JP2007240282A - Nucleic acid molecule screening method and protein detecting/quantifying method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for efficiently obtaining a desired nucleic acid molecule from the nucleic acid molecules in a nucleic acid sample solution using a target substance stably. <P>SOLUTION: A target nucleic acid molecule is sorted by this nucleic acid molecule screening method including the step of fixing the fragmented peptide, which is obtained by subjecting protein including a selected peptide molecule, for example, to enzymatic treatment, on a support with the fixed fragmented peptide in a contact with the nucleic acid molecules in the nucleic acid sample solution while analysis utilizing the near field light on the surface of the support is performed to obtain the interaction amount of the fixed fragmented peptide with the nucleic acid molecules and the target nucleic acid molecule is sorted from the nucleic acid molecules in the nucleic acid sample solution. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a nucleic acid molecule screening method and a nucleic acid molecule screening apparatus, and in particular, screens a target nucleic acid molecule that is a nucleic acid or a nucleic acid derivative that specifically interacts with a selected peptide molecule from nucleic acid molecules in a nucleic acid sample solution. The present invention relates to a nucleic acid molecule screening method and a protein detection / quantification method using a nucleic acid molecule obtained thereby.

Nucleic acids and / or derivatives thereof that bind to specific proteins and peptides have been selected from the various candidates since 1990, and the binding nucleic acids and / or derivatives thereof are commonly used as aptamers. Has been called. Aptamers are not only able to bind to specific target substances like antibodies, but also have high affinity and are easier to produce than antibodies, so aptamers specific to specific physiologically active sites. Is expected to be used in the medical field. As methods for obtaining such aptamers, techniques such as separation using an affinity column and amplification by PCR are used (for example, Patent Document 1 and Patent Document 2).
As a method for evaluating the binding property between an aptamer, a specific protein, and a peptide, there is a method using near-field light such as surface plasmon resonance due to the necessity of labeling and high sensitivity (for example, Non-Patent Document 1). ). In addition, there is a method using near-field light, for example, near-field light fluorescence, from the viewpoint that background noise can be suppressed without being affected by other than the surface. (For example, Non-Patent Document 2) Since the binding property is evaluated particularly during the screening operation and only the aptamer having an arbitrary binding ability can be recovered based on the evaluation, it has the advantage of high screening efficiency. ing. (For example, Non-Patent Document 3)
JP 2001-61475 A JP 2005-245254 A Journal of Biological Chemistry (2001) Vol.276, pp.21476-21481 Analytical Chemistry, (1998) Vol.70, pp.3419-3425 Analytical Biochemistry, (2005) Vol.342, pp.312-317

  However, the method of screening only the protein itself or its specific partial sequence and screening its binding aptamer may not obtain the desired aptamer depending on the instability of the target protein or peptide. . In addition, when performing analysis using near-field light (hereinafter referred to as near-field light analysis), it is necessary to immobilize the target protein on the outermost surface of the measurement surface. Problems such as breakage due to insolation operation and immobilization due to insolubility occur.

  Therefore, the present invention is to provide a nucleic acid molecule screening method and protein detection / quantification method capable of efficiently obtaining a desired nucleic acid molecule from a nucleic acid molecule in a nucleic acid sample solution by stably using a target substance.

  The nucleic acid molecule screening method of the present invention is a nucleic acid molecule screening method for screening a target nucleic acid molecule that is a nucleic acid or a nucleic acid derivative that specifically interacts with a selected peptide molecule from nucleic acid molecules in a nucleic acid sample solution, A degradation treatment step of degrading a protein containing the peptide molecule to obtain a fragment peptide; an immobilization step of immobilizing the fragment peptide on a support to obtain an immobilized fragment peptide; and Obtaining the amount of interaction between the immobilized fragment peptide and the nucleic acid molecule by performing a contact step of bringing the nucleic acid molecule into contact with the immobilized fragment peptide and performing an analysis using near-field light on the surface of the support. A selection step of selecting the target nucleic acid molecule from the nucleic acid molecule in the nucleic acid sample solution based on the interaction amount obtained. .

Here, it is preferable that the decomposition treatment step is to decompose the protein using an enzyme.
The nucleic acid molecule screening method preferably further includes an amplification step of amplifying the target nucleic acid molecule selected in the selection step by a polymerase chain reaction (PCR) method.
When an amplification step is provided, the target nucleic acid molecule obtained by the amplification step is collected, and at least the contact step and the selection step, preferably the contact step, the selection step and the amplification step are repeated at least once. It is preferable.

In the nucleic acid molecule screening method, the immobilized fragment peptide may be composed of two or more fragment peptides obtained from the same protein, and the target nucleic acid molecule is composed of DNA, RNA, and derivatives thereof. It may be selected from a group.
In the nucleic acid molecule screening method, the near-field light analysis is preferably surface plasmon resonance (SPR) analysis.
Moreover, before performing the said contact process, it is more preferable to immerse the member which a nucleic acid sample solution contacts in the solution containing DEPC (diethyl pyrocarbonate) or hydrogen peroxide.

  The protein detection / quantification method of the present invention uses the target nucleic acid molecule obtained by the above-described nucleic acid molecule screening method to detect or quantify the peptide that interacts with the target nucleic acid molecule, thereby It is characterized by detecting or quantifying.

  According to the present invention, since a fragment peptide is used to obtain a target nucleic acid molecule, a nucleic acid molecule capable of efficiently obtaining a desired nucleic acid molecule from a nucleic acid molecule in a nucleic acid sample solution by stably using a target substance. A screening method and a protein detection / quantification method can be provided.

