JP2011055770A - Nucleic acid aptamer, and method for screening the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a nucleic acid aptamer having high specific binding force to a low-molecular-weight molecule such as an amino acid, and to provide a method for efficiently screening the same. <P>SOLUTION: The method for screening a nucleic acid aptamer using an atomic force microscope is provided, including: (a) a step of fixing a target substance onto the probe of the atomic force microscope; (b) a step of fixing a nucleic acid via a covalent bond onto a surface of a flat plate; (c) a step of scanning the surface of the flat plate obtained in the step (b) using the probe obtained in the step (a); (d) a step of retrieving the nucleic acid flying a space apart from the surface of the flat plate and caught on the probe, in the step (c); (e) a step of measuring the binding force to the target substance, for the nucleic acid obtained in the step (d); and (f) a step of repeating the steps (b) to (e) until a nucleic acid aptamer where the measured binding force in the step (e) exhibits a given value is found. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a nucleic acid aptamer and a method for screening a nucleic acid aptamer.

  Aptamers refer to nucleic acid molecules and peptides that specifically bind to a target substance. So far, various aptamers for target substances such as organic compounds, proteins, nucleic acids, cells, cellular tissues, microorganisms and the like are known. Nucleic acid aptamers are expected to be applied to drugs and the like as biological substances capable of specific molecular recognition such as antibodies. Nucleic acid aptamers have the advantage that antibodies can be synthesized chemically and in a short time in vitro, with little or no immunogenicity.

  A method for obtaining a nucleic acid aptamer that specifically binds to a target substance of interest is known as the SELEX method. Usually, screening is performed from a large library of random sequences using the principle of affinity chromatography. However, since the binding power and specificity of the nucleic acid obtained by one screening to the target substance are not sufficient, it is necessary to repeat the screening from the nucleic acid library obtained by the screening. Usually, the number of times of repeating the screening is several to a dozen times although it depends on the target substance. For this reason, in order to obtain the nucleic acid aptamer which has sufficient binding force and specificity, much labor and time are required.

  By the way, when an atomic force microscope (AFM) is used, a force acting between the probe and the sample can be detected. The probe is also called a cantilever. When the cantilever is moved closer to the sample, the cantilever bends due to the weak force generated between the cantilever and the sample, and the amount of deflection is detected by the optical lever method. The optical lever method is a method of irradiating the back surface of a cantilever with a semiconductor laser and detecting the reflected laser light with an optical sensor divided into four parts vertically and horizontally. By analyzing a force curve in which the relationship between the deflection amount of the cantilever and the distance between the probe and the sample is analyzed, information on the force acting between the probe and the sample and the hardness of the sample can be obtained. The detected deflection amount is fed back to the sample stage, and the shape of the sample surface is scanned by scanning the cantilever horizontally along the sample surface while controlling the height of the sample stage so that the deflection amount is constant. Can know. AFM can be used in a variety of environments such as air, liquid, high temperature and low temperature, without distinguishing between samples such as conductors and insulators. It is also possible to use a biological sample or the like in a state close to nature. From these characteristics, AFM is expected as a new biosensing tool (Non-patent Documents 1 and 2).

  In the SELEX method for obtaining a nucleic acid aptamer, the present inventors tried a screening method using AFM instead of affinity chromatography (Non-patent Document 3). That is, single-stranded DNA with biotinylated 5 ′ end was immobilized on an AFM probe (cantilever) via avidin, and the cantilever was used to scan the surface of a metal plate on which thrombin was immobilized as a target substance. If the aptamer is present in the single-stranded DNA immobilized on the cantilever, the aptamer is surely bound in the process of scanning on a large amount of thrombin, and the binding between the aptamer and thrombin exceeds the binding force between avidin and biotin. It is thought that it is released from the cantilever and captured on the metal plate by force. Therefore, the single-stranded DNA captured by the cantilever was recovered, amplified, further fixed to the cantilever, and the same operation was repeated. As a result, it was found that the binding strength of the obtained single-stranded DNA to thrombin increases each time the screening operation is repeated, that is, aptamers are screened.

  However, in the method described in Non-Patent Document 3, since the number of single-stranded DNAs that can be immobilized on the AFM cantilever is small, in order to obtain a nucleic acid aptamer having a high specific binding force, as in the conventional SELEX method, Several screenings were necessary.

