JP4706019B2 - Aptamer identification method - Google Patents

Description

  The present invention relates to a method for identifying aptamers.

  Aptamers are nucleic acid ligands that bind to specific molecules. Aptamers were first reported in 1990 by Gold et al. And acquired using a method called Systematic Evolution of Ligands by EXponential Enrichment (SELEX) (Tuerk, C. and Gold L. (1990), Science, 249, 505-510). In this method, one type of target molecule is usually immobilized on a carrier, a nucleic acid library is added thereto, a nucleic acid that binds to the target molecule is recovered, this is amplified, and the target molecule is added again to the immobilized carrier. . By repeating this step about 10 times, aptamers having high binding power to the target molecule are concentrated, the base sequence thereof is determined, and the aptamer that recognizes the target molecule is obtained.

  In principle, the conventional SELEX method should be able to search for aptamers that bind to all target molecules, but in practice it excludes aptamers that adsorb nonspecifically to the carrier used for immobilization. Can not do it.

  The SELEX method basically includes (1) addition of a library to a carrier on which a target molecule is immobilized, (2) removal of unbound nucleic acid, (3) recovery of an aptamer bound to the target molecule, (4) This is a technique of repeating the four steps of amplification of the collected aptamer by PCR or the like. The amplification process of (4) is usually performed by PCR, but since nucleic acids that form stable higher-order structures are difficult to be amplified by PCR, a large amount of aptamers that are easily amplified are amplified and strongly bound to the target molecule. Aptamers are not necessarily amplified. This is probably because aptamers that bind to the target molecule often have a stable higher-order structure. Therefore, aptamers that strongly bind to the target molecule are excluded at the PCR stage, and are not specific to the immobilized carrier. This is thought to be because only the nucleic acid bound to is amplified. In fact, in principle, aptamers should be able to be acquired for all target molecules, but the types of aptamers acquired so far are not necessarily many.

  That is, the problems of SELEX of the prior art are that it is difficult to eliminate aptamers that bind non-specifically to the immobilized carrier and that the non-specific adsorption aptamers are easily amplified in the amplification stage.

Prior art document information related to the present invention includes the following.
Patent 2763958 Tuerk, C. and Gold L. (1990), Science, 249, 505-510

  An object of the present invention is to solve the above-mentioned problems of SELEX and to provide a method for efficiently obtaining an aptamer that can specifically bind to a target molecule.

  The present inventors prepare a carrier in which a target molecule and a non-target molecule are immobilized at different locations, add a nucleic acid library to the carrier, and separate aptamers bound to the target molecule. It has been found that aptamers with high specificity can be obtained.

In a first aspect, the present invention is a method for identifying an aptamer that can bind to a target molecule comprising:
(A) preparing a first carrier on which a target molecule is immobilized and a second carrier on which at least one non-target molecule is immobilized;
(B) contacting a library of single-stranded nucleic acids with the first and second carriers under conditions where the first carrier and the second carrier compete for binding with the single-stranded nucleic acid. ,
(C) recovering the single-stranded nucleic acid bound to the target molecule immobilized on the first carrier, and (d) determining the base sequence of the recovered single-stranded nucleic acid.
The method including each of these steps is provided.

  Preferably, in the method of the present invention, the first carrier is used as the second carrier between the step of bringing the single-stranded nucleic acid library into contact with the carrier and the step of recovering the single-stranded nucleic acid bound to the target molecule. Further comprising the step of separating from.

  Preferably, the step of recovering the single-stranded nucleic acid is performed using a ligand that is known to bind to the target molecule.

In another aspect, the present invention provides a method for identifying an aptamer that can bind to a target molecule comprising:
(A) preparing a first carrier on which a target molecule is immobilized and a second carrier on which at least one non-target molecule is immobilized;
(B) contacting a library of single-stranded nucleic acids with the first and second carriers under conditions where the first carrier and the second carrier compete for binding with the single-stranded nucleic acid. ,
(C) recovering the single-stranded nucleic acid bound to the target molecule immobilized on the first carrier;
(D) amplifying the recovered single-stranded nucleic acid;
(E) contacting the amplified single-stranded nucleic acid with the first carrier and the second carrier under conditions where the first carrier and the second carrier compete for binding with the single-stranded nucleic acid. ,
(F) recovering the single-stranded nucleic acid bound to the target molecule immobilized on the first carrier;
(G) Repeat steps (d)-(f) as necessary, and (h) determine the base sequence of the recovered single-stranded nucleic acid.
The method including each of these steps is provided.