  The nucleic acid molecule screening method of the present invention is a nucleic acid molecule screening method for screening a target nucleic acid molecule that is a nucleic acid or a nucleic acid derivative that specifically interacts with a selected peptide molecule from nucleic acid molecules in a nucleic acid sample solution, A degradation treatment step of degrading a protein containing the peptide molecule to obtain a fragment peptide; an immobilization step of immobilizing the fragment peptide on a support to obtain an immobilized fragment peptide; and Obtaining the amount of interaction between the immobilized fragment peptide and the nucleic acid molecule by performing a contact step of bringing the nucleic acid molecule into contact with the immobilized fragment peptide and performing an analysis using near-field light on the surface of the support. A selection step of selecting the target nucleic acid molecule from the nucleic acid molecule in the nucleic acid sample solution based on the interaction amount obtained. .

In this method, a target nucleic acid molecule that specifically interacts with a selected specific peptide molecule is selected from nucleic acid molecules in a nucleic acid sample solution using a fragment peptide obtained by decomposing the protein.
Such a fragment peptide has higher structural stability than a protein itself including a fragment peptide molecule, and is less likely to be destroyed by an immobilization operation on a support. In the case of a protein itself, even if it exhibits insoluble properties due to a three-dimensional structure or hydrophilicity / hydrophobicity, a fragment peptide can be easily dissolved in a solution, and screening using a nucleic acid sample solution is facilitated. Furthermore, even if it is a peptide molecule at the inner site in a stable state, it can be easily screened for aptamers that can interact with this site without performing protein denaturation treatment, etc., by using a fragment peptide. . As a result, a desired nucleic acid molecule can be efficiently and reproducibly obtained by a stable target substance, that is, a fragment peptide.
The interaction between the target nucleic acid molecule and the fragment peptide in the present invention refers to a physical, chemical, or biochemical binding / association reaction.

  Examples of the nucleic acid molecule in the present invention include a nucleic acid or a derivative, and in addition to DNA and RNA, these derivatives, that is, naturally-modified DNA and RNA partially modified. Examples of the nucleic acid derivative include PNA (peptide nucleic acid) which is a compound having a structure similar to DNA and forming a skeleton by peptide bond. Among such nucleic acid molecules, the target nucleic acid molecule in the present invention has a property of specifically interacting with a selected specific peptide molecule, and is generally called an aptamer.

The solution constituting the nucleic acid sample solution is not particularly limited as long as it can stably hold nucleic acid molecules, and is generally used as a buffer solution, for example, a known buffer solution (Chemical Handbook Application Edition 2nd edition). , Pages 1312 to 1320), to which salt, metal ions (magnesium ions, potassium ions, calcium ions, etc.), stabilizers (DTT, etc.) and the like are added can be used. In particular, PBS buffer (phosphate buffer saline), Tris buffer, HEPES buffer and the like are preferably used.
In addition, it does not specifically limit as a protein containing the peptide molecule which the said aptamer can interact.

In the decomposition treatment step in the present screening method, the protein molecule is decomposed in order to obtain a fragment peptide as a peptide molecule to be immobilized on the support.
The degradation treatment may be any treatment that can degrade the protein into a peptide of a predetermined length, but it must be a chemical treatment that can degrade the protein under relatively mild conditions, particularly a degradation treatment using an enzyme. Is preferred. In addition, since a plurality of fragment peptides can be easily obtained from one protein by the enzymatic degradation treatment, various aptamers can be obtained more easily than the synthesis and screening of the target peptide molecule. Furthermore, the enzymatic decomposition treatment is more preferable from the viewpoint of the reproducibility of the fragment peptide obtained because it is possible to control the cleavage site in the protein.

  As the enzyme used for the decomposition treatment, various proteases can be preferably used. As such protease, any protease can be used as long as it can be used for peptide hydrolysis, and examples thereof include trypsin, chymotrypsin, papain, subtilisin, and dispase. These enzymes may be used alone or in combination of two or more. It can select suitably according to the kind of protein (peptide molecule) used, the kind of desired aptamer, and the purpose of screening.

  The fragment peptide obtained by the degradation treatment step is immobilized on the support in the immobilization step. The immobilized fragment peptide can be immobilized on a support in a single type or a plurality of types, but in the case of fragment peptides derived from the same protein, there are multiple types in order to increase the efficiency of screening. It is preferable.

The support for immobilizing the fragment peptide may be composed of a transparent substrate or an opaque substrate in accordance with the suitability for near-field optical analysis. Further, an immobilization film having a metal part on the side of the fragment peptide immobilization side of the substrate corresponding to the suitability of near-field optical analysis, and a functional group that is arranged directly on the metal part or on the substrate and can immobilize the fragment peptide. Can be mentioned.
As the transparent substrate, generally used is an optical glass such as BK7, or a synthetic resin, specifically, a material made of a material transparent to laser light such as polymethyl methacrylate, polyethylene terephthalate, polycarbonate, or cycloolefin polymer. it can. Such a substrate is preferably made of a material that does not exhibit anisotropy with respect to polarized light and has excellent processability.

As a metal which comprises a metal part, what can produce a plasmon resonance can be mentioned, for example. Preferred examples include free electron metals such as gold, silver, copper, aluminum, and platinum, with gold being particularly preferred. These metals can be used alone or in combination. In consideration of adhesion to the substrate, an intervening layer made of chromium or the like may be provided between the substrate and the layer made of metal.
Although the shape of the metal part is arbitrary, for example, when considering the use for a surface plasmon resonance biosensor, it is preferably in a film shape and has a film thickness of 0.1 nm to 500 nm, particularly 1 nm to 200 nm. preferable. If it exceeds 500 nm, the surface plasmon phenomenon of the medium cannot be sufficiently detected. Moreover, when providing the intervening layer which consists of chromium etc., it is preferable that the thickness of the intervening layer is 0.1 to 10 nm.