T. Kato et al., “In vitro selection of DNA aptamers which bind to cholic acid”, Biochimica et Biophysica Acta, 2000, 1493, p. 12-18 V. Dupres et al., “Probing molecular recognition sites on biosurfaces using AFM”, Biomaterials, 2007, 28, p. 2393-2402. Miyaji et al., “Selection of DNA Atamer Using Atomic Force Microscope”, Proceedings of the 88th Annual Meeting of the Chemical Society of Japan, The Chemical Society of Japan, March 12, 2008, 4A4-32

  An object of the present invention is to provide a nucleic acid aptamer having a high specific binding force to a small molecule such as an amino acid, and to provide a method for efficiently screening a nucleic acid aptamer having a high specific binding force.

  As a result of intensive studies to solve the above problems, the present inventors fixed a target substance on the probe of an atomic force microscope in a method for screening a nucleic acid aptamer using an atomic force microscope, and The inventors have found that a method for efficiently screening a nucleic acid aptamer having a high specific binding force can be provided by immobilizing a nucleic acid on the surface of a flat plate scanned with a probe, thereby completing the present invention. By this method, a nucleic acid aptamer having a high specific binding force to a small molecule such as an amino acid is obtained.

  The present invention provides a method for screening a nucleic acid aptamer using an atomic force microscope, which comprises (a) a step of fixing a target substance to a probe of an atomic force microscope, and (b) a surface of a flat plate. A step of immobilizing a nucleic acid via a non-covalent bond, (c) a step of scanning the surface of the flat plate obtained in step (b) with the probe obtained in step (a), (d) step (c) Recovering the nucleic acid released from the surface of the flat plate and captured by the probe, (e) measuring the binding force of the nucleic acid obtained in step (d) to the target substance, and (f ) It includes a step of repeating steps (b) to (e) until a nucleic acid aptamer having a constant binding force measured in step (e) appears.

  In one embodiment, the nucleic acid aptamer is a DNA aptamer.

  In one embodiment, the target substance is an amino acid, a peptide or a protein.

  In one embodiment, the target substance is leucine.

  In one embodiment, the flat plate is a gold plate.

  In one embodiment, the step (b) is obtained in (b1) a step of fixing avidin on the surface of a flat plate, (b2) a step of preparing a nucleic acid having a biotin terminal, and (b3) a step (b1). A step of fixing the nucleic acid obtained in the step (b2) to the surface of the flat plate.

  In one embodiment, step (c) above is performed in a solution containing methionine.

  In one embodiment, the binding force is a dissociation constant for the target substance measured by a surface plasmon resonance method.

In one embodiment, the constant value is 1.55 × 10 ⁻⁶ M or less.

The present invention provides a DNA aptamers obtained by the above method, the DNA aptamer, the dissociation constant for L- leucine as measured by surface plasmon resonance exhibits less 1.55 × ^{10 -6} M.

  The present invention relates to a base sequence from position 1 to position 64 of the base sequence described in SEQ ID NO: 3 or a complementary sequence thereof, and a base sequence from position 1 to position 26 of the base sequence described in SEQ ID NO: 4 or a complementary sequence thereof A DNA aptamer having at least one base sequence selected from the group consisting of:

  ADVANTAGE OF THE INVENTION According to this invention, the nucleic acid aptamer which has the high specific binding power with respect to small molecules, such as an amino acid, and the method of screening efficiently the nucleic acid aptamer which has a high specific binding power can be provided. According to the present invention, the possibility of applying a nucleic acid aptamer as a sensor element of a biosensor or a targeting molecule of a drug delivery system is expanded.

It is a schematic diagram which shows the outline of the method of this invention. It is a graph (c) which shows the histogram (a) and (b) which show the binding power of L-leucine and single-stranded DNA, and the average value. It is a graph which shows the average value of the binding force of L-leucine or L-methionine, and single stranded DNA. It is a schematic diagram which shows the fixation procedure of L-leucine to a SPR sensor chip. Sensorgram (a) showing the time-dependent change of SPR signal when LA-1 is injected into the SPR sensor chip to which L-leucine is immobilized, and a graph showing the relationship between the concentration of LA-1 and the equilibrium value of SPR signal ( b). Sensorgram (a) showing the time-dependent change of the SPR signal when LB-1 is injected into the SPR sensor chip to which L-leucine is immobilized, and a graph showing the relationship between the concentration of LB-1 and the equilibrium value of the SPR signal ( b).

