JP7629641B2 - Affinity chromatography based on artificial nucleic acids - Google Patents

Description

The present invention relates to an affinity chromatography packing material based on an artificial nucleic acid, and an affinity chromatography column packed with the packing material. The present invention also relates to a method and a kit for detecting a target nucleic acid using a column packed with an affinity chromatography packing material based on an artificial nucleic acid.

One technique for separating and identifying target molecules is affinity chromatography, which uses binding affinity to separate target molecules. For example, target molecules can be separated by binding affinity, such as antigen-antibody reactions, binding between proteins and their receptors, and complementary binding of nucleic acids. Specifically, an affinity column is filled with a carrier to which a ligand that specifically binds to the target molecule is immobilized, and a sample is passed through the affinity column. The target molecule contained in the sample binds to the ligand, while other molecules pass through the affinity column, resulting in a difference in elution from the affinity column between the target molecule and other molecules. It is possible to separate or identify the target molecule by utilizing this difference.

On the other hand, artificial nucleic acids are nucleic acids whose physical properties, such as hydrogen bonding pattern, higher-order structure, and polarity, have been changed by adding chemical modifications to the nucleic acid base, sugar, and phosphate diester moiety. Specific functions can be chemically introduced into artificial nucleic acids, making it possible to achieve functions that are not possible with natural nucleic acids. For example, it is possible to create artificial nucleic acids that have high resistance to nucleases and fluorescent properties that respond to the microscopic environment. Examples of such artificial nucleic acids include peptide nucleic acid (PNA), locked nucleic acid (LNA), phosphorothioates (S-oligonucleotides, S-oligos), and morpholino oligonucleotides.

For example, PNA is a compound with a structure similar to that of nucleic acids, with the backbone formed by peptide bonds instead of phosphate bonds. PNA has excellent hybridization affinity with DNA, is not repulsed by negative charges caused by phosphate groups, is not affected by salt concentration, and has good biological stability and heat resistance.

In the prior art, there is a report of immobilizing PNA, a type of artificial nucleic acid, on a carrier with a His tag and packing it into a column (Non-Patent Document 1). This method shows the potential of affinity chromatography using PNA, but no actual examples have been reported of how a specific target nucleic acid could be concentrated and purified, and it is questionable whether target DNA can actually be purified using this affinity chromatography.

Orum H., Nielsen P.E., Jorgensen M., Larsson C., Stanley C., Koch T. BioTechniques Vol. 19 No. 3, pp. 472-480 (1995)

The present invention aims to prepare an affinity column with excellent nucleic acid affinity and stability, and to use it to detect and identify nucleic acids.

As a result of intensive research to solve the above problems, the inventors discovered that by binding a nucleic acid, including an artificial nucleic acid as a ligand, to a carrier via a peptide bond, a disulfide bond or a linker, it is possible to obtain a filler for affinity chromatography whose nucleic acid affinity is not affected by temperature and pH and which has excellent storage stability, and thus completed the present invention.

