MODE FOR CARRYING OUT THE INVENTION
Hereinafter, the present invention will be described in detail.
[0015]
The present invention is based on the finding that the binding affinity between a nucleic acid aptamer and a ligand is enhanced in the presence of a compound having a large dipole moment (ie, a compound having a large polarity). Therefore, the present invention is useful in various methods involving the binding reaction between such a nucleic acid aptamer and a ligand, but typically, the target base sequence is determined using the binding affinity between the nucleic acid aptamer and the ligand as an index. It can be suitably applied to a detection method (aptamer method).
[0016]
Hereinafter, the method for detecting a target base sequence of the present invention, and the detection sensitivity enhancing reagent and kit will be described.
[0017]
1. Target nucleotide sequence detection method
The method for detecting a target nucleotide sequence of the present invention is based on the aptamer method, and determines the binding affinity between a target nucleotide sequence in a test sample and a nucleic acid aptamer formed by a probe capable of hybridizing to the nucleotide sequence with a ligand. A method for detecting the target base sequence as an indicator, characterized in that a compound having a dipole moment of 1.8 or more in the reaction solution is added to the reaction solution. The aptamer method is a phenomenon in which the structure of an aptamer formed by a specific base sequence and a probe changes depending on whether or not the target base sequence is included, which also changes the binding affinity between the aptamer and the ligand. Is used to detect a target base sequence. A method for detecting a target base sequence using such an aptamer method is known, and for details, refer to Patent Document 1, for example.
[0018]
Therefore, the method for detecting a target base sequence of the present invention includes, for example, the following steps:
(A) mixing a test sample with a probe capable of hybridizing with a target base sequence to form a nucleic acid aptamer in which the target base sequence and the probe in the test sample have hybridized;
(B) reacting the nucleic acid aptamer and the ligand in a reaction solution in the presence of a compound having a dipole moment of 1.8 or more; and
(C) detecting a target base sequence using the binding affinity between the nucleic acid aptamer and the ligand as an index.
[0019]
In the present invention, “nucleic acid aptamer” or “aptamer” is a nucleic acid having a binding activity to a specific ligand, and has at least one set of complementary base sequences within a molecule or between molecules. Refers to nucleic acid molecules made up by sequence hybridization. The nucleic acid aptamer having a suitable structure in the present invention includes, for example, a double-stranded portion (hybrid) formed by hybridization of the complementary base sequence, and a single-stranded portion that does not form a hybrid. Those having a higher-order structure unique to the aptamer in which the moiety is formed by hydrogen bonding, stacking, hydrophobic interaction, and the like. Such higher-order structures include, in addition to generally known stem-loop, stem-bulge, and pseudoknot structures, the features of both the stem-loop and stem-bulge described in Patent Document 1. It refers to a stem-junction structure (such as a three-way junction) and the like, and the nucleic acid aptamer in the present invention is not particularly limited as long as it has these higher-order structures, but a nucleic acid aptamer having a three-way junction structure is preferable.
[0020]
This conformation is important for the binding affinity of the nucleic acid aptamer to the ligand, and under conditions that do not conform to the nucleic acid aptamer (ie, do not form a nucleic acid hybrid), the binding affinity is lost.
[0021]
On the other hand, “ligand” refers to any component other than nucleic acid bound by the higher-order structure of a nucleic acid aptamer. To date, proteins and various low molecular weight compounds having binding affinity to nucleic acid aptamers have been reported. For example, amino acids (Proc. Natl. Acad. Sci. USA 90, 3680-3684, 1993), nucleotides (Nature 364, 550-553, 1993; Biochemistry 34, 656-665, 1995), coenzyme A (Biochemistry, 53). 4663, 1998), theophylline (Science 263, 1425-1429, 1994), aminoglycoside antibiotics (Biochemistry 35, 12338-12346, 1996), organic dyes (Nature 355, 850-852, 19992), porphyrin derivatives (Biochem 35). , 6911-6922, 1996), and cholic acid (Patent Document 1).
[0022]
In step (a), a test sample and a probe capable of hybridizing with a target base sequence are mixed. Thereby, when the target base sequence is present in the test sample, a nucleic acid aptamer in which the target base sequence is hybridized with the probe is formed.