  The atomic force microscope (AFM) used in the present invention is a type of scanning probe microscope, and refers to a microscope that obtains an image by detecting the force acting between atoms of a probe and a sample. Examples thereof include atomic force microscopes manufactured by Seiko Instruments Inc., Olympus Corporation, and Shimadzu Corporation.

  The nucleic acid aptamer referred to in the present invention refers to a nucleic acid molecule that specifically binds to a target substance. The binding force of the nucleic acid aptamer with the target substance needs to show a constant value as described later. Examples of the nucleic acid aptamer include a DNA aptamer and an RNA aptamer, and a DNA aptamer is preferable from the viewpoint of stability. The length of the base sequence of the nucleic acid is not particularly limited, but is preferably 20 to 100 bases. As shown in FIG. 1, those having a hairpin type, a bulge type, a G-quartet type, or the like are known as the secondary structure, but the secondary structure is not particularly limited.

  The material of the atomic force microscope (AFM) probe (hereinafter sometimes referred to as a cantilever) is not particularly limited. Gold or gold coated materials are preferred. A gold monomolecular crystal specifically binds to a molecule having a thiol group or a disulfide group to form a monomolecular film (self-assembled film formation).

  The target substance may be immobilized on the probe of the atomic force microscope as long as it can be immobilized with a force stronger than the force acting by the method of immobilizing nucleic acids on the surface of the flat plate (detailed below). For example, the method of fixing through a covalent bond is mentioned. Examples of a method for fixing via a covalent bond include a method using a cross-linking agent. Examples of the crosslinking agent include DTSSP (3,3′-dithiobis [sulfosuccinimidylpropionate]), SPDP (N-succinimidyl 3- [2-pyridyldithio] propionate), EMCS (N- (6- Maleimidocaproyloxy) succinimide).

  The target substance used in the present invention is not particularly limited. It doesn't matter whether inorganic or organic. Examples of organic substances include organic compounds, amino acids, peptides, proteins, nucleic acids, lipids, and sugars. It may be a cell, cell tissue, microorganism or the like.

  The flat plate used in the present invention is not particularly limited as long as it can fix nucleic acids. A flat plate is preferable, and gold or gold-coated material is preferable.

  The nucleic acid may be immobilized on the surface of the flat plate as long as it can be immobilized with a force weaker than the force acting by the method of immobilizing the target substance on the probe of the atomic force microscope, and preferably noncovalent bonding is performed. It is a method of fixing via. Examples of the method for fixing via a non-covalent bond include a method for fixing via an ionic bond and a method for fixing via an intermolecular force. A method of fixing via intermolecular force is preferred. Examples of immobilization via intermolecular forces include, for example, immobilization via antibody-antigen binding force, immobilization via ligand-receptor binding force, and avidin-biotin binding force. The method of fixing via is mentioned.

  The method of immobilizing via the binding force between avidin and biotin is preferably a step of immobilizing avidin on the surface of the plate, a step of preparing a nucleic acid having biotin at the end, and a surface of biotin terminated on the surface of the plate on which avidin is immobilized. A step of immobilizing the nucleic acid contained in

  Examples of avidin include egg white-derived avidin and Streptomyces-derived streptavidin.

  Examples of the method for fixing avidin on the surface of the flat plate include a method using a crosslinking agent. Examples of the crosslinking agent include DTSSP, SPDP, and EMCS.

  The method for preparing a nucleic acid having biotin at its end is not particularly limited. For example, a method of chemically synthesizing nucleic acid and covalently binding biotin to the 5 'end or 3' end can be mentioned. Alternatively, a method in which a primer having biotin at the 5 'end is prepared in advance and a nucleic acid is synthesized by PCR or the like using this primer.

  The method for immobilizing a nucleic acid having biotin at its end on the surface of a plate on which avidin is immobilized is not particularly limited. For example, a method of immersing a flat plate on which avidin is fixed in a solution of nucleic acid having a biotin end is mentioned. The concentration of the nucleic acid in the solution of the nucleic acid having biotin at the end is preferably 1 μM.

  The method of scanning the surface of the flat plate on which the nucleic acid is immobilized with the probe on which the target substance is immobilized is not particularly limited. For example, there are a method of scanning in the atmosphere and a method of scanning in a liquid. In consideration of physiological conditions, a method of scanning in a liquid is preferable. From the viewpoint of increasing the efficiency of screening, a method of scanning in a solution containing a substance having a structure similar to the target substance is more preferable. For example, when the target substance is leucine, it is more preferable that methionine having a structure similar to leucine is contained in the solution. For example, when the target substance is valine, it is more preferable to contain alanine and isoleucine having a structure similar to valine in the solution.