For example, the present invention includes the following embodiments:
[1] A packing material for affinity chromatography comprising a carrier and a nucleic acid ligand immobilized on the carrier,
The nucleic acid ligand comprises at least one artificial nucleic acid selected from the group consisting of peptide nucleic acid (PNA), locked nucleic acid (LNA) and S-oligo;
A packing material for affinity chromatography, wherein the nucleic acid ligand is immobilized on the carrier by a peptide bond, a disulfide bond or a bond via a linker.
[2] The affinity chromatography packing material according to [1], wherein the nucleic acid ligand comprises PNA.
[3] The affinity chromatography packing material according to [1] or [2], wherein the nucleic acid ligand is an 8- to 30-mer.
[4] The affinity chromatography packing material according to any one of [1] to [3], comprising a carrier having immobilized thereon a nucleic acid ligand having a sequence complementary to a first sequence, and a nucleic acid ligand having a sequence complementary to a second sequence.
[5] The affinity chromatography packing material according to any one of [1] to [3], comprising a first carrier to which a nucleic acid ligand having a sequence complementary to a first sequence is immobilized, and a second carrier to which a nucleic acid ligand having a sequence complementary to a second sequence is immobilized.
[6] The affinity chromatography packing material according to [4] or [5], wherein the first sequence and the second sequence have sequences complementary to different regions of the target nucleic acid.
[7] The affinity chromatography packing material according to [4] or [5], wherein the first sequence and the second sequence have sequences that differ by 1 to 3 bases.
[8] The affinity chromatography packing material according to any one of [1] to [7], wherein the linker is selected from the group consisting of SMCC, EMCS, LC-SMCC, SM(PEG)2, SIA, Sulfo-SANPAH, MBS, and an epoxy resin curing agent.
[9] A plurality of linkers are immobilized on the surface of the support,
The affinity chromatography packing material according to any one of [1] to [8], wherein the nucleic acid ligand is bound to some of the plurality of linkers.
[10] The affinity chromatography packing material according to [9], wherein a capping agent is bound to a linker among the plurality of linkers to which the nucleic acid ligand is not bound.
[11] The affinity chromatography packing material according to any one of [1] to [10], wherein the carrier is selected from the group consisting of resins, porous particles, gels, and magnetic beads.
[12] A column for affinity chromatography packed with the affinity chromatography packing material according to any one of [1] to [11].
[13] The affinity chromatography column according to [12], which is provided with a heating means.
[14] A method for detecting or identifying a target nucleic acid in a sample, comprising:
A step of introducing a sample into an affinity chromatography column packed with the affinity chromatography packing material according to any one of [1] to [11];
detecting or identifying a target nucleic acid based on elution from the affinity chromatography column.
[15] The method according to [14], wherein the affinity chromatography packing comprises a carrier having immobilized thereon a nucleic acid ligand having a sequence complementary to one sequence of a target nucleic acid.
[16] The method according to [14], wherein the affinity chromatography packing comprises a support having immobilized thereon a nucleic acid ligand having a sequence complementary to a first sequence of a target nucleic acid, and a nucleic acid ligand having a sequence complementary to a second sequence of the target nucleic acid.
[17] The method according to [14], wherein the affinity chromatography packing comprises a first support to which a nucleic acid ligand having a sequence complementary to a first sequence of a target nucleic acid is immobilized, and a second support to which a nucleic acid ligand having a sequence complementary to a second sequence of the target nucleic acid is immobilized.
[18] The method according to [16] or [17], wherein the first sequence and the second sequence have sequences that differ by 1 to 3 bases.
[19] A kit for detecting a target nucleic acid in a sample, comprising the affinity chromatography packing material according to any one of [1] to [11].

The present invention provides a packing material and column for affinity chromatography. The packing material and column for affinity chromatography of the present invention are based on artificial nucleic acids and therefore have excellent nucleic acid affinity and storage stability. Therefore, by using such a packing material and column for affinity chromatography, it is possible to detect and identify a target nucleic acid in a short time with high sensitivity. Therefore, the present invention is useful in fields such as detection and identification of nucleic acids, diagnostic agents, and molecular biology research.

The chemical structure of Fmoc-PNA12mer-C ₁₀ -Cys-NH ₂ (1) is shown. The chemical structure of LNA 12mer (GacAatGtaGct)-C ₆ -SH (3) is shown. The chemical structure of S-oligo 12mer (GacAatGtaGct)-C ₃ -SH (4) is shown. 1 shows a synthetic scheme for immobilizing the ligand PNA (1) on an affinity column. 1 shows a synthetic scheme for immobilizing the ligand PNA (2) on an affinity column. 1 shows a synthetic scheme for immobilizing ligands PNA(1) and PNA(2) on an affinity column. 1 shows a synthetic scheme for immobilizing the ligand LNA (3) on an affinity column. 1 shows a synthetic scheme for immobilizing ligand S-oligo (4) on an affinity column. An outline of an affinity column evaluation method is given below. FIG. 1 shows conceptual diagrams of variations in the form of immobilizing a ligand on an affinity column. 1 is a graph showing the evaluation results of an affinity column using the ligand PNA (1). 1 is a graph showing the evaluation results of an affinity column using the ligand PNA (2). 1 is a graph showing the evaluation results of an affinity column containing two types of carriers onto which two types of ligands, PNA (1) and PNA (2), are immobilized. 1 is a graph showing the evaluation results of an affinity column containing a carrier onto which two types of ligands, PNA (1) and PNA (2), are immobilized. 1 is a graph showing the outline and results of evaluation of an affinity column for DNA having base mismatches.