[0023]
In the present invention, the “test sample” refers to a sample for which it is desired to determine whether or not a target base sequence is present, and is typically a biological sample (eg, whole blood, plasma, serum, urine, body fluid, etc.). , Saliva, etc.) and animal or plant cells or tissue cultures as long as they contain nucleic acids. The test sample can be prepared by appropriately using a method well known in the art. Generally, a test sample is prepared by extracting a nucleic acid (DNA or RNA). For example, when extracting DNA, a method of performing phenol extraction and ethanol precipitation, a method using glass beads, etc., and when extracting RNA, a guanidine-cesium chloride ultracentrifugation method, a hot phenol method, or an AGPC method is used. Etc. can be used.
[0024]
The term “target base sequence” refers to a base sequence containing a specific sequence for which detection is desired, and specific sequences for which such detection is desired include, for example, mutant sequences, polymorphic sequences, and the like. "Polymorphism" refers to the presence of one or more forms of a gene or a part thereof at the same time, and a portion of a gene in which at least two different forms, that is, two different base sequences are present, is defined as a polymorphic region. Called. Polymorphic regions include, for example, genetic polymorphisms such as single nucleotide polymorphisms (SNPs), which differ in various alleles. In the present invention, the target base sequence is preferably a sequence containing a single nucleotide polymorphism (SNP).
[0025]
In the present invention, “probe” or “nucleic acid probe” refers to a nucleic acid that can hybridize with a target base sequence contained in a test sample. In the present invention, “hybridize” or “hybridization” refers to specific binding of two or more nucleic acids based on their complementarity. Therefore, a “hybridizable probe” can be said to be a nucleic acid having a base sequence complementary to the target base sequence. "Complementary" or "complementarity" is a concept used for nucleic acids having a base-pairing relationship, and complementarity is defined as "partial", that is, only a certain number of bases in a nucleic acid are paired. It may be such that they match according to a rule, or the whole may be completely complementary. Other nucleic acid analogs (eg, IsoC / IsoG base pairs) or nucleic acid analogs used to base pair with natural nucleic acids are also included within the above definitions of “complementary” and “complementary”. Thus, it can be said to be complementary to the base-pairing partner.
[0026]
The probe is designed so that the structure of the nucleic acid aptamer formed by the target base sequence and the probe changes depending on the presence or absence of a specific sequence of the target base sequence, and the binding affinity with the ligand changes. For example, when the structure of the nucleic acid aptamer to be formed is a three-way junction structure, it is necessary that all three base pair bonds at the junction are completely complementary. Those skilled in the art can design an appropriate probe in consideration of the type of the target base sequence and the aptamer structure.
[0027]
For example, the design of a probe in the case where the target base sequence contains an SNP and the nucleic acid aptamer to be formed has a three-way junction structure will be briefly described with reference to FIG. Three-way junction refers to a structure in which three nucleic acid strands form a double strand with each other, resulting in three double-stranded nucleic acids interleaving at one site (FIG. 1A). That is, three stems (double strands) intersect at one place, and three complementary base pairs of six bases constitute a three-way junction. In the detection of the target base sequence, an aptamer structure (ie, a three-way junction structure) is formed by using one of the three nucleic acid strands as the target base sequence and two as probes. Then, when the binding of the complementary base pairs at the junction portion is completely complementary to all three, the ligand can bind to the three-way junction structure. Therefore, the probe is designed so that the binding of the complementary base pair at the junction is completely complementary.
[0028]
As shown in FIG. 1A, when an SNP (base G) of a positive target base sequence is to be detected, a probe A and a probe a that are complementary to the target base sequence are designed. At this time, it should be noted that the base G corresponding to the SNP in the target base sequence constitutes a junction part of the three-way junction, and that all the junctions are completely complementary. Probe A is composed of a portion complementary to the target base sequence and a portion complementary to probe a. Probe a is composed of a part complementary to the target base sequence and a part complementary to probe A. The probe A and the probe a may be separate nucleic acid molecules, or may be, for example, a single nucleic acid molecule having a hairpin structure. Those skilled in the art can determine, based on the base sequence of the target base sequence, the base sequence of a probe that can hybridize under specific conditions manually or by using known probe design software. .