  There is no particular limitation on the method for recovering the nucleic acid captured by the probe fixed with the target substance released from the surface of the flat plate fixed with the nucleic acid. An example is the PCR method.

  A method for measuring the binding strength of the obtained nucleic acid to the target substance is not particularly limited. For example, a method of measuring by an AFM method and a method of measuring a dissociation constant by a surface plasmon resonance (SPR) method can be mentioned. The SPR method is preferable in that kinetic analysis can be performed in addition to the analysis of the binding force.

In the present invention, the above steps are repeated until a nucleic acid aptamer having a constant binding force appears. The constant value is preferably 2 × 10 ⁻⁶ M or less, more preferably 1.55 × 10 ⁻⁶ M or less when the binding force is measured as a dissociation constant by the SPR method.

A DNA aptamer can be obtained by the above method. The thus obtained DNA aptamer of the present invention has, for example, the base sequence shown in SEQ ID NO: 3 or 4 or a complementary sequence thereof. DNA aptamers of the invention, the dissociation constant for L- leucine as measured by SPR method show the following 1.55 × ^{10 -6} M.

  EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited thereto. In the following examples,% represents% (w / v).

Example 1: Fixation of L-leucine to AFM cantilever AFM (Seiko Instruments Co., Ltd.) cantilever (Bio-level; gold coating; spring constant: 0.03 N / m; resonance frequency: 37 kHz; lever length : 60 μm; lever height: 7 μm) was left in an ozone generator (Waken Pharmaceutical Co., Ltd.) for 1 hour to remove organic matter on the cantilever surface. The cantilever was immersed in 100 μL of 4 mg / mL DTSSP (3,3′-dithiobis [sulfosuccinimidyl propionate]; manufactured by PIERCE), left to stand at room temperature for 30 minutes, and then ultrapure. Washed with 10 mL of water. In this process, the thiol group (—SH) formed by breaking the disulfide bond (S—S) in the DTSSP molecule is bonded to the gold monomolecular crystal on the surface of the cantilever. Next, the sample was immersed in 100 μL of a 20 mg / mL L-leucine (manufactured by Nacalai Tesque) solution prepared with a phosphate buffer (PBS; 100 mM phosphate, 150 mM NaCl; pH 7.5), and light-shielded at room temperature for 1 hour after location, and washed folding buffer (50mM Tris-HCl, 300mM NaCl , 30mM KCl, 5mM MgCl 2) at 10 mL. In this process, the succinimide group at the end of the DTSSP molecule is bonded to the amino group in the L-leucine molecule. In this way, an AFM cantilever in which L-leucine was immobilized was prepared.

Example 2: Immobilization of single-stranded DNA on the surface of a metal plate (round 1 screening)
(Preparation of double-stranded DNA library)
Using a mixed solution (N60 solution) of single-stranded DNA having a random sequence of 60 bases as a template DNA solution, PCR reaction was performed under the following reaction solution composition and reaction conditions.

<Reaction solution composition>
Template DNA solution 1μL
P1 primer (SEQ ID NO: 1) 2 μL
P2 primer (SEQ ID NO: 2) 2 μL
dNTPs (2 mM each) 5 μL
10 x buffer 5 μL
Distilled water 3.5μL
Polymerase 0.5 μL
50μL in total
As dNTPs (2 mM each), 10 × buffer and polymerase, those of Expand Long Template PCR System (Roche Diagnostics Co., Ltd.) were used.

<Reaction conditions>
95 ° C .: 1 minute → 95 ° C .: 2 minutes → (95 ° C .: 15 seconds → 72 ° C .: 30 seconds) × 20 → 72 ° C .: 10 minutes → 4 ° C.

  Subsequently, 10 μL of 6 × sample buffer (30% glycerol, 0.25% bromophenol blue, 1 mM EDTA) was mixed with 50 μL of the reaction solution, and the mixture was electrophoresed in a 9% polyacrylamide gel. A TBE buffer (10 mM Tris-HCl, 10 mM boric acid, 2 mM EDTA) was used as the electrophoresis buffer. The electrophoresed gel is immersed in a 0.1% ethidium bromide solution, and the gel taken out from the ethidium bromide solution is irradiated with 365 nm UV to contain double-stranded DNA having a length of about 80 to 120 base pairs. The gel was cut out. Double-stranded DNA was purified from the excised gel according to a conventional method, and dissolved in 100 μL of TE buffer (10 mM Tris-HCl, 1 mM EDTA; pH = 8.0) to prepare a double-stranded DNA solution.