The present invention is described in detail below. This application claims priority from Japanese Patent Application No. 2020-015458, filed on January 31, 2020, the entire contents of which are incorporated herein by reference.
In one aspect, the present invention provides a packing material for affinity chromatography comprising a carrier and a nucleic acid ligand immobilized on the carrier, the packing material comprising:
The nucleic acid ligand comprises at least one artificial nucleic acid selected from the group consisting of peptide nucleic acid (PNA), locked nucleic acid (LNA) and S-oligo;
The present invention relates to a packing material for affinity chromatography, in which the nucleic acid ligand is immobilized on the carrier by a peptide bond, a disulfide bond, or a bond via a linker.

The affinity chromatography packing material according to the present invention includes a carrier on which nucleic acids, including artificial nucleic acids, are immobilized as ligands that bind to target nucleic acids. An artificial nucleic acid is a nucleic acid whose physical properties, such as hydrogen bond pattern, higher-order structure, and polarity, have been changed by adding chemical modifications to the nucleic acid base, sugar, and phosphate diester moiety. Examples of artificial nucleic acids are shown below.

LNA (Locked Nucleic Acid) is an artificial nucleic acid in which the sugar conformation of the nucleic acid is completely fixed to N-type (RNA type) (the arrow part in the structure below is fixed to completely fix the N-type). It forms a strong double strand with the target single-stranded DNA/RNA through Watson-Crick hydrogen bonds. Since the sugar conformation is fixed in advance, it is thought that the entropy loss during double strand formation with RNA is greatly reduced. This enables LNA to form an unprecedentedly stable double strand.

The ability of artificial nucleic acids to form double strands is generally evaluated by measuring the melting temperature (Tm) of the double strand. A detailed analysis of the Tm value of a double strand consisting of LNA and target RNA revealed an increase in the Tm value of +4 to +6 degrees per LNA modification (Obika S et al., Tetrahedron Lett, 39:5401-5404, 1998).

LNA also has higher enzyme resistance than natural oligonucleotides. It has been tested for the activity of RNaseH, a ribonuclease that cleaves RNA forming a DNA/RNA hybrid double strand to produce single-stranded DNA, and it has been reported that LNA has higher enzyme resistance than natural nucleotides (Wahlestedt et al., (2000) PNAS 97, 5633-5638). It is also known that LNA shows higher enzyme resistance in mixmers than in gapmers. It has been found that phosphorothioates, another artificial nucleic acid, also show similar enzyme resistance. Phosphorothioates (also called S-oligonucleotides or S-oligos) are oligoDNAs in which all phosphate groups at the binding sites between nucleotides have been sulfurized (structure below), and as mentioned above, they have nuclease resistance (Wahlestedt et al., (2000) PNAS 97, 5633-5638).

Another type of artificial nucleic acid is peptide nucleic acid (PNA). PNA is a compound with a structure similar to that of nucleic acids, but with a backbone made up of peptide bonds instead of phosphate bonds. PNA has excellent hybridization affinity with DNA, is not repelled by negative charges due to phosphate groups, is not affected by salt concentration, and has good biological stability and heat resistance.

Another type of artificial nucleic acid is the morpholino oligonucleotide. Morpholinos are oligonucleotides that contain a morpholine ring in place of the ribose or deoxyribose of nucleic acids (RNA, DNA).

The nucleic acid ligand contains at least one artificial nucleic acid, and may be entirely made up of artificial nucleic acids, or may contain artificial nucleic acids in part. When it contains artificial nucleic acids in part, for example, it may contain artificial nucleic acids every few bases (2, 3, 4, 5, etc.), or it may contain artificial nucleic acids in the region at the end opposite to the end of the nucleic acid ligand immobilized on the carrier.

The nucleic acid ligand has an appropriate affinity for the target nucleic acid to be isolated or detected by affinity chromatography. Preferably, the nucleic acid ligand has a sequence that binds (hybridizes) to the target nucleic acid, for example, a sequence that is complementary to one sequence of the target nucleic acid (target sequence). In this specification, "sequence" means a base sequence unless otherwise specified.

In one embodiment, the affinity chromatography packing material of the present invention comprises a support having immobilized thereon a nucleic acid ligand having a sequence complementary to a first sequence and a nucleic acid ligand having a sequence complementary to a second sequence.

In another embodiment, the affinity chromatography packing material of the present invention comprises a first support having a nucleic acid ligand fixed thereto, the nucleic acid ligand having a sequence complementary to a first sequence, and a second support having a nucleic acid ligand fixed thereto, the nucleic acid ligand having a sequence complementary to a second sequence.