[0029]
When the probe designed as described above is brought into contact with the target base sequence, they hybridize to form the aptamer structure shown in FIG. 1A. The junction of the aptamer structure binds with perfect complementarity, and the ligand can bind to this structure because the higher order structure of the aptamer is retained in a specific structure. On the other hand, a base sequence (negative base sequence) having a SNP (base C) different from the target SNP (base G) has an aptamer structure different from the aptamer structure formed by the target base sequence, as shown in FIG. 1B. To form That is, at the junction of the three-way junction structure, the base C corresponding to the SNP is not bonded to the probe due to complementarity. The binding affinity of the ligand for such aptamer structure differs from that of FIG. 1A above, and the affinity is low.
[0030]
In addition, the length (size) of the designed probe must be appropriately changed according to the type of the target base sequence, the type of the nucleic acid aptamer structure, the hybridization conditions, and the like. For example, when the target base sequence contains an SNP and two probes are designed so that the nucleic acid aptamer takes a three-way junction structure, the length of each probe is 8 to 60 bases, preferably 16 to 30 bases. It can be base length.
[0031]
Whether or not the designed probe appropriately hybridizes to the target nucleotide sequence can be confirmed using known probe design software. Examples of such software include GENETYX (manufactured by Genetics). Is mentioned.
[0032]
The probe may be any nucleic acid molecule or a derivative thereof as long as it can hybridize with the target base sequence to form a nucleic acid aptamer. Specifically, nucleic acid molecules include nucleic acid molecules isolated from natural sources (eg, DNA, RNA, etc.) or chemically synthesized nucleic acid molecules (eg, amplification products, etc.), derivatives of nucleic acids including synthetic nucleotide derivatives, Alternatively, it may be a derivative of a nucleic acid whose skeleton is composed of a peptide bond or an alkyl chain. Probes can be generated by any technique known in the art, for example, chemical synthesis, DNA replication, reverse transcription, PCR, or a combination thereof.
[0033]
The method of mixing the test sample and the probe prepared as described above is not particularly limited. For example, a method of adding a test sample to a solution containing a probe or a method of mixing the test sample with a solution containing a probe can be used. The mixing condition is not particularly limited as long as the target base sequence in the test sample and the probe can hybridize. Hybridization depends on the complementarity between the nucleic acids, the stringency of the conditions employed, and the T of the hybrid formed. _m The hybridization conditions are appropriately determined in consideration of these factors. “Stringency” is a concept used for conditions such as temperature, ionic strength, and the presence of other compounds when performing hybridization. Under "high stringency" conditions, base pairing occurs only between nucleic acids consisting of sequences having a high frequency of complementary bases. For example, when it is necessary to detect a single base difference such as SNP, the “high stringency condition” is adopted. Hybridization conditions, including stringency, and their determination are well known in the art and are described in general experimental protocols.
[0034]
As described above, the nucleic acid aptamer is formed by mixing the test sample and the probe and hybridizing the probe with the target base sequence in the test sample.
[0035]
In step (b), the nucleic acid aptamer and the ligand are reacted in a reaction solution in the presence of a compound having a dipole moment of 1.8 or more.
[0036]
Although the present invention is not limited to a particular principle or mechanism, the present inventors speculate on one of the following reaction mechanisms. The binding mode between the nucleic acid aptamer and the ligand in the aptamer method is based on a hydrophobic interaction, and the strength of the hydrophobic interaction is considered to depend on the surrounding environment. In particular, it strongly depends on the polarity of the solvent. The lower the polarity of the solvent, the weaker the hydrophobic interaction, and the higher the polarity, the higher the hydrophobic interaction. Therefore, the present inventor has found that by adding a polar substance having a high molecular polarity to a sample solution, the polarity around the binding site between the nucleic acid aptamer and the ligand is increased, and the binding affinity is increased by increasing the hydrophobic interaction. Guessed to improve.