In another aspect, the present invention provides a method for identifying an aptamer that can bind to a target molecule comprising:
(A) preparing a first carrier on which a target molecule is immobilized and a second carrier on which at least one non-target molecule is immobilized;
(B) contacting a library of single-stranded nucleic acids with the first and second carriers under conditions where the first carrier and the second carrier compete for binding with the single-stranded nucleic acid. ,
(C) recovering the single-stranded nucleic acid bound to the target molecule immobilized on the first carrier;
(D) determining the base sequence of the recovered single-stranded nucleic acid;
(E) preparing a second library of single-stranded nucleic acids based on the determined base sequence, wherein the second library of single-stranded nucleic acids has a partial sequence of the determined base sequence and The remaining sequence is composed of a plurality of single-stranded nucleic acids having a different base sequence from the determined base sequence,
(F) the first and second carriers of the second library of single-stranded nucleic acids under conditions where the first and second carriers compete for binding to the single-stranded nucleic acid. Contact with
(G) recovering the single-stranded nucleic acid bound to the target molecule immobilized on the first carrier;
(H) Repeat steps (e)-(g) if necessary, and (i) determine the base sequence of the recovered single-stranded nucleic acid.
The method including each of these steps is provided.

In another aspect, the present invention provides a method for identifying an aptamer that can bind to a first target molecule and an aptamer that can bind to a second target molecule, comprising:
(A) preparing a first carrier on which a first target molecule is immobilized and a second carrier on which a second target molecule is immobilized;
(B) contacting a library of single-stranded nucleic acids with the first and second carriers under conditions where the first carrier and the second carrier compete for binding with the single-stranded nucleic acid. ,
(C) separating the first carrier and the second carrier;
(D) Separate the single-stranded nucleic acid bound to the first target molecule immobilized on the first carrier and the single-stranded nucleic acid bound to the second target molecule immobilized on the second carrier. And (e) determining the base sequence of each single-stranded nucleic acid recovered;
The method including each of these steps is provided.

  The present invention provides a method for identifying aptamers that can bind to a target molecule. As used herein, “aptamer” refers to a nucleic acid ligand that binds to a specific molecule. The aptamer may be DNA or RNA, and may be a nucleic acid mimic such as PNA. Aptamers may also contain various modifications in bases, sugars, and / or internucleotide linkages.

  In the first aspect of the present invention, in the first step, a first carrier on which a target molecule is immobilized and a second carrier on which at least one non-target molecule is immobilized are prepared.

  “Target molecule” refers to a target molecule for which it is desired to identify an aptamer that binds to the molecule, and includes, for example, nucleic acids, proteins, peptides, saccharides, natural or synthetic chemicals, and the like. It is not limited. “Non-target molecule” refers to any molecule other than the target molecule. The number of non-target molecules used in the method of the present invention is not limited, but it is preferable to use a large number of non-target molecules in order to obtain highly specific aptamers. “Carrier” refers to a substance capable of immobilizing a target molecule on the surface thereof, and examples thereof include cellulose, plastic, glass, and resin. The carrier can be in the form of a tube, a membrane, a porous, a bead or the like. Preferred carriers include nitrocellulose membranes, PVDF membranes, PES (polyethylsulfonate) membranes, latex beads, polystyrene beads, agarose gel beads, CPG (controlled porous glass) beads, gold nanoparticles, magnetic microparticles, silicon chips, etc. . The carrier may be provided on the microchannel. Techniques for immobilizing target molecules on a carrier are well known in the art.