The metal part is preferably arranged on the substrate. Here, “arranged on the substrate” means not only when the metal part is arranged so as to be in direct contact with the substrate, but also through other layers without the metal part being in direct contact with the substrate. This also includes the case where they are arranged.
The metal part may be formed by a conventional method, for example, sputtering, vapor deposition, ion plating, electroplating, electroless plating, or the like.

Examples of the immobilized membrane include hydrogels such as agarose, dextran, carrageenan, alginic acid, starch, and cellulose, or derivatives thereof such as carboxymethyl derivatives, or water-swellable organic polymers such as polyvinyl alcohol, polyacrylic acid, polyacrylamide, Polyethylene glycol or the like can be used. In particular, polyethylene glycol derivatives and dextran derivatives are preferably used from the viewpoint of maintaining physiological activity.
Although the thickness of the immobilization film is arbitrary, for example, when considering the use for a surface plasmon resonance biosensor, it is preferably 0.1 nm to 500 nm, and particularly preferably 1 nm to 300 nm.

  The immobilization method may be any method as long as the fragment peptide can be immobilized on the support surface, but the amino coupling method in which the carboxyl group on the support surface and the amino group of the fragment peptide are immobilized by a covalent bond. Is preferable from the viewpoint of simplicity and stability. The amino coupling method used for this purpose may be carried out according to a conventional method, and examples thereof include a method using N-ethyl- (dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS). .

  In the contact step, the nucleic acid molecule in the sample solution is brought into contact with the immobilized fragment peptide obtained in the immobilization step. By this contact, many of the nucleic acid molecules present in the nucleic acid sample solution interact specifically or non-specifically with the immobilized fragment peptide.

In the selection step, the nucleic acid sample is obtained based on the obtained interaction amount while performing the analysis using the near-field light on the support surface to obtain the interaction amount between the immobilized fragment peptide and the nucleic acid molecule. The target nucleic acid molecule is selected from nucleic acid molecules in the liquid.
Immediately after the contact in the contacting step, in addition to aptamers that specifically interact with the immobilized fragment peptide, nucleic acids that interact with the immobilized fragment peptide non-specifically with low affinity and do not interact physically A nucleic acid molecule (hereinafter referred to as a “non-specific nucleic acid molecule”) that is in a weakly bound or associated state, interacts with the immobilized fragment peptide. Thereafter, such non-specific nucleic acid molecules are dissociated from the immobilized fragment peptide, thereby being excluded from the group of nucleic acid molecules interacting with the immobilized fragment peptide, and aptamer as a nucleic acid molecule interacting with the immobilized fragment peptide. Can be sorted out.

  Near-field light analysis methods applicable to this screening method include surface plasmon resonance (SPR) analysis, local plasmon resonance analysis, grating coupled resonance analysis, two-plane polarization interference analysis, and near-field fluorescence measurement. And surface enhanced Raman scattering measurement. Among these, it is preferable that the SPR analysis is capable of monitoring the amount of interaction over time easily and with high sensitivity.

SPR analysis is based on the fact that the intensity of monochromatic light reflected from the boundary between an optically transparent substance such as glass and the metal thin film layer depends on the refractive index of the sample on the opposite side of the metal reflecting surface. Yes, therefore, the molecules in the sample can be analyzed by measuring the intensity of the reflected monochromatic light. A surface plasmon measuring apparatus using the above system basically includes a dielectric block formed in a prism shape, for example, and a metal film formed on one surface of the dielectric block and brought into contact with a substance to be measured such as a sample liquid. A light source that generates a light beam; an optical system that causes the light beam to enter the dielectric block at various angles so that a total reflection condition is obtained at an interface between the dielectric block and the metal film; and It comprises light detection means for detecting the surface plasmon resonance state, that is, the state of total reflection attenuation by measuring the intensity of the light beam totally reflected at the interface (see, for example, JP-A-6-167443). .
By using this apparatus, the amount of interaction between the immobilized fragment peptide on the support and the nucleic acid molecule can be obtained from the detected state of attenuated total reflection. The amount of interaction varies depending on the amount of nucleic acid molecule that interacts with the immobilized fragment peptide. That is, when the immobilized fragment peptide is brought into contact with the nucleic acid molecule, the amount of interaction increases to a saturation amount, and the non-specifically interacting nucleic acid molecule is dissociated from the immobilized fragment peptide by the subsequent washing step. The amount of interaction decreases.

As described above, the amount of interaction decreases when the non-specific nucleic acid molecule is dissociated from the immobilized fragment peptide. Therefore, the selection of the nucleic acid molecule can be easily evaluated as the amount of change in the amount of interaction.
The evaluation as to whether or not the sorting process has been completed may be performed by setting a specific interaction amount serving as a threshold value in advance, or from the maximum interaction amount immediately after the contact process. You may perform using the specific change rate used as the threshold value when using a change rate. Moreover, when performing a repeated sorting process as will be described later, it may be evaluated as a comparison with the rate of change obtained in the previous sorting process.

  The values used for these evaluations can be appropriately set according to the desired amount of the target nucleic acid molecule obtained at the end of the selection process. For example, since the target nucleic acid molecule can be efficiently selected by repeatedly performing the selection step as described later, it is not necessary to extremely reduce the content of non-specific nucleic acid molecules in one selection step. For this reason, when performing the repeated selection process, the target nucleic acid molecule can be efficiently selected even if a value that increases the amount of the non-specific nucleic acid molecule contained is set.