[0037]
The polarity strength of a polar substance can generally be represented by a dipole moment. A "dipole" is said to have a dipole if the centers of gravity of the positive and negative charges in an atom or molecule do not match. The dipole moment of a polar substance is defined as follows. First, the quantity of electricity equal to the center of gravity of each of the positive and negative charges in the material molecule is calculated, and the product (ql) of the distance l between the centers of gravity (unit: strong ohm) and the quantity of electricity ± q (unit: coulomb) is calculated. The value obtained is the dipole moment (the unit is Debye). Therefore, a substance having no polarity has a dipole moment of zero. In general, the position of the center of gravity of positive and negative charges and the quantity of electricity can be obtained by calculating the atomic arrangement and electronic state of a substance molecule by a semi-empirical electron orbital method. When this calculation method is applied, for example, the dipole moment of water is obtained as 1.860 Debye, which is in good agreement with the measured value of 1.855 Debye. However, the above calculation method of the dipole moment is only an approximate value that is predicted, and when a truly accurate value is required, the dielectric constant of the substance is measured by experiment, and the exact dipole moment of the substance is calculated from the dielectric constant. It is possible to determine the value.
[0038]
In the present invention, a compound having a large dipole moment in a solution for reacting a nucleic acid aptamer with a ligand can be used. As such a compound, a compound having a larger dipole moment in the solution than in the solution is preferable. Since the dipole moment of the solution in which the nucleic acid aptamer is reacted with the ligand is considered to be almost equal to that of water, the dipole moment of the compound preferred in the present invention is about 1.8 or more, more preferably about 2 0.0 to about 6.0. In this specification, the terms “compound having a dipole moment”, “compound having a large dipole moment”, and “polar compound” all indicate a compound having a dipole moment of about 1.8 or more in a solution.
[0039]
Compounds having a large dipole moment include those shown below, and the measured value of the dipole moment is shown in parentheses: tetraalkylammonium fluoride (tetramethylammonium fluoride (TMAF), tetraethylammonium fluoride (TEAF)) , Tetrabutylammonium fluoride (TBAF), tetraoctylammonium fluoride (TOAF), dimethyl sulfone (4.43), pyridazine (4.22), nitrobenzene (4.21), β-propiolactone (4). .18), benzonitrile (4.14), cis-2-butenenitrile (4.08), propionitrile (4.02), acetonitrile (3.93), acrylonitrile (3.89), methyl isocyanide (3.83), malononitrile (3.7 ), Cyanoacetylene (3.72), formamide (3.71), nitroethylene (3.70), nitromethane (3.46), pyrvonitrile (3.45), cyclopentanone (3.30), 1, 3,4-thiadiazole (3.28), s-trans-formylcyclopropane (3.26), methyl isocyanate (3.18), methyl thiocyanate (3.16), acrylaldehyde (3.12), Methyl nitrate (3.08), acetophenone (3.00), acetyl fluoride (2.96), isoxazole (2.95), camphor (2.95), acetone (2.90), benzaldehyde (2.9%). 76), acetaldehyde (2.75), s-cis-formylcyclopropane (2.74), isoquinoline (2.53), propionaldehyde Hyd (2.46), cis-1,2-difluoroethylene (2.42), 1,1,1-trifluoroethane (2.35), pyrimidine (2.33), formaldehyde (2.33), Trifluoroacetic acid (2.3), quinoline (2.22), t-butyl bromide (2.21), pyridine (2.15), propyl bromide (2.13), t-butyl chloride (2.2). 12), propyl chloride (2.10), formyl fluoride (2.08), indole (2.08), chlorocyclopentane (2.08), 1,3,5-trioxane (2.08), chloride Ethyl (2.05), trans-propyl fluoride (2.05), ethyl bromide (2.01), propyl iodide (2.01), propylene oxide (2.00), trans-ethyl formate (1 .98), difluorometa (1.98), ethyl fluoride (1.96), methylthiirane (1.95), oxetane (1.93), gauche-propyl fluoride (1.90), methyl chloride (1.89), aziridine (1.89), cis-1,2-dichloroethylene (1.89), ethylene oxide (1.88), methyl fluoride (1.86).
[0040]
As the compound having a dipole moment, it is preferable to use tetraalkylammonium fluoride (TMAF, TEAF, TBAF, TOAF, etc.), dimethyl sulfoxide (DMSO), methanol, polyethylene glycol (PEG), and glycerin.