  In the second step, the single-stranded nucleic acid library is brought into contact with the first carrier and the second carrier described above. A “single-stranded nucleic acid library” is a collection of single-stranded nucleic acids having a random sequence, and the nucleic acid may be DNA or RNA. The length of the nucleic acid varies depending on the size of the aptamer desired to be obtained and the size and structure of the target molecule, and is not particularly limited, but is generally 10-80 bases, preferably 15-60 bases, more preferably 20 -40 bases. A random sequence refers to the sequence of a collection of single-stranded nucleic acids, which is a mixture of different types of bases at all or part of the base sequence. For example, a base sequence of a collection of nucleic acids in which A, G, C, and T are present at a probability of 25% at specific positions in the sequence is a random sequence. The probability of the presence of each base is not limited to 25% and can vary. Moreover, the base sequence of a specific position may be fixed among the base sequences of the single-stranded nucleic acid. For example, all single stranded nucleic acids of a library may contain sequences that are convenient for amplification and sequencing at the same position in the sequence. In addition, as seen in the case of the offspring library described later, the library is such that only a specific position of the base sequence is random based on the sequence information obtained in the previous round, and the sequence of the remaining positions is fixed. May be created.

  A library of single-stranded nucleic acids can be easily produced by a nucleic acid synthesizer using a mixture of appropriate nucleic acid monomer units by a method well known in the art. In addition, the single-stranded nucleic acid may be labeled with a detectable label, which facilitates monitoring of the binding between the target and the nucleic acid and recovery in a later step.

  The contact between the single-stranded nucleic acid and the carrier in the second step is performed under conditions such that the first carrier and the second carrier compete for binding with the single-stranded nucleic acid. That is, it does not carry out with a separate tube, For example, it contacts in the same container simultaneously. Thereby, the probability that an aptamer having an affinity for a carrier or a non-target target molecule binds to the target molecule is reduced, and an aptamer that specifically binds to the target molecule can be obtained more selectively. Specifically, the contact under competing conditions can be performed as follows. A target molecule and a non-target molecule are spotted and immobilized on different locations on a single membrane carrier (PVDF, nitrocellulose, etc.), and a library of single-stranded nucleic acids dissolved in an appropriate solution is added to the membrane. Incubate for an appropriate time. Alternatively, a carrier (membrane, beads, etc.) on which a target molecule is immobilized and a carrier on which a non-target molecule is immobilized may be placed in the same container, and a single-stranded nucleic acid library may be added to this container and incubated. Alternatively, a column on which target molecules are immobilized and a column on which non-target molecules are immobilized may be prepared, and a single-stranded nucleic acid library dissolved in an appropriate solution may be repeatedly passed through these columns. Alternatively, a single-stranded nucleic acid library may be added to a protein array or bead array and incubated.

  After the second step, the carrier is preferably washed to remove single-stranded nucleic acid that has not bound to any carrier. For washing, a solution in which the single-stranded nucleic acid library is dissolved in the second step may be used, a solution obtained by adding a surfactant or the like to this solution, or an appropriate solution having a different pH or salt concentration. May be used. Washing is usually performed once to several times. Next, preferably, the first carrier and the second carrier are separated. In the separation, for example, when the target molecule and the non-target molecule are spotted at different locations on a single film-like carrier, the target molecule and the non-target molecule are separated from each other by cutting off the portion where the target molecule is spotted. In the case where the target molecule is immobilized, it can be carried out by taking out only the first carrier on which the target molecule is immobilized.

  In the third step, the single-stranded nucleic acid bound to the target molecule immobilized on the first carrier is recovered. As is well known in the art, the recovery can be performed by eluting the aptamer using a solution that can dissociate the bond between the target molecule and the nucleic acid. Alternatively, the aptamer may be eluted by applying a known ligand known to bind to the target molecule. The single-stranded nucleic acid recovered here can be a mixture of a plurality of single-stranded nucleic acids having different base sequences.

  In the fourth step, the base sequence of the recovered single-stranded nucleic acid is determined. The determination of the base sequence can be easily performed using a method well known in the art, for example, the dideoxy method. Alternatively, the sequence may be determined using an automated sequencer. In this way, one or more aptamers that can bind to the target molecule can be identified.

  According to a second aspect of the method of the present invention, in the method for identifying an aptamer that can bind to a target molecule, after collecting a single-stranded nucleic acid that binds to the target molecule, this is amplified and contacted with the carrier again. Features. The first to third steps of this method are the same as in the first aspect. In the fourth step, the recovered single-stranded nucleic acid is amplified. Amplification means increasing the copy number of the recovered single-stranded nucleic acid, and may be performed by subcloning into an appropriate vector and introducing it into an appropriate host, or by PCR. Since PCR is considered to have low amplification efficiency of nucleic acids having a higher order structure, it is preferable to reduce the number of reaction cycles when PCR is used.