  In order to select efficiently in the selection step, a buffer solution or the like may be flowed to actively dissociate weakly interacting nucleic acid molecules. The supply of such a buffer solution can adjust the selection time, flow rate, etc., depending on the desired screening accuracy, thereby reducing the amount of nucleic acid molecules interacting with the immobilized fragment peptide. Can be adjusted.

  The nucleic acid molecule selected by interacting with the immobilized fragment peptide by the selection step can then be recovered by forcibly separating it from the immobilized fragment peptide. In order to separate the interacting nucleic acid molecules, separation means for eliminating the interaction between the two can be used. As such a separation means, it is preferable to select a separation solution having little influence on the aptamer and the fragment peptide. As such a separation solution, a chaotropic reagent such as a 7M urea solution or a high solution such as a 1M sodium chloride solution is used. Mention may be made of a salt concentration solution.

  In the screening method of the present invention, it is preferable to perform an amplification step in which the aptamer selected in the selection step is amplified by the PCR method. Thereby, the quantity of the aptamer which interacts with a fragment peptide can be increased. The PCR used in the amplification step may be performed by any method capable of synthesizing and amplifying aptamers by polymerase chain reaction using polymerases and nucleotides according to the type of aptamer and primers prepared according to the aptamer. .

  When performing the amplification step, depending on the type of the nucleic acid molecule, a conversion step for converting the nucleic acid into a type of nucleic acid suitable for efficient PCR treatment may be provided. As such a conversion step, for example, when the collected aptamer is RNA, a reverse transcription step to DNA using reverse transcriptase can be mentioned.

In the screening method of the present invention, the aptamer selected in the selection step is recovered, and again the contact step and the selection step (hereinafter, these two steps are collectively referred to as “screening step”). In addition to the above, it is preferable to repeat the three steps including the amplification step at least once, and in some cases, multiple times.
FIG. 1 shows a conceptual diagram when a screening process including an amplification process is repeatedly performed. As shown in FIG. 1, in the first contact step, various nucleic acid molecules are mixed in the nucleic acid sample solution, but aptamers that interact specifically with the fragment peptides are selected through the selection step. Is done. At this time, even if non-specific nucleic acid molecules are present together with aptamers in the nucleic acid molecules recovered in the first selection step, the second screening step results in selection and elimination again in the second screening step. The ratio of aptamers in the subsequent nucleic acid molecules is higher than the ratio in the nucleic acid molecules after the first screening step.
Therefore, by repeating this, the target aptamer can be obtained more efficiently and in large quantities.
In addition, since aptamers can be selected more efficiently by repeating the screening process, it is not necessary to remove nonspecific nucleic acid molecules in the selection process over a long period of time, and aptamers can be efficiently extracted in a shorter time. Obtainable.

  The number of repetitions of the screening process can be changed as appropriate depending on the type of fragment peptide, aptamer, etc., but it depends on the amount of aptamer obtained and the amount of nonspecific nucleic acid molecules present in the nucleic acid sample solution after the screening process. It can be judged based on.

  For members and parts that come into contact with nucleic acid molecules, such as devices, flow paths, and support surfaces used in this screening method, a treatment for eliminating the degradation of the nucleic acid molecules is performed in advance before the contact step. It is preferable to keep it. Examples of such treatment include nuclease removal treatment. In the nuclease removal treatment, a solution such as diethyl procarbonate (DEPC) or hydrogen peroxide may be brought into contact with a member and a part that come into contact with the nucleic acid molecule. Further, it is more preferable to immerse a member, preferably at least the flow path member, with which the nucleic acid sample solution can come into contact with a solution containing DEPC (diethyl pyrocarbonate) or hydrogen peroxide. In this case, the treatment liquid used for the nuclease removal treatment may be volatilized and removed by heating the member to a high temperature after the nuclease removal treatment.

Commercially available SPR devices and PCR devices can be directly applied to this screening method. Moreover, the target aptamer can be obtained more efficiently by using a screening apparatus equipped with both SPR and PCR. FIG. 2 shows a screening apparatus 100 to which the screening method of the present invention can be applied. The screening apparatus 100 includes an operation unit 106 including an SPR unit 102 and a PCR unit 104, and a control unit 60 that controls driving of these units. Moreover, the screening method can be continuously executed by providing a transport unit and a collection unit (not shown).
An example of such an SPR unit 104 will be described below with reference to the drawings.

FIG. 3 shows a biosensor 10 suitable as an SPR unit in the above-described screening apparatus.
The biosensor 10 is a so-called surface plasmon sensor that measures the interaction between a fragment peptide P and a nucleic acid molecule using surface plasmons generated on the surface of a metal film.
As shown in FIG. 3, the biosensor 10 includes a tray holding unit 12, a transport unit 14, a container mounting table 16, a liquid suction / discharge unit 20, an optical measurement unit 54, and a control unit 60.
The tray holding unit 12 includes a mounting table 12A and a belt 12B. The mounting table 12A is attached to a belt 12B spanned in the arrow Y direction, and is movable in the arrow Y direction by the rotation of the belt 12B. Two trays T are positioned and mounted on the mounting table 12A. In the tray T, eight sensor sticks 40 are stored. The sensor stick 40 is a chip to which the fragment peptide P is fixed, and details will be described later. Under the mounting table 12A, a push-up mechanism (not shown) for pushing up the sensor stick 40 to a position of a stick holding member 14C described later is arranged.