[0041]
In the method for detecting a target base sequence of the present invention, the form of use of the compound having a dipole moment is not particularly limited as long as the compound is present in the reaction solution at the time of reacting the nucleic acid aptamer with the ligand. For example, a compound having a dipole moment can be added to the reaction solution before, simultaneously with, or after the addition of the ligand to the reaction solution containing the nucleic acid aptamer. Alternatively, step (b) can be performed before, simultaneously with, or after step (a), in which a dipole moment is added to the reaction solution in advance, and then the nucleic acid aptamer formation of step (a) is performed. To react the nucleic acid aptamer with the ligand. Further, a reaction solution may be prepared by mixing together a sample containing a target base sequence, a probe, a ligand, and a compound having a dipole moment.
[0042]
The optimal amount of the compound having a dipole moment can be determined, for example, by measuring the strength of the binding affinity in the presence of various concentrations of the compound, that is, the dependence of the binding affinity on the concentration of the compound. it can.
[0043]
The ligand to be reacted is not particularly limited as long as it is a component other than nucleic acid that is bound by the higher-order structure of the nucleic acid aptamer as described above, and any ligand can be used. In the present invention, it is preferable to use cholic acid, which is known to suitably bind to a nucleic acid aptamer having a three-way junction structure, and a compound having the same steroid skeleton as the structure of cholic acid. Such compounds having a steroid skeleton include, but are not limited to, cholic acid and derivatives thereof, such as lithocholic acid, chenodeoxycholic acid, cholic acid methyl ester, and lithocholic acid methyl ester, and other compounds having a steroid skeleton. For example, 5β-pregnane-3α-ol-20-one, trans-androsterone and the like can be mentioned.
[0044]
The amount of the ligand can be appropriately determined in consideration of the amount of the nucleic acid aptamer to be reacted. Further, the ligand may be labeled with a labeling substance for detecting a target base sequence using the binding affinity to a nucleic acid aptamer described later as an index. Such labeling substances include fluorescent molecules (fluorescein (FITC), sulforhodamine (TR), tetramethylrhodamine (TRITC), etc.), luminescent substances (luciferin, etc.), enzymes (alkaline phosphatase, etc.), complex systems (biotin- Avidin type).
[0045]
By reacting the nucleic acid aptamer with the ligand in the presence of a compound having a dipole moment of 1.8 or more in the reaction solution, the binding between them is enhanced, and a change in the binding affinity described below can be favorably observed. become.
[0046]
After step (a) and step (b), the target base sequence is detected using the binding affinity between the nucleic acid aptamer and the ligand as an index (step (c)). In the detection method of the present invention, the hybridization between the target base sequence and the probe, that is, the target base sequence can be detected by using the binding affinity between the nucleic acid aptamer and the ligand as an index. In the present invention, “detection” refers to determining whether or not a target base sequence is present in a test sample, and in some cases, the amount (or ratio) of the target base sequence contained in the test sample. Also refers to measuring
[0047]
To detect a change in the binding affinity between the nucleic acid aptamer and the ligand, any known binding analysis principle can be applied. Examples thereof include, but are not limited to, a heterogeneous system, a homogeneous system, and surface plasmon resonance. Is mentioned.
[0048]
In a heterogeneous system, a nucleic acid aptamer resulting from hybridization of a labeled probe to a target base sequence can be captured by a ligand immobilized on a solid phase. By detecting the labeled probe captured on the solid phase (or remaining in the liquid phase), a change in the binding affinity between the aptamer and the ligand, that is, the target base sequence can be detected.
[0049]
In a homogeneous system, a phenomenon called fluorescence polarization caused by the binding of a fluorescent molecule to another molecule can be used. Specifically, the fluorescence polarization is obtained by labeling a probe or ligand with a fluorescent molecule and measuring the fluorescence polarization resulting from the binding between the nucleic acid aptamer and the ligand to detect a change in the binding affinity, that is, the target base sequence. Can be detected. As the fluorescent molecule, any fluorescent molecule known in the art can be used, such as fluorescein isothiocyanate. When the ligand is a compound that emits fluorescence by itself (for example, a porphyrin derivative), the binding to the aptamer can be detected using the fluorescence.