  In the fifth step, the single-stranded nucleic acid amplified in this manner is subjected to the same conditions as in the second step under the conditions in which the first carrier and the second carrier compete for binding to the single-stranded nucleic acid. In contact with the first carrier and the second carrier. In the sixth step, the single-stranded nucleic acid bound to the target molecule is recovered in the same manner as in the third step. As described above, washing is preferably performed between the fifth step and the sixth step, and preferably, the first carrier and the second carrier are separated.

  If necessary, the fourth to sixth steps can be repeated. The repetition is generally 2-10 rounds, preferably 3-6 rounds. This makes it possible to concentrate single-stranded nucleic acids having high binding affinity for the target molecule. Next, the base sequence of the recovered single-stranded nucleic acid is determined in the same manner as in the fourth step of the first aspect. In this way, one or more aptamers that can bind to the target molecule can be identified.

  According to a third aspect of the method of the present invention, in the method for identifying an aptamer capable of binding to a target molecule, after determining the base sequence of the recovered single-stranded nucleic acid, the second library is prepared based on the sequence information. It is characterized by creating. The first to fourth steps of this method are the same as in the first aspect. In the fifth step, a second library of single-stranded nucleic acids is prepared based on the base sequence determined in the fourth step. This second library is a so-called offspring library. Usually, there are a plurality of base sequences determined in the fourth step, and this progeny library is designed by the following method based on the base sequences of the plurality of aptamers. First, a plurality of those determined base sequences are divided into sequence blocks of about 3 to 5 bases, for example, and these sequence blocks are shuffled among a plurality of individuals on a computer, and a new aptamer obtained by hybridizing them is obtained. A large number of base sequences are generated. Further, one or a plurality of mutations are generated on a random site of the generated new base sequence on a computer, and a large number of new aptamer base sequences are generated. A large number of new aptamers are synthesized based on these base sequences to form a progeny library.

  In the sixth step and the seventh step, using the second library thus obtained, the library is brought into contact with the first carrier and the second carrier in the same manner as in the second and third steps. The single-stranded nucleic acid bound to the target molecule is recovered.

  If necessary, the fifth to seventh steps can be repeated. The repetition is generally 2-10 rounds, preferably 3-6 rounds. This selectively concentrates single-stranded nucleic acids having high binding affinity for the target molecule. Next, the base sequence of the recovered single-stranded nucleic acid is determined in the same manner as in the fourth step of the first aspect. In this way, one or more aptamers that can bind to the target molecule can be identified.

  According to a fourth aspect of the method of the present invention, in the method for identifying an aptamer that can bind to a target molecule, a plurality of aptamers that specifically bind to a plurality of target molecules are identified. In the first step of this method, a first carrier on which a first target molecule is immobilized and a second carrier on which a second target molecule is immobilized are prepared. In the second step, the library of single-stranded nucleic acids is contacted with the first carrier and the second carrier under conditions where the first carrier and the second carrier compete for binding to the single-stranded nucleic acid. Let In the third step, the first carrier and the second carrier are separated. Specific examples of the carrier and the procedures for immobilization, contact, and separation in the first to third steps are the same as those in the first to third steps of the first aspect. By performing the contacting step under conditions where multiple target molecules compete for binding to single-stranded nucleic acid, it has a higher binding affinity for each target molecule and more than for other target molecules. Aptamers having a low binding affinity can be efficiently obtained.

  Next, in the fourth step, the single-stranded nucleic acid bound to the first target molecule immobilized on the first carrier and the first target molecule bound to the second target molecule immobilized on the second carrier 1 Single-stranded nucleic acids are collected separately, and in the fifth step, the base sequence of each collected single-stranded nucleic acid is determined. In this way, one or more aptamers that can bind to the target molecule can be identified.

  After recovering the single-stranded nucleic acid in the fourth step, it is amplified in the same manner as in the second embodiment of the method of the present invention, and the next round in which the target molecule and the non-target molecule are contacted with the immobilized carrier. By performing, aptamers that can specifically bind to the target molecule may be concentrated. Further, after determining the base sequence of the single-stranded molecule in the fifth step, a progeny library may be created in the same manner as in the third aspect of the method of the present invention, and the next round may be performed.