  As shown in FIGS. 4 and 5, the sensor stick 40 includes a dielectric block 42, a flow path member 44, a holding member 46, an adhesive member 48, and an evaporation preventing member 49.

  The dielectric block 42 is made of a transparent resin or the like that is transparent to the light beam, and has a prism portion 42A having a trapezoidal cross section, and is integrated with the prism portion 42A at both ends of the prism portion 42A. The to-be-held part 42B formed in this is provided. As shown in FIG. 6, a metal film 50 is formed on the upper surface on the wide side of the two parallel surfaces of the prism portion 42A. The dielectric block 42 functions as a so-called prism. When the measurement is performed by the biosensor 10, a light beam is incident from one of two opposing non-parallel sides of the prism portion 42A, and a metal film is formed from the other. The light beam totally reflected at the interface with 50 is emitted.

  As shown in FIG. 6, an immobilization film 50 </ b> A is formed on the surface of the metal film 50. The immobilization film 50 </ b> A is for immobilizing the fragment peptide P on the metal film 50. The immobilization membrane 50A is selected according to the type of fragment peptide P to be immobilized.

On the immobilization film 50A, a measurement region E1 in which the fragment peptide P is immobilized and a reference region E2 in which the fragment peptide P is not immobilized and a reference signal for signal measurement in the measurement region E1 is formed. . That is, the reference region E2 is a region provided for correcting data obtained from the measurement region E1 to which the fragment peptide P is fixed. The reference region E2 is formed when the above-described immobilization film 50A is formed. As a formation method, for example, a surface treatment is performed on the immobilization film 50A to deactivate a binding group that binds to the fragment peptide P. Thereby, a part of the fixed film 50A becomes the measurement area E1, and the remaining part becomes the reference area E2.
As shown in FIG. 7, light beams L2 and L1 are incident on the reference region E2 and the measurement region E1 located upstream of the reference region E2, respectively.

  On both side surfaces of the prism portion 42A, engaging convex portions 42C engaged with the holding member 46 along the upper side edge are virtual surfaces perpendicular to the upper surface of the prism portion 42A along the lower side edge. Vertical protrusions 42D configured on the extension are formed at seven locations, respectively. Further, an engagement groove 42E is formed in the center portion along the longitudinal direction of the lower surface of the dielectric block 42.

  The flow path member 44 has a rectangular parallelepiped shape slightly narrower than the dielectric block 42, and is arranged in parallel on the metal film 50 of the dielectric block 42 as shown in FIG. A channel groove 44 </ b> A is formed on the lower surface of each channel member 44, communicated with the supply port 45 </ b> A and the discharge port 45 </ b> B formed on the upper surface, and the liquid channel 45 between the metal film 50. Composed. Accordingly, six independent liquid channels 45 are formed in one sensor stick 40. On the side wall of the flow path member 44, a convex portion 44 </ b> B is formed that is press-fitted into a concave portion (not shown) inside the holding member 46 to ensure adhesion with the holding member 46.

  The holding member 46 is long and includes an upper plate 46A and two side plates 46B. The side plate 46B is formed with an engagement hole 46C that is engaged with the engagement convex portion 42C of the dielectric block 42. The holding member 46 is attached to the dielectric block 42 with the engagement holes 46 </ b> C and the engagement protrusions 42 </ b> C engaged with the six flow path members 44 interposed therebetween. Thereby, the flow path member 44 is attached to the dielectric block 42. In the upper surface plate 46A, a tapered pipette insertion hole 46D that narrows toward the flow path member 44 is formed at a position facing the supply port 45A and the discharge port 45B of the flow path member 44. A positioning boss 46E is formed between the adjacent pipette insertion hole 46D and the pipette insertion hole 46D.

  An evaporation preventing member 49 is bonded to the upper surface of the holding member 46 via an adhesive member 48. A pipette insertion hole 48D is formed at a position facing the pipette insertion hole 46D of the adhesive member 48, and a positioning hole 48E is formed at a position facing the boss 46E. Further, a slit 49D, which is a cross-shaped cut, is formed at a position facing the pipette insertion hole 46D of the evaporation preventing member 49, and a positioning hole 49E is formed at a position facing the boss 46E. By inserting the boss 46E into the holes 48E and 49E and bonding the evaporation preventing member 49 to the upper surface of the holding member 52, the slit 49D of the evaporation preventing member 49 and the supply port 45A and the discharge port 45B of the flow path member 44 are formed. Configured to face each other. When the pipette tip CP is not inserted, the slit 49D covers the supply port 45B, and the liquid supplied to the liquid channel 45 is prevented from evaporating.

  As shown in FIG. 3, the transport unit 14 of the biosensor 10 includes an upper guide rail 14A, a lower guide rail 14B, and a stick holding member 14C. The upper guide rail 14 </ b> A and the lower guide rail 14 </ b> B are horizontally disposed in the arrow X direction perpendicular to the arrow Y direction, above the tray holding unit 12 and the optical measurement unit 54. A stick holding member 14C is attached to the upper guide rail 14A. The stick holding member 14C can hold the held parts 42B at both ends of the sensor stick 40 and can move along the upper guide rail 14A. The engagement groove 42E of the sensor stick 40 held by the stick holding member 14C and the lower guide rail 14B are engaged, and the stick holding member 14C moves in the arrow X direction, so that the sensor stick 40 is placed on the optical measurement unit 54. To the measurement unit 56.