(Preparation of single-stranded DNA library with biotin at the end)
Using the double-stranded DNA solution as a template DNA solution, a PCR reaction was performed under the following reaction solution composition and reaction conditions. The biotinylated P1 primer having biotin at the 5 ′ end was prepared by consigning to Life Technologies Japan Co., Ltd.

<Reaction solution composition>
Template DNA solution 1μL
Biotinylated P1 primer (SEQ ID NO: 1) 2 μL
dNTPs (2 mM each) 5 μL
10 x buffer 5 μL
Distilled water 36.5μL
Polymerase 0.5 μL
50μL in total
As dNTPs (2 mM each), 10 × buffer and polymerase, those of Expand Long Template PCR System (manufactured by Roche Diagnostics) were used.

<Reaction conditions>
95 ° C .: 1 minute → 95 ° C .: 2 minutes → (95 ° C .: 15 seconds → 72 ° C .: 30 seconds) × 45 → 72 ° C .: 10 minutes → 4 ° C.

  Subsequently, ethanol was mixed with the reaction solution to cause precipitation. The precipitate was washed with 70% ethanol and then dissolved in 45 μL of TE buffer to prepare a single-stranded DNA solution.

(Immobilization of single-stranded DNA on the surface of a metal plate)
5 μL of a mixed solution of 150 μL of 377 part A (epoxy resin; manufactured by Epoxy Technology) and 150 μL of 377 part B (hardening agent: manufactured by Epoxy Technology) is dropped onto a slide glass (manufactured by Matsunami Glass Industrial Co., Ltd.), and then a gold-deposited wafer (stock) Company SUMCO). This was put into a muffle furnace (manufactured by Denken Co., Ltd.), heated at 150 ° C. for 1 hour to harden the epoxy resin, and then naturally cooled. The wafer was peeled off from the slide glass to obtain a slide glass having a gold surface as a gold plate.

  To this metal plate, 100 μL of a 4 mg / mL DTSSP solution prepared with a 20 mM acetate buffer (pH 5.0) was carefully added dropwise, and then the metal plate was allowed to stand at room temperature for 30 minutes, and then ultrapure water. Washed with 10 mL. In this process, the thiol group (—SH) formed by breaking the disulfide bond (S—S) in the DTSSP molecule is bonded to the gold monomolecular crystal on the surface of the gold plate. To this metal plate, 100 μL of a 1 mg / mL streptavidin (manufactured by SIGMA) solution prepared in PBS was carefully added dropwise, and then the metal plate was left to stand at room temperature for 1 hour, and then washed with 10 mL of PBS. In this process, the succinimide group at the end of the DTSSP molecule is bonded to the amino group in the streptavidin molecule. To this metal plate, 100 μL of a single-stranded DNA solution prepared so that OD (260 nm) = 0.4 with a folding buffer was carefully dropped, and then the metal plate was left to stand at room temperature for 1 hour in the dark. , And washed with 10 mL of folding buffer containing 0.01% Tween 20 and then with 10 mL of folding buffer. In this way, a gold plate on which single-stranded DNA was immobilized was prepared.

Example 3: Scanning of metal plate surface with AFM cantilever (round 1 screening)
The surface of the metal plate produced in Example 2 was scanned using the AFM cantilever prepared in Example 1 according to a conventional method under the following scanning conditions, and the force (L (L) acting between the AFM cantilever and the metal plate surface) -The binding force between leucine and single-stranded DNA was measured. The results are shown in FIG.

<Scanning conditions>
AFM: Contact mode, FCM (Force Curve Mapping) measurement mode Resolution: 20 μm × 20 μm → 512 × 512 pixels Scanning frequency: 1 Hz
Scanning solution: 100 mM methionine (manufactured by Nacalai Tesque) solution prepared in folding buffer

Example 4: Recovery of single-stranded DNA trapped in an AFM cantilever (round 1 screening)
The cantilever which finished the scan of Example 3 was washed with 10 mL of folding buffer containing 0.01% Tween 20, then immersed in 200 μL of TE buffer containing 1% DMSO, heated at 98 ° C. for 5 minutes, Cool on ice for 5 minutes. The solution was collected, ethanol was mixed with the solution to cause precipitation. The precipitate was washed with 70% ethanol, and then dissolved in 20 μL of TE buffer to obtain a single-stranded DNA eluate.