[0050]
Surface plasmon resonance (SPR) is a method that can directly detect the binding of a substance optically, and is well known in the art. A surface plasmon resonance sensor is known as a technique for measuring the interaction of biomolecules by measuring a change in the refractive index of a medium near a metal thin film. SPR occurs when surface plasmons are excited at the metal / liquid interface (sensor surface), and irradiates light to the side of the surface that is not in contact with the sample, and reflects light from it, which gives the SPR a specific combination. The reflected light intensity decreases with the angle and wavelength (SPR signal generation). Molecules bound to the sensor surface cause a change in the refractive index near the surface layer, which is detected as a change in the SPR signal. Further changes in the refractive index and the SPR signal are caused by the interaction of the biomolecules, and by detecting the change in the signal, the interaction of the biomolecules can be measured. In the present invention, for example, by immobilizing a ligand captured by a nucleic acid aptamer on a chip, the amount (binding amount) of a nucleic acid aptamer formed by hybridization between a target base sequence and a probe can be changed by changing the SPR signal. Can be measured from By using SPR, detection can be performed without using a labeling substance.
[0051]
The effect of the compound having the above-mentioned dipole moment will be specifically described with reference to a preferred embodiment of the present invention. FIG. 2 shows that a nucleic acid aptamer containing a target base sequence or a nucleic acid aptamer containing a base sequence having a difference of one base from the target base sequence was treated with cholic acid labeled with a fluorescent molecule (fluorescein isothiocyanate: FITC) in the presence of TMAF at different concentrations. The figure shows the difference in the degree of fluorescence polarization between an aptamer containing a target base sequence and a nucleic acid aptamer containing a base sequence that differs from the target base sequence by one base at each TMAF concentration by measuring the polarization degrees of the fluorescent molecules. It is. The degree of polarization of the fluorescent molecule tends to depend on the molecular weight of the substance to which the fluorescent molecule is bound. When the value of the degree of polarization is large, it means that the fluorescent molecule is bound to a substance having a large molecular weight. Therefore, when the ligand to which the fluorescent molecule is bound binds to a nucleic acid aptamer having a three-way junction structure, the degree of polarization of the fluorescent molecule increases because the molecular weight of the nucleic acid aptamer is sufficiently larger than the molecular weight of the ligand. Become. On the other hand, when the nucleic acid aptamer does not have a three-way junction structure and the ligand does not bind, the change in the degree of polarization of the fluorescent molecule is small. As shown in FIG. 2, the difference in the degree of fluorescence polarization between a nucleic acid aptamer containing a target base sequence and a nucleic acid aptamer containing a base sequence having a difference of one base from the target base sequence is greatly exhibited by the addition of TMAF. That is, according to the present invention, it is possible to detect a change in the binding affinity between a nucleic acid aptamer and a ligand caused by a mutation (for example, a single base substitution) in a target base sequence as a remarkable difference in the degree of fluorescence polarization. .
[0052]
2. Detection sensitivity enhancement reagent
The compound having a dipole moment of 1.8 or more in the above-mentioned binding reaction solution between the nucleic acid aptamer and the ligand can be prepared by using the compound as it is or as a solution containing the compound or in the form of a buffer solution. It can be provided as a reagent for enhancing the binding affinity and enhancing the detection sensitivity of the change in the binding affinity.
[0053]
Further, the detection sensitivity enhancing reagent of the present invention can be applied not only to the method for detecting a target base sequence described in detail in the present specification, but also to various methods involving a binding reaction between a nucleic acid aptamer and a ligand. For example, in the above-mentioned SELEX method, the detection sensitivity enhancing reagent of the present invention can be used when screening a nucleic acid aptamer having a high binding affinity for a specific ligand. By performing the reaction between the ligand and the nucleic acid aptamer in the presence of the detection sensitivity enhancing reagent, the binding affinity at the time of screening can be measured with high sensitivity. In addition, by using the detection sensitivity enhancing reagent of the present invention, even when a small amount of a ligand or a nucleic acid aptamer is used, its binding affinity can be detected with high sensitivity.
[0054]
3. Kit for detection of target nucleotide sequence
The detection kit of the present invention is characterized by including a nucleic acid probe, a ligand, and a detection sensitivity enhancing reagent. The present detection kit is useful for detecting a target nucleotide sequence using the aptamer method.