  This method can be used to simultaneously identify aptamers for multiple target molecules as well as two types of target molecules. The more target molecules that compete for binding to single-stranded nucleic acids, the more specific aptamers that bind to each target are considered to be. Therefore, the method of the present invention is particularly suitable for use with, for example, a protein array in which a large number of proteins are immobilized on a plate or a chip, or a bead array in which a large number of beads each having a different protein immobilized thereon.

  Hereinafter, specific procedures of the method of the present invention will be described with reference to the drawings.

  FIG. 1 shows a specific example of a method for identifying aptamers using a membrane as a carrier. A target molecule and a non-target molecule are spotted and immobilized on a PVDF membrane, and a single-stranded nucleic acid library is added to the membrane and incubated. After washing the membrane, the portion spotted with the target molecule is cut out, and the single-stranded nucleic acid bound to this portion is extracted. Next, the extracted single-stranded nucleic acid is amplified and added to a new PVDF membrane on which target molecules and non-target molecules are immobilized, and a second round of selection is started. After performing several rounds of selection in this way, by determining the base sequence of the obtained single-stranded nucleic acid, it is possible to identify an aptamer that specifically binds to the target molecule without binding to the non-target molecule. it can.

  FIG. 2 shows another embodiment of a method for identifying aptamers that specifically bind to a plurality of target molecules, respectively, using a membrane as a carrier. A plurality of target molecules are spotted and immobilized on the PVDF membrane, and a single-stranded nucleic acid library is added to the membrane and incubated in the same manner as in the embodiment of FIG. After washing the membrane, single-stranded nucleic acids bound to multiple target molecules are extracted together, amplified, and then added to a new PVDF membrane to initiate a second round of selection. After several rounds of selection in this way, single-stranded nucleic acids bound to each target molecule are isolated separately and each base sequence is determined to specifically bind to multiple target molecules. A plurality of aptamers can be identified.

  FIG. 3 shows a specific example of a method for identifying an aptamer using a bead array. A bead array is an array in which a large number of beads on which proteins are immobilized are arranged on a plate on which depressions are formed. The beads array is made from Nagai H, Murakami Y, Morita Y, Yokoyama K, Tamiya E. Development of a microchamber array for picoliter PCR, Anal Chem. 2001 Mar 1; 73 (5): 1043-7. The method can be prepared by adding beads to a microchamber array and developing them. Multiple target molecules and optionally non-target molecules are immobilized on each bead and placed in place on the array. A single-stranded nucleic acid library is added to the array and incubated. Next, each bead is recovered, and single-stranded nucleic acid bound to the target molecule is recovered, and its sequence can be determined. Alternatively, beads are randomly placed on the array, and after binding to a single-stranded nucleic acid library, the beads are collected, the sequence of the single-stranded nucleic acid on the beads is determined, and the protein on the same bead is subjected to mass spectrometry. It is also possible to identify by a conventional method such as. In this way, aptamers that bind to each of a large number of target molecules can be identified. The beads do not necessarily have to be arranged on the array. One type of target molecule is immobilized on one bead, and these are mixed and placed in a microtube. At the same time, a single-stranded nucleic acid library is added to compete. It can also be made. If color-coded fluorescent beads are used, they can be separated by FACS. Further, as described above, the protein may be identified by a method such as mass spectrometry.

  FIG. 4 shows a specific example of a method for identifying an aptamer using a microchannel. A microchannel is a channel provided on a substrate of glass, metal, plastic, or the like with a width of about 10 μm to about 1 mm, and a pump for controlling the flow of liquid on the channel on the substrate, A valve or the like is provided. A plurality of target molecules are immobilized on the microchannel, and the single-stranded nucleic acid library is flowed while circulating through the channel. After washing the channel with an appropriate washing solution, a ligand known to bind to one of these target molecules is passed through the channel, and the single-stranded nucleic acid bound to the target molecule is eluted. After washing the channel again, a ligand known to bind to another target molecule is passed through the channel. In this way, single-stranded nucleic acids that bind to a large number of target molecules can be sequentially recovered. By determining the base sequence of the recovered single-stranded nucleic acid, aptamers that bind to each of a large number of target molecules can be identified.