Example 5: Round 2 screening In the same manner as in Examples 2 to 4, except that the single-stranded DNA eluate obtained in Example 4 was used instead of the N60 solution as the template DNA solution in Example 2. A single-stranded DNA eluate was obtained.

Example 6: Round 3 screening As in Example 2, except that the single-stranded DNA eluate obtained in Example 5 was used as the template DNA solution instead of the N60 solution. A single-stranded DNA eluate was obtained. The result of measuring the binding force between L-leucine and single-stranded DNA is shown in FIG.

Example 7: Round 4 screening In Example 2, the single-stranded DNA eluate obtained in Example 6 was used as the template DNA solution instead of the N60 solution. A single-stranded DNA eluate was obtained.

  The average value of the binding strength between L-leucine and single-stranded DNA measured in Examples 5, 6 and 7 is shown in FIG. As shown in FIG.2 (c), the average value of the binding force of L-leucine and single strand DNA is shown in Examples 2 to 4 (Round 1), Example 5 (Round 2), and Example 6 (Round). As the rounds of 3) and screening progressed, they increased to 66.2 pN, 89.5 pN, and 134.8 pN, respectively, but in Example 7 (Round 4), they decreased to 129.0 pN despite the progress of the round. . Finally, the highest value of the average binding force between L-leucine and single-stranded DNA was the same as that in Example 6 (Round 3), and the elution of single-stranded DNA obtained in Example 5 (Round 2). It was 134 pN when the liquid was used. Thus, it was found that single-stranded DNA having a high binding force for L-leucine can be obtained by screening in only two rounds. Therefore, the method of the present invention can greatly reduce the number of rounds of screening compared with the conventional SELEX method.

Example 8: Evaluation of single-stranded DNA obtained in Example 5 (Round 2 screening) In Example 2, the single-stranded DNA eluate obtained in Example 5 was used as the template DNA solution instead of the N60 solution. A metal plate on which single-stranded DNA was immobilized was prepared in the same manner as in Example 2 except that it was used.

(Evaluation of binding force to L-leucine)
The L-leucine prepared in Example 1 was immobilized under the same scanning conditions as in Example 3 except that the folding buffer was used instead of the 100 mM methionine solution prepared in the folding buffer as the scanning solution. Using the AFM cantilever, the surface of the prepared metal plate was scanned, and the force (binding force between L-leucine and single-stranded DNA) acting between the AFM cantilever and the metal plate surface was measured. The results are shown in FIG. The binding force between L-leucine and single-stranded DNA was 67.2 pN.

(Evaluation of binding strength to L-methionine)
An AFM cantilever in which L-methionine was immobilized was prepared in the same manner as in Example 1 except that L-methionine was used instead of L-leucine.

  The AFM cantilever with the L-methionine prepared above was immobilized under the same scanning conditions as in Example 3 except that the folding buffer was used instead of the 100 mM methionine solution prepared in the folding buffer as the scanning solution. The surface of the metal plate produced as described above was scanned to measure the force (binding force between L-methionine and single-stranded DNA) acting between the AFM cantilever and the metal plate surface. The results are shown in FIG. The binding force between L-methionine and single-stranded DNA was 16.6 pN.

  As shown in FIG. 3, when the metal plate on which the single-stranded DNA obtained in Example 5 was immobilized was used, the binding force between L-leucine and the single-stranded DNA was L-methionine and one. It was larger than the binding force with the strand DNA. From this, it is considered that in Example 5, a single-stranded DNA having a high specific binding force to L-leucine was obtained.

Example 9: Isolation and nucleotide sequencing of single-stranded DNA obtained in Example 5 (Round 2 screening) (Isolation of single-stranded DNA)
A PCR reaction was carried out in the same manner as in Example 1 except that the single-stranded DNA eluate obtained in Example 5 was used as the template DNA solution.

  Subsequently, ethanol was mixed with the reaction solution to cause precipitation. The precipitate was washed with 70% ethanol, and then dissolved in 20 μL of TE buffer to prepare a single-stranded DNA solution.

  Next, the ligation reaction was performed at 16 ° C. for 6 hours or more with the following reaction solution composition.