[0055]
"Kit" refers to a supply system for supplying a substance, and is a system that can store, transport, or supply a reaction reagent and / or an auxiliary substance contained in a container. In the detection kit of the present invention, the reaction reagent includes a nucleic acid probe, a ligand, and a detection sensitivity enhancing reagent. The auxiliary substance may include, but is not limited to, a labeling substance, a buffer, an instruction manual, and the like. By using the detection kit of the present invention, the target base sequence can be detected very easily.
[0056]
【Example】
Hereinafter, the present invention will be described more specifically with reference to examples. However, the technical scope of the present invention is not limited to the following examples.
[0057]
Example 1 Measurement of Binding Affinity of DNA Aptamer and Ligand in the Presence of Compound with Large Dipole Moment by Fluorescence Polarization
In this example, changes in the binding between a ligand and an aptamer formed by a target base sequence and a probe in the presence of a compound having a large dipole moment were examined. That is, a condition in which a nucleic acid aptamer containing a three-way junction structure is formed by the following positive or negative target base sequence (the underlined portion corresponds to SNP) and the following two molecules of a probe, and a compound having a large dipole moment is added: Below, the difference in the binding affinity between the nucleic acid aptamer and the ligand when using the positive target base sequence and the negative target base sequence was observed by measuring the degree of fluorescence polarization. The purpose of this example was to detect SNPs contained in the positive target base sequence.
[0058]
Positive target base sequence (for positive control): 5'-CCTAGCAGC G GGAGCGGTGG-3 ′ (SEQ ID NO: 1)
Negative target base sequence (for negative control): 5'-CCTAGCAGC C GGAGCGGTGG-3 ′ (SEQ ID NO: 2)
Probe a: 5′-CCACCGCTCCACTCAACTGG-3 ′ (SEQ ID NO: 3)
Probe A: 5'-CCAGTTGAGTCGCTGCTAGG-3 '(SEQ ID NO: 4)
[0059]
Briefly describing the operation, a buffer (composition: pH = 7.6, 50 mM Tris-HCl, 1M) containing 40 nM each of the above-mentioned positive target base sequence or negative target base sequence and DNA of three molecules of probe a and probe A was used. NaCl, 30 mM KCl, 5 mM MgCl ₂ A) A solution was prepared, the DNA was denatured at 95 ° C. for 5 minutes, and gradually cooled to 20 ° C. for 30 minutes to form an aptamer having a three-way junction structure (FIG. 1). To a DNA solution containing 40 nM of the nucleic acid aptamer, 1 nM of a fluorescein isothiocyanate (FITC) -labeled ligand and 0, 0.1, 0.3, 1.0 of tetramethylammonium fluoride (TMAF; manufactured by Wako) as a polar substance were added. The final DNA concentration was 20 nM, in addition to a concentration of 3.0, 10.0 or 20.0% (w / v). When the fluorescence polarization of 100 μl of the mixed solution was measured, the results shown in Table 1 below were obtained.
[0060]
The FITC labeling of the ligand was performed as follows. First, 1 mmol of cholic acid (manufactured by Wako) or lithocholic acid (manufactured by Wako) is mixed with 1 mmol of tert-butyl N- (3-aminopropyl) carbamide and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC). ) 2 mmol, 2 mmol of 1-hydroxybenzotriazole, and 0.2 ml of diisopropylethylamine, and stirred at room temperature for 24 hours. The mixture was extracted with ethyl acetate / 1N sodium hydroxide, and the organic layer was washed with 1N hydrochloric acid and dried. The obtained organic layer was dissolved in methylene chloride and trifluoroacetic acid and stirred for 1 hour. After evaporating the solvent, the mixture was extracted with ethyl acetate / 1N sodium hydroxide, and the organic layer was concentrated to dryness. After purification, the crude product was dissolved in acetonitrile in an equimolar amount to FITC (manufactured by Molecular Probes), stirred for 1 hour, and concentrated to dryness to obtain a FITC-labeled ligand.