On the container mounting table 16, a compound solution plate 17, a buffer liquid stock container 18, a waste liquid container 19 and a separation liquid container (not shown) are mounted. The compound solution plate 17 is partitioned into 96 so that a nucleic acid sample solution can be stocked. The buffer liquid stock container 18 is composed of containers 18A to 18E, and the containers 18A to 18E are formed with openings K into which pipette tips CP to be described later can be inserted.
The waste liquid container 19 includes a plurality of containers 19A to 19E, and an opening K into which the pipette tip CP can be inserted is formed in the same manner as the containers 18A to 18E.

  The liquid suction / discharge portion 20 includes an upper guide rail 14A, a crossing rail 22 that extends above the guide rail 16B and extends in the arrow Y direction, and a head 24. The crossing rail 22 can be moved in the arrow X direction by a drive mechanism (not shown). The head 24 is attached to the cross rail 22 and is movable in the arrow Y direction. The head 24 is also movable in the vertical direction (arrow Z direction) by a drive mechanism (not shown). As shown in FIG. 8, the head 24 includes two pipette portions 24A and 24B. Pipette tips CP are attached to the tip portions of the pipette portions 24A and 24B, and the length in the Z direction can be individually adjusted (see FIGS. 8A to 8B). Many pipette tips CP are stocked in a pipette tip stocker (not shown) and can be replaced as necessary.

  In the biosensor 10, the liquid is supplied to the sensor stick 40 by the pipette tip CP. Instead of the pipette tip, one end is connected to each solution plate and the other end can be connected to the sensor stick 40. A liquid injection tube may be provided, and the liquid may be supplied by a liquid feed pump.

  As shown in FIG. 9, the optical measurement unit 54 includes a light source 54A, a first optical system 54B, a second optical system 54C, a light receiving unit 54D, and a signal processing unit 54E. A divergent light beam L is emitted from the light source 54A. The light beam L becomes two light beams L1 and L2 via the first optical system 54B, and is incident on the measurement region E1 and the reference region E2 of the dielectric block 42 arranged in the measurement unit 56. In the measurement region E1 and the reference region E2, the light beams L1 and L2 include various incident angle components with respect to the interface between the metal film 50 and the dielectric block 42 and are incident at an angle greater than the total reflection angle. The light beams L 1 and L 2 are totally reflected at the interface between the dielectric block 42 and the metal film 50. The totally reflected light beams L1 and L2 are also reflected with various reflection angle components. The totally reflected light beams L1 and L2 are received by the light receiving unit 54D through the second optical system 54C, are photoelectrically converted, and a light detection signal is output to the signal processing unit 54E. In the signal processing unit 54E, predetermined processing is performed based on the input light detection signal, and measurement data G1 in the measurement region E1 and reference data G2 in the reference region E2 are obtained. The measurement data G1 and reference data G2 are output to the control unit 60.

  The control unit 60 has a function of controlling the entire biosensor 10 that is the SPR unit 102 together with the PCR unit 104. As shown in FIG. 9, the light source 54A, the signal processing unit 54E, and the biosensor 10 are not shown. Connected to the system. As shown in FIG. 2, the control unit 60 is connected to a display unit 62 for displaying various types of information and an input unit 64 for inputting various types of instructions and information.

The control unit 60 includes a memory (not shown), and stores various programs for controlling the biosensor 10, various data, and a screening program.
The reference data G2 stored in the control unit 60 is obtained when the running buffer is supplied onto the immobilized membrane 50A on which the fragment peptide P is not immobilized, and when the nucleic acid molecule A is supplied. It is indicated by the amount of change RU (Resonance Unit, corresponding to approximately 1 RU = 1 pg / mm ² ) from the signal.

  As described above, in the biosensor 10, when the sensor stick 40 is conveyed to the measurement unit 56 and a measurement start instruction is input from the input unit 64, the control unit 60 performs measurement processing. In this interaction amount measurement process, a nucleic acid sample solution is supplied to the liquid channel 45, the light source 54A, the signal processing unit 54E, and a drive system (not shown) are driven to acquire measurement data G1 from the measurement region E1, and the reference region Reference data G2 is acquired from E2. Next, the measurement data G1 is corrected with the reference data G2, and the interaction amount data G3 is calculated. When the supply of the nucleic acid sample solution is completed, the supply of the buffer solution is started. Meanwhile, the calculation of the interaction amount data G3 is continued, and the obtained interaction amount data G3 is stored over time. Here, since the nonspecific nucleic acid molecules are eliminated by supplying the buffer solution and the selection proceeds, the interaction amount data G3 becomes a small value with time.

In the control unit 60, when the obtained interaction amount data G3 reaches a predetermined measurement end value or when a stop signal is input from the outside, the measurement process is ended, and the separation solution is replaced with the buffer solution. While supplying to the liquid flow path 45, the nucleic acid molecule isolate | separated from the measurement area | region E1 is collect | recovered. When the collection for a predetermined time is completed, the process in the biosensor 10 is completed and an instruction to switch to the PCR process in the PCR unit 104 is given.
Note that the predetermined measurement end value used for instructing the end of the measurement process may be a threshold for the interaction amount data G3 calculated over time, or a threshold for the rate of change of the interaction amount data G3. It may be a value.

  By using the screening apparatus 100 including the biosensor 10 as the SPR unit 102 and the PCR unit 104, the target aptamer can be efficiently screened from the nucleic acid molecules in the nucleic acid solution.

Since the aptamer obtained by the above screening method interacts with the fragment peptide of the target protein, it is preferably used for detection or quantification of the target protein.
That is, the protein detection / quantification method of the present invention uses the aptamer obtained by the above screening method to detect or quantify the peptide that interacts with the target nucleic acid molecule, thereby detecting the protein containing the peptide.・ Quantitative.
For the detection or quantification of the protein, the aptamer obtained by this screening method may be labeled and detected and quantified without using near-field light analysis based on the label. As in the case of the fragment peptide, it is preferably immobilized on a support and detected and quantified based on near-field optical analysis. By using near-field optical analysis, the target protein can be detected or quantified with high sensitivity, stability and simplicity based on the amount of interaction.