<Reaction solution composition>
pTA2 vector (Toyobo Co., Ltd.) 1μL
Single-stranded DNA solution 4μL
Solution I (DNA Ligation Kit Ver.2.1; manufactured by Takara Bio Inc.) 5 μL
10μL in total

  Next, 10 μL of the reaction solution was mixed with 100 μL of E. coli competent cells (Nova blue; manufactured by Takara Bio Inc.), and Escherichia coli was transformed with the mixed solution according to a conventional method. Transformants were screened on LB agar medium containing 50 μg / mL ampicillin.

  Subsequently, the plasmid was extracted from the obtained Escherichia coli transformant according to a conventional alkaline SDS method. An equal amount of 13% polyethylene glycol solution was mixed with the plasmid extract to cause precipitation, and the precipitate was dissolved in 50 μL of TE buffer to prepare a plasmid solution.

  Subsequently, in order to confirm whether or not the obtained plasmid had an insert, the obtained plasmid solution was used as a template DNA solution, and a PCR reaction was performed under the following reaction solution composition and reaction conditions.

<Reaction solution composition>
Template DNA solution 0.4μL
P1 primer (SEQ ID NO: 1) 0.4 μL
P2 primer (SEQ ID NO: 2) 0.4 μL
dNTPs (2 mM each) 1.0 μL
10 x Buffer 1.0 μL
Distilled water 6.78μL
Polymerase 0.02 μL
10μL in total
As dNTPs (2 mM each), 10 × buffer and polymerase, those of Expand Long Template PCR System (Roche Diagnostics Co., Ltd.) were used.

<Reaction conditions>
95 ° C .: 2 minutes → (95 ° C .: 15 seconds → 72 ° C .: 30 seconds) × 20 → 72 ° C .: 10 minutes → 4 ° C.

  Next, in the same manner as in Example 2, 10 μL of the reaction solution was mixed with 2 μL of 6 × sample buffer, and the mixture was electrophoresed in a 9% polyacrylamide gel. The electrophoresed gel is immersed in a 0.1% ethidium bromide solution, and the gel taken out of the ethidium bromide solution is irradiated with 365 nm UV to determine the presence or absence of double-stranded DNA of about 80 to 120 base pairs in length. confirmed. In this way, plasmid clones having inserts were screened.

(Decoding the base sequence of single-stranded DNA)
About the plasmid clone which has insert, PCR reaction was performed on the following reaction liquid composition and reaction conditions, using a plasmid solution as a template DNA solution.

<Reaction solution composition>
Template DNA solution 1μL
M13 Rv primer (Takara Bio Inc.) 1μL
DTCS Quick Start Master Mix (BECKMAN) 4μL
4μL of distilled water
10μL in total

  Subsequently, the base sequence of the obtained reaction solution was decoded using BECKMAN CEQ8000 (manufactured by BECKMAN). The base sequences of the decoded clones LA-1 (64mer) and LB-1 (26mer) are shown in SEQ ID NOs: 3 and 4, respectively.

  LA-1 contains the sequence of “GGGGTGGGGG” inside, suggesting the presence of a G quartet structure (see FIG. 1).

Example 10: Evaluation by SPR method of single-stranded DNA isolated in Example 9 (Immobilization of L-leucine and L-methionine to SPR sensor chip)
50 μL of 10 mg / mL L-leucine solution prepared with 25 mM borate buffer (pH 8.0) and 15 mg / mL EMCS (N- (6-maleimidocaproyloxy) succinimide prepared with DMSO; Dojin Corporation (Manufactured by Chemical Laboratories) 50 μL of the solution was mixed and allowed to react at room temperature under light shielding for 1.5 hours. The reaction solution (100 μL) was mixed with 50 mM borate buffer (pH 8.0) (900 μL) to prepare a 1 mg / mL L-leucine-EMCS solution.

  Similarly, a 1 mg / mL L-methionine-EMCS solution was prepared.

  Then, according to the following fixing procedure, L-leucine or L-methionine as a control was fixed to the sensor chip of the SPR measuring device Biacore 3000 (manufactured by GE Healthcare Biosciences). In this fixation, as shown in FIG. 4, cystamine and EMCS were interposed between the SPR sensor chip and L-leucine so that the single-stranded DNA could sufficiently interact with L-leucine.