[0061]
[Table 1]

[0062]
Aptamers formed by positive or negative target nucleotide sequences are shown in FIGS. 1A and 1B, respectively. In the case of a positive target base sequence, binding to the probe occurs at the base of the SNP, and a ligand can bind to this three-way junction structure. On the other hand, in the case of a negative target base sequence, the base does not bind to the probe at the SNP base, and the ligand cannot bind. Therefore, the binding affinity of the ligand should be different between the positive target base sequence and the negative target base sequence. As is clear from Table 1, when the positive sequence or the negative sequence was used, the change in the binding affinity between the nucleic acid aptamer and the ligand was proved by the difference in the degree of polarization of the fluorescence labeled with the ligand. That is, it was shown that the difference in single base (difference between the positive sequence and the negative sequence) changed the binding affinity between the nucleic acid aptamer and the ligand. In addition, it was confirmed that the difference in the degree of polarization was dependent on the amount of the polar substance added, and it was shown that the detection sensitivity of the binding affinity between the nucleic acid aptamer and the ligand was improved in the presence of the polar substance. As shown in Table 1, the optimum range of the addition amount when TMAF is used as the polar substance is estimated to be about 1 to 3%. Back calculation from the data in Table 1 shows that adding a polar substance to the reagent as an additive improves sensitivity by more than two orders of magnitude.
[0063]
【The invention's effect】
According to the present invention, a method for specifically detecting a target base sequence with high sensitivity in an aptamer method, and a detection sensitivity enhancer and a detection kit are provided. In the present invention, detection can be performed with high sensitivity even for a small amount of a target base sequence, so that it is useful for detecting a target base sequence in any sample.
[0064]
[Sequence list]

[0065]
[Sequence List Free Text]
SEQ ID NOS: 1-4: synthetic oligonucleotides
[Brief description of the drawings]
FIG. 1 is a diagram showing the structure of an aptamer formed by a target base sequence and a probe. A shows the structure of the aptamer in the case of the positive target base sequence, and B shows the structure of the aptamer in the case of the negative target base sequence.
FIG. 2 is a graph showing changes in binding affinity between aptamer and ligand (difference in polarization) when tetramethylammonium fluoride (TMAF) is added at various concentrations (%) as a compound having a large dipole moment. It is.

Claims (14)

A method for detecting a target base sequence in a test sample, comprising the following steps:
(A) mixing a test sample with a probe capable of hybridizing with a target base sequence to form a nucleic acid aptamer in which the target base sequence and the probe in the test sample have hybridized;
(B) reacting the nucleic acid aptamer and the ligand in a reaction solution in the presence of a compound having a dipole moment of 1.8 or more, and (c) indicating the binding affinity between the nucleic acid aptamer and the ligand Detecting the target base sequence as
The above-described detection method, comprising: The detection method according to claim 1, wherein the compound is a tetraalkylammonium fluoride. The tetraalkylammonium fluoride is selected from the group consisting of tetramethylammonium fluoride (TMAF), tetraethylammonium fluoride (TEAF), tetrabutylammonium fluoride (TBAF), and tetraoctylammonium fluoride (TOAF). The detection method according to claim 2, wherein The detection method according to any one of claims 1 to 3, wherein the target base sequence contains a single nucleotide polymorphism (SNP). The detection method according to any one of claims 1 to 4, wherein the ligand is a compound having a steroid skeleton. The detection method according to any one of claims 1 to 5, wherein the detection is performed by a change in a signal generated from a labeling substance that labels the ligand. The detection method according to claim 6, wherein the labeling substance is a fluorescent molecule. 7. The detection method according to claim 6, wherein the change in the signal is polarization of fluorescence. A detection sensitivity enhancing reagent comprising a compound having a dipole moment of 1.8 or more in a solution. The detection sensitivity enhancing reagent according to claim 9, wherein the compound is a tetraalkylammonium fluoride. The tetraalkylammonium fluoride is selected from the group consisting of tetramethylammonium fluoride (TMAF), tetraethylammonium fluoride (TEAF), tetrabutylammonium fluoride (TBAF), and tetraoctylammonium fluoride (TOAF). The detection sensitivity-enhancing reagent according to claim 10, which is a reagent. A kit for detecting a target base sequence, comprising a nucleic acid probe, a ligand, and the detection sensitivity enhancing reagent according to any one of claims 9 to 11. The detection kit according to claim 12, wherein the ligand is a compound having a steroid skeleton. The detection kit according to claim 12, wherein the ligand is labeled with a fluorescent molecule.

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