The protein used in this detection / quantification method is preferably a fragment peptide obtained by fragmentation by chemical treatment. By using a fragmented peptide, as described above, even an unstable protein or a water-insoluble protein can be detected or quantified easily and reproducibly.
In the present invention, “detection or quantification” may mean both detection and quantification of protein, and any of these may be used, and detection is made by using interaction amount data. And quantitative determination can be performed simultaneously.

EXAMPLES Hereinafter, although an Example demonstrates this invention concretely, this invention is not limited by this.
In this embodiment, a case where SPR is used for near-field light analysis will be described as an example.

[Example 1]
Preparation of various solutions and immobilization of fragment peptides (1) Preparation of random RNA mixed solution A nucleic acid solution to be screened is prepared as a random RNA solution as follows.
First, 114 bp oligo DNA having an arbitrary sequence for obtaining random RNA is obtained by chemical synthesis. This DNA has the 5 ′ primer binding sequence shown below from the 5 ′ end to 40 bp, the following 3 ′ primer binding sequence from the 3 ′ end to 24 bp, and the internal 50 bp has a random sequence. .

Subsequently, Taq polymerase (manufactured by Takara Bio Inc.), primer (5 ′ primer: the above binding sequence, 3 ′ primer: complementary strand of the above binding sequence), and dNTP mixed solution were added, and 60 ° C. (50 seconds), 72 ° C. (70 Second) and 95 ° C. (70 seconds) PCR reaction is performed for 15 cycles. Details of the PCR reaction follow the recommended conditions of Takara Bio.
After completion of the PCR reaction, extraction is performed by isopropanol precipitation, the PCR product is extracted, and an in vitro transcription reaction is performed at 37 ° C. for 3 hours using T7 RNA polymerase (manufactured by Promega). The details of the in vitro transcription reaction follow the Promega recommended conditions. After the reaction, a phenol / chloroform treatment and isopropanol precipitation are performed to obtain a random RNA mixed solution of the transcription product.

(2) Preparation of fragment peptide Calmodulin is reacted at 37 ° C. for 30 minutes using trypsin (manufactured by Worthington Biochemical Corp.). The liquid after the reaction is injected into an ultrafiltration column (MilliPore, Ultrafree-MC 10,000 NMWL Filter Unit), and centrifuged at 14,000 g for 5 minutes. The obtained filtrate is used as a fragment peptide solution, and the peptide concentration is estimated using the absorbance at 280 nm as an index.

(3) Immobilization of fragment peptide The flow path member 44 and the holding member 46 provided in the sensor stick 40 in the biosensor 10 are immersed in 1 mg / ml DEPC (diethyl pyrocarbonate) at 37 ° C. for 2 hours, and then sterilized with water. After rinsing several times, heat at 100 ° C. for 15 minutes.
As the immobilization film 50A, a threshold for forming a well for spatially dividing the measurement region E1 and the reference region E2 is set on a sensor stick that has been subjected to a CMD (carboxymethyl dextran) coating process. Fill wells with HBS-P buffer (Biacore). The buffer is removed and 100 μl of the activation solution (equal mixture of 0.2 M EDC and 0.05 M NHS) is injected into the well for 1 second and left for 30 minutes. Subsequently, the activation solution is removed, 100 μl of HBS-P buffer is injected into the well for 1 second, and then the buffer is removed immediately, and the fragment peptide solution (0.1 mg / ml, acetate buffer pH 6. 100 μl) HBS-P is injected into the well in the reference region E2 for 1 second and left for 30 minutes. Subsequently, the peptide solution is removed, and 100 μl of HBS-P buffer is injected into the well in 1 second. Thereafter, the buffer is removed, and 100 μl of blocking solution (1M ethanolamine · HCl pH 8.5) is injected into the well in 1 second. Leave for 30 minutes. Subsequently, the blocking solution is removed, 100 μl of HBS-P buffer is injected into the well for 1 second, and then the well is washed sequentially with 100 μl of 10 mM NaOH solution and HBS-P. By this operation, a peptide-immobilized membrane is obtained.

[Example 2]
Screening A threshold for forming a well is removed from a sensor stick on which a peptide is immobilized, and the biosensor 10 is set. Fill the flow path with HBS-P buffer, and in that state, 100 μl of the random RNA mixed solution prepared in Example 1 is injected into the flow path for 1 second, brought into contact with the fragment peptide-immobilized membrane, and left for 5 minutes. At this time, since the immobilized fragment peptide and the nucleic acid molecule start to interact, the amount of interaction increases. Next, 100 μl of HBS-P buffer is injected into the flow channel in 1 second, and then 30 μl of HBS-P buffer is slowly injected. At this time, the interaction is measured by the SPR signal. When the non-specific nucleic acid molecule is dissociated until the SPR signal reaches an intermediate value between the baseline and the dissociation start signal, 100 μl of HEPES buffer containing 7M urea is supplied to elute the RNA molecule (aptamer) bound to the fragment peptide. And collect.
Thereby, an aptamer that specifically interacts with a calmodulin fragment peptide can be obtained.