In the above embodiment, the first sequence and the second sequence may have sequences complementary to different regions of the same target nucleic acid, or may have sequences complementary to regions of different target nucleic acids. In a specific embodiment, the first sequence and the second sequence have sequences that differ by 1 to 3 bases (e.g., 1 base), that is, the first sequence and the second sequence each have a sequence complementary to a region containing a 1 to 3 base polymorphism (e.g., a single nucleotide polymorphism).

"Complementary" or "complementarity" refers to the correspondence of bases in nucleic acids that base pair, such as the correspondence between adenine (A) and thymine (U) or uracil (U), and guanine (G) and cytosine (C). In this specification, it refers not only to perfect complementarity, but also to partial complementarity, which has partial complementarity to a certain sequence and therefore retains the ability to bind to that sequence.

The length of the nucleic acid ligand is selected so that it has an appropriate affinity for the target nucleic acid to be isolated or detected in affinity chromatography. Taking into consideration factors such as affinity retention and ease of synthesis, the length of the nucleic acid ligand is generally 8-30 mers, preferably 10-25 mers, and more preferably 10-23 mers.

A person skilled in the art can appropriately design the nucleic acid ligand, i.e., determine the overall length, the type and arrangement of the artificial nucleic acid contained therein, etc., taking into consideration the type and length of the target nucleic acid, the purpose, the type of elution solution used, etc. Furthermore, the nucleic acid ligand can also be prepared by conventional methods, for example, using a synthesis device capable of synthesizing artificial nucleic acids.

The carrier for immobilizing the ligand may be any carrier commonly used in affinity chromatography, and the material and form may be appropriately selected depending on the application, the type of eluent, the amount of elution, etc. For example, carriers in the form of porous particles, gels, resin magnetic beads, etc. may be used. It is preferable to use a carrier that is resistant to treatments such as heating, elution, and washing, and has excellent storage stability.

Ligands can be immobilized on supports by peptide bonds, disulfide bonds, or bonds via linkers. When using peptide bonds, peptide bonds can be obtained by converting the reactive end of the support to a carboxylic acid using a peptide condensing agent such as HATU to an active ester and then reacting with a primary amino group on the ligand. When the reactive end of the support is a primary amino group, it is also possible to obtain peptide bonds by converting the reactive end of the support to an active ester and then reacting with the carboxylic acid on the ligand to an active ester in advance. In the case of disulfide bonds, it is possible to use support and ligand with SH groups at both reactive ends and bond via an oxidation reaction.

When a ligand is fixed to a support via a linker, the linker can be, for example, a heterocrosslinker, an epoxy resin hardener, a homocrosslinker, etc. As hetero-crosslinkers, for example, reagents that share both N-hydroxysuccinimide ester and maleimide on both sides of a hydrocarbon backbone and whose active groups crosslink amino and sulfhydryl groups, respectively, can be used. Specific examples include SMCC (succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate), EMCS (N-ε-malemidocaproyl-oxysuccinimide ester), LC-SMCC (succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxy-(6-amidocaproate)), SM(PEG)2 (PEGylated SMCC crosslinker), SIA (succinimidyl iodoacetate), Sulfo-SANPAH (sulfosuccinimidyl 6-(4'-azido-2'-nitrophenylamino)hexanoate), and MBS (3-Maleimidobenzoic Acid N-Succinimidyl Ester). Epoxy resin curing agents can also be used as reagents for crosslinking amino groups or sulfhydryl groups, as with heterocrosslinkers; for example, jER Cure (registered trademark) (Mitsubishi Chemical) is commercially available. As homocrosslinkers, for example, reagents that have the same N-hydroxysuccinimide ester, imide ester, or maleimide at both ends of a hydrocarbon chain and crosslink between amino groups or sulfhydryl groups can be used, specifically DSG (disuccinimidyl glutarate), DMP (dimethyl pimelimidate), BMOE (bismaleimidoethane), etc. can be mentioned. As another homocrosslinker, a reagent that is composed of a polyethylene glycol chain in addition to a hydrocarbon chain and crosslinks between amino groups or sulfhydryl groups can also be used, specifically BS(PEG)5 (PEGylated bis(sulfosuccinimidyl)suberate), BM(PEG)3 (1,11-bismaleimido-triethyleneglycol), etc. can be used.