<Fixing procedure>
1. EDC (1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride; manufactured by GE Healthcare Biosciences) and NHS (N-hydroxysuccinimide; manufactured by GE Healthcare Biosciences) are mixed in equal amounts 100 μL of the solution was injected at a flow rate of 10 μL / min (fixation of NHS on the SPR sensor chip).
2. 70 μL of 500 mM cystamine solution was injected 3 times at a flow rate of 5 μL / min (binding of cystamine to NHS).
3. 300 μL of a 100 mM dithiothreitol (DTT) solution prepared with ultrapure water was injected at a flow rate of 10 μL / min (reduction of cystamine).
30 μL of a 4.1 mg / mL L-leucine-EMCS solution or L-methionine-EMCS solution was injected 10 times at a flow rate of 5 μL / min (binding of L-leucine-EMCS or L-methionine-EMCS to cystamine).

  The amount immobilized on the SPR sensor chip was 252 RU for L-leucine and 167 RU for L-methionine.

(Evaluation of single-stranded DNA isolated in Example 9 by surface plasmon resonance method)
The concentration of the LA-1 and LB-1 solutions isolated in Example 9 was adjusted to 1 μM with TE buffer, and 20 μL of this solution was introduced into the SPR sensor chip on which L-leucine had been immobilized at a flow rate of 5 μL / min. It was ejected. Similarly, solutions adjusted to 0.5 and 0.4 μM were injected. The results are shown in FIGS. 5 and 6, respectively.

As shown in FIG. 5B, for LA-1, a value of dissociation constant K _D = 1.33 × 10 ⁻⁷ was obtained. As shown in FIG. 6B, a value of K _D = 1.55 × 10 ⁻⁶ was obtained for LB-1. These values are considered sufficient to function as aptamers for small molecules such as amino acids. In addition, when the SPR sensor chip which fixed L-methionine was used, the SPR signal which shows the coupling | bonding of LA-1 or LB-1 and L-methionine was not obtained (data not shown). Thus, a DNA aptamer could be obtained by two screenings.

  ADVANTAGE OF THE INVENTION According to this invention, the nucleic acid aptamer which has the high specific binding power with respect to small molecules, such as an amino acid, and the method of screening efficiently the nucleic acid aptamer which has a high specific binding power can be provided. According to the present invention, the possibility of applying a nucleic acid aptamer as a sensor element of a biosensor or a targeting molecule of a drug delivery system is expanded.

Claims (11)

A method for screening nucleic acid aptamers using an atomic force microscope,
(A) a step of fixing the target substance to the probe of the atomic force microscope,
(B) a step of immobilizing a nucleic acid on the surface of a flat plate via a non-covalent bond;
(C) scanning the surface of the flat plate obtained in step (b) with the probe obtained in step (a);
(D) collecting the nucleic acid released from the surface of the flat plate and captured by the probe in step (c);
(E) a step of measuring the binding force of the nucleic acid obtained in step (d) to the target substance, and (f) a step until a nucleic acid aptamer whose binding force measured in step (e) shows a constant value appears. A method comprising the steps of repeating (b) to (e).   The method according to claim 1, wherein the nucleic acid aptamer is a DNA aptamer.   The method according to claim 1 or 2, wherein the target substance is an amino acid, a peptide, or a protein.   The method according to any one of claims 1 to 3, wherein the target substance is leucine.   The method according to claim 1, wherein the flat plate is a gold plate. The step (b)
(B1) a step of fixing avidin on the surface of the flat plate,
(B2) preparing a nucleic acid having biotin at its end;
(B3) The method according to any one of claims 1 to 5, comprising a step of immobilizing the nucleic acid obtained in step (b2) on the surface of the flat plate obtained in step (b1).   The method according to claim 4, wherein the step (c) is performed in a solution containing methionine.   The method according to claim 1, wherein the binding force is a dissociation constant for the target substance measured by a surface plasmon resonance method. The method according to claim 8, wherein the constant value is 1.55 × 10 ⁻⁶ M or less. A DNA aptamer obtained by the method according to claim 8 or 9, DNA aptamers dissociation constant for L- leucine as measured by surface plasmon resonance exhibits less 1.55 × ^{10 -6} M.   From the group consisting of the base sequence from position 1 to position 64 of the base sequence shown in SEQ ID NO: 3 or its complementary sequence, and the base sequence from position 1 to position 26 of the base sequence shown in SEQ ID NO: 4 or its complementary sequence A DNA aptamer having at least one selected base sequence.

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