Next, PCR is performed to amplify the aptamer of the recovered nucleic acid molecule.
First, isopropanol precipitation is performed on the collected nucleic acid molecule solution to concentrate the nucleic acid molecule solution. A reverse transcriptase (Improm-II ™ Reverse Transcriptase, manufactured by Promega) is added to the concentrated nucleic acid molecule (RNA) and reacted for 30 minutes to convert it into DNA. Details of the reverse transcription reaction are performed under the conditions recommended by Promega. PCR15Cycle is performed on the solution containing the obtained DNA under the same conditions as in Example 1. Under the same conditions as the PCR in the examples. After completion of the PCR reaction, the product is extracted by isopropanol precipitation to extract the PCR product. Thereby, a large amount of aptamers obtained by the initial screening can be obtained.

  The above-described series of operations of the transcription step, the contact step, the recovery step, the reverse transcription step and the PCR step are repeated three times for the DNA mixed solution containing the amplified aptamer obtained by the PCR. Thereby, screening can be performed while efficiently amplifying aptamers that interact with the calmodulin fragment peptide, and the target aptamer can be efficiently obtained.

[Example 3]
Detection and quantification of protein by aptamer As in Example 1, the aptamer is immobilized on a sensor stick subjected to CMD coating treatment as the immobilization film 50A. The immobilization method follows the peptide immobilization method of Example 1. However, instead of the fragment peptide solution, 100 μl of the RNA mixed solution (0.1 mg / ml HBS-P) recovered in Example 2 is reacted with the measurement region E1. By this operation, an aptamer-immobilized membrane is obtained. Calmodulin is then fragmented by the same procedure as in Example 1 to obtain a fragment peptide solution.

  The obtained fragment peptide solution is supplied through the flow path while contacting the aptamer-immobilized membrane while measuring the amount of interaction using the SPR signal. Among the calmodulin fragment peptides, when there is a fragment peptide capable of binding to the immobilized aptamer, it binds to the aptamer on the sensor surface membrane and the SPR signal rises. As a result, an SPR signal of 15 RU was obtained in the measurement region E1 of the aptamer-immobilized membrane as compared with the reference region E2. Therefore, it can be seen that the peptide peptide having affinity for the immobilized aptamer is contained in the fragment peptide solution in an amount of 15 RU.

[Comparative Example 1]
The operations of Examples 1 to 3 are performed in the same manner as a whole protein without fragmenting calmodulin. As a result, in the measurement region E1 of the aptamer-immobilized membrane obtained in this comparative example, a signal difference was obtained only about 0 to 3 RU compared with the reference region E2, and it was hidden in the background noise.

It is a conceptual diagram explaining the screening method of this invention. It is a schematic block diagram of the screening apparatus applicable to this invention. It is a whole perspective view of the SPR part which can be used in this invention. It is a perspective view of the sensor stick which can be used in this invention. It is a disassembled perspective view of the sensor stick which can be used in this invention. It is sectional drawing of one liquid flow-path part of the sensor stick which can be used in this invention. It is a figure which shows the state in which the light beam is injecting into the measurement area | region of a sensor stick which can be used in this invention, and a reference area | region. (A)-(C) are side views of the pipette part which comprises the liquid supply part which can be used in this invention. It is the schematic of the optical measurement part vicinity of the SPR part which can be used in this invention.

Explanation of symbols

DESCRIPTION OF SYMBOLS 10 Biosensor 40 Sensor stick 50 Metal film 50A Immobilization film | membrane 54 Optical measurement part 60D Memory 60 Control part 62 Display part 64 Input part P Fragment peptide E1 Measurement area E2 Reference area 100 Screening apparatus 102 SPR part 104 PCR part

Claims (9)

A nucleic acid molecule screening method for screening a target nucleic acid molecule which is a nucleic acid or a nucleic acid derivative that specifically interacts with a selected peptide molecule from nucleic acid molecules in a nucleic acid sample solution,
A degradation treatment step of degrading a protein containing the peptide molecule to obtain a fragment peptide;
An immobilization step of immobilizing the fragment peptide on a support to obtain an immobilized fragment peptide;
Contacting the nucleic acid molecule in the nucleic acid sample solution with the immobilized fragment peptide;
The nucleic acid in the nucleic acid sample solution is obtained based on the obtained interaction amount while performing the analysis using the near-field light on the surface of the support to obtain the interaction amount between the immobilized fragment peptide and the nucleic acid molecule. A selection step of selecting the target nucleic acid molecule from the molecule;
A nucleic acid molecule screening method comprising:   The screening method according to claim 1, wherein the decomposition treatment step is to decompose a protein using an enzyme.   The screening method according to claim 1 or 2, further comprising an amplification step of amplifying the target nucleic acid molecule selected in the selection step by a polymerase chain reaction (PCR) method.   The screening method according to claim 3, wherein the nucleic acid molecule to be amplified obtained in the amplification step is collected, and the contact step and the selection step are repeated at least once.   The screening method according to any one of claims 1 to 4, wherein the immobilized fragment peptide is composed of two or more fragment peptides obtained from the same protein.   The screening method according to any one of claims 1 to 5, wherein the target nucleic acid molecule is selected from the group consisting of DNA, RNA, and derivatives thereof.   The screening method according to any one of claims 1 to 6, wherein the analysis using near-field light is surface plasmon resonance (SPR) analysis.   The member which can contact the nucleic acid sample solution is immersed in a solution containing DEPC (diethyl pyrocarbonate) or hydrogen peroxide before the contacting step, according to any one of claims 1 to 3. Screening method.   Detection of a protein containing the peptide by detecting or quantifying the peptide that interacts with the target nucleic acid molecule using the target nucleic acid molecule obtained by the screening method according to any one of claims 1 to 7. Alternatively, a protein detection / quantification method that performs quantification.

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