  EXAMPLES The present invention will be described below in more detail with reference to examples, but the present invention is not limited to these examples.

  Using a PVDF (polyvinylidene difluoride) membrane as an immobilization carrier, DNA aptamers that bind to a plurality of proteins (insulin, glucagon, PQQGDF (pyrroloquinoline quinone glucose dehydrogenase)) were searched.

  The PVDF membrane was cut to 2 cm × 2 cm and immersed in ethanol for 3 minutes. The membrane was washed with about 30 ml of MilliQ® water for 5 minutes and then with about 30 ml of binding buffer (10 mM Tris-HCl, pH 7.4, 100 mM KCl) for 15 minutes. 5 μg / 10 μl of insulin, glucagon and PQQGDH were added to one surface of the washed PVDF membrane, respectively, and immobilized. The membrane was washed with a binding buffer and then stained with CBB to confirm that the protein was immobilized.

  A random library of FITC-modified 30-mer ssDNA (5′-gTACCAgCTTATTCAATTNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNAgATAgTATgTTCATCAg-3 ′) was produced by request synthesis. ssDNA was prepared to 1 nmol / 100 μl with a binding buffer, heated at 95 ° C. for 3 minutes, and then gradually cooled to 25 ° C. for 30 minutes to fold. The prepared ssDNA solution was diluted to 3000 μl with a binding buffer, added to the PVDF membrane on which the protein was immobilized, and incubated at 25 ° C. for 1 hour.

  The membrane was washed with binding buffer containing approximately 30 ml of 0.05% Tween. A portion of the membrane on which each protein was blotted was cut out, and 400 μl of phenol and 200 μl of 7M urea were added thereto and shaken for 30 minutes. This was centrifuged for 5 minutes (15000 rpm, 4 ° C.), and the resulting supernatant was treated with chloroform. SsDNA was recovered by ethanol precipitation, dried and dissolved in 20 μl MilliQ® water. The ssDNA concentration was calculated from the fluorescence intensity of FITC.

  The obtained ssDNA was PCR amplified using a FITC-modified primer (GTACCAGCTTATTCAATT) and a biotin-modified primer (CTGATGAACATACTATCT) (20 cycles, 94 ° C, denatured at 34 s, annealed at 46 ° C, 30 s, 72 ° C, 30 s) Extension reaction), the obtained dsDNA was added to avidin-immobilized beads, and ssDNA was prepared by eluting with 0.15 M NaOH. Next, libraries that bind to insulin, glucagon and PQQGDH collected in the first round were added to separate PVDF membranes, and the second round was started. In the same manner, a total of 6 rounds of concentration were performed.

  The ratio of moles of bound library to moles of library added in each round is shown in the table.

  Although the proportion of the library eluted in the first to third rounds did not increase markedly, it was observed that the proportion of aptamer bound to PQQGDH was slightly higher. In the fourth round, an increase in the recovery rate of aptamers bound to PQQGDH was observed, and it is considered that a DNA aptamer that recognizes PQQGDH to some extent was obtained.

  So far, PQQGDH has been adsorbed alone on agarose beads and microtiter plates, and SELEX has been attempted. However, only aptamers exhibiting non-specific adsorption are amplified, and aptamers exhibiting specificity to PQQGDH can be obtained. could not. In accordance with the present invention, it is considered that an aptamer that specifically binds to PQQGDH was obtained by immobilizing a plurality of proteins on the same immobilization carrier and competing for binding with ssDNA.

  Aptamers identified according to the present invention are useful for the detection and quantification of target molecules such as nucleic acids, proteins, saccharides, and analysis of functions and interactions.

FIG. 1 shows a specific example of the method of the present invention for identifying aptamers using a membrane as a carrier. FIG. 2 shows another embodiment of a method for identifying aptamers using a membrane as a carrier. FIG. 3 shows a specific example of the method of the present invention for identifying aptamers using a bead array. FIG. 4 shows a specific example of the method of the present invention for identifying aptamers using microchannels.

Claims (13)

A method for identifying an aptamer that can bind to a target molecule, comprising:
(A) preparing a first carrier on which a target molecule is immobilized and a second carrier on which at least one non-target molecule is immobilized;
(B) contacting a library of single-stranded nucleic acids with the first and second carriers under conditions where the first carrier and the second carrier compete for binding with the single-stranded nucleic acid. ,
(C) recovering the single-stranded nucleic acid bound to the target molecule immobilized on the first carrier, and (d) determining the base sequence of the recovered single-stranded nucleic acid.
The method including each process of these. The method of claim 1, further comprising separating the first carrier from the second carrier between step (b) and step (c). A method for identifying an aptamer that can bind to a target molecule, comprising:
(A) preparing a first carrier on which a target molecule is immobilized and a second carrier on which at least one non-target molecule is immobilized;
(B) contacting a library of single-stranded nucleic acids with the first and second carriers under conditions where the first carrier and the second carrier compete for binding with the single-stranded nucleic acid. ,
(C) recovering the single-stranded nucleic acid bound to the target molecule immobilized on the first carrier;
(D) amplifying the recovered single-stranded nucleic acid;
(E) contacting the amplified single-stranded nucleic acid with the first carrier and the second carrier under conditions where the first carrier and the second carrier compete for binding with the single-stranded nucleic acid. ,
(F) recovering the single-stranded nucleic acid bound to the target molecule immobilized on the first carrier;
(G) Repeat steps (d)-(f) as necessary, and (h) determine the base sequence of the recovered single-stranded nucleic acid.
The method including each process of these. 4. The method of claim 3, further comprising separating the first carrier from the second carrier between step (b) and step (c). The method according to claim 3 or 4, further comprising the step of separating the first carrier from the second carrier between step (e) and step (f). A method for identifying an aptamer that can bind to a target molecule, comprising:
(A) preparing a first carrier on which a target molecule is immobilized and a second carrier on which at least one non-target molecule is immobilized;
(B) contacting a library of single-stranded nucleic acids with the first and second carriers under conditions where the first carrier and the second carrier compete for binding with the single-stranded nucleic acid. ,
(C) recovering the single-stranded nucleic acid bound to the target molecule immobilized on the first carrier;
(D) determining the base sequence of the recovered single-stranded nucleic acid;
(E) preparing a second library of single-stranded nucleic acids based on the determined base sequence, wherein the second library of single-stranded nucleic acids has a partial sequence of the determined base sequence and The remaining sequence is composed of a plurality of single-stranded nucleic acids having a different base sequence from the determined base sequence,
(F) the first and second carriers of the second library of single-stranded nucleic acids under conditions where the first and second carriers compete for binding to the single-stranded nucleic acid. Contact with
(G) recovering the single-stranded nucleic acid bound to the target molecule immobilized on the first carrier;
(H) Repeat steps (e)-(g) if necessary, and (i) determine the base sequence of the recovered single-stranded nucleic acid.
The method including each process of these. 7. The method of claim 6, further comprising separating the first carrier from the second carrier between step (b) and step (c). The method according to claim 6 or 7, further comprising the step of separating the first carrier from the second carrier between step (f) and step (g). A method for identifying an aptamer that can bind to a first target molecule and an aptamer that can bind to a second target molecule, comprising:
(A) preparing a first carrier on which a first target molecule is immobilized and a second carrier on which a second target molecule is immobilized;
(B) contacting a library of single-stranded nucleic acids with the first and second carriers under conditions where the first carrier and the second carrier compete for binding with the single-stranded nucleic acid. ,
(C) separating the first carrier and the second carrier;
(D) Separate the single-stranded nucleic acid bound to the first target molecule immobilized on the first carrier and the single-stranded nucleic acid bound to the second target molecule immobilized on the second carrier. And (e) determining the base sequence of each single-stranded nucleic acid recovered;
The method including each process of these.   The method according to any one of claims 1 to 9, wherein the step of recovering single-stranded nucleic acid is performed using a ligand known to bind to a target molecule.   The method according to any one of claims 1 to 10, wherein the carrier is a membrane capable of immobilizing a protein.   The method according to any one of claims 1 to 10, wherein the carrier is a bead capable of immobilizing a protein. The method according to claim 1, wherein the carrier is provided on a microchannel.