One of the preferred linkers is SMCC, which is a crosslinker having an active ester group and a maleimide group. For example, the _NH2 group bound to the carrier is reacted with the active ester group of SMCC, and the SH group introduced into the ligand is reacted with the maleimide group of SMCC, thereby immobilizing the ligand on the carrier via SMCC.

One or more linkers can be fixed to the surface of the carrier. One ligand can be fixed to a carrier to which one linker is fixed. On the other hand, when multiple linkers are fixed to the surface of the carrier, the ligand may be bound to a part of the multiple linkers, or the ligand may be bound to all of the multiple linkers. When a ligand is bound to a part of the multiple linkers, it is preferable to bind a capping agent to a linker to which no ligand is bound. Examples of such capping agents include 1,4-diaminobutane (amino group), H-Arg-NH ₂ ·2HCl (guanidino group), 5-aminovaleric acid (carboxyl group), amylamine (methyl group), 4-phenylbutylamine (phenyl group), and Fmoc-Lys-OH·HCl (amino group·carboxyl group). In addition, when multiple linkers are fixed to the surface of the carrier, one type of ligand (i.e., a ligand having the same sequence) may be bound, or two or more types of ligands (i.e., multiple ligands having different sequences) may be bound.

A spacer may be introduced into the nucleic acid ligand and/or carrier in consideration of ease of chemical reaction, stability of the chemical structure, improvement of solubility, etc. For example, C3, C6, C10, etc. can be used as the spacer.

The affinity chromatography packing material according to the present invention is capable of binding to a target nucleic acid with excellent binding affinity by introducing an artificial nucleic acid into the ligand, and is not affected by salt concentration, and has excellent biological stability (resistance to degradation by nucleases and proteases) and thermal stability. It can also be used repeatedly, and has the advantage of being low cost.

In another aspect, the present invention relates to an affinity chromatography column packed with the above-mentioned affinity chromatography packing material. The affinity chromatography column is not particularly limited as long as it is one that is conventionally used in affinity chromatography. Various types of affinity chromatography columns are commercially available, and a person skilled in the art can appropriately select the material, shape, and volume depending on the application, type of eluent, elution amount, equipment to be used, etc.

The affinity chromatography column according to the present invention may further include a heating means. The heating means may be integrated with the column or may be removable from the column. The heating means may heat the entire column or may heat only a portion of the column. The nucleic acid ligand used in the affinity chromatography packing according to the present invention can be used without being thermally denatured by heating with the heating means.

In a further aspect, the present invention provides a method for detecting or identifying a target nucleic acid in a sample, comprising the steps of:
Introducing the sample into an affinity chromatography column packed with the affinity chromatography packing material;
detecting or identifying a target nucleic acid based on elution from the affinity chromatography column.

The nucleic acid ligand used in the affinity chromatography packing material of the present invention can bind to a target nucleic acid. By utilizing this binding affinity, when a sample is introduced into an affinity chromatography column packed with the packing material, the target nucleic acid binds to the nucleic acid ligand while other molecules pass through, and the target nucleic acid in the sample can be detected or identified by examining the elution from the column.

The sample is not particularly limited as long as it is a sample for which the target nucleic acid is to be detected or identified. Examples include biological fluid samples (blood, serum, plasma, urine, etc.), tissue or cell samples, and environmental samples (soil, rivers, etc.). In the case of blood samples, it is preferable to store the blood on ice or in a refrigerator after collection. In addition, when using plasma, it is preferable to use EDTA as an anticoagulant, but heparin, sodium citrate, and other anticoagulants known or commonly used in the field can be used.

In the present invention, "detecting or identifying a target nucleic acid in a sample" means determining whether or not a target nucleic acid is present in a sample, as well as measuring the amount or concentration of the target nucleic acid in a sample.

In the method according to the present invention, a sample is introduced into an affinity chromatography column packed with the affinity chromatography packing material according to the present invention. The introduction of the sample into the column is not particularly limited. For example, the sample can be introduced into the column by gravity or by using a syringe, either as is or after dilution with an appropriate solution or buffer.

Next, the elution of the sample from the affinity chromatography column is examined, and the target nucleic acid is detected or identified according to the eluted fraction. The elution of the sample can be examined using a method or device known in the art, such as liquid chromatography/mass spectrometry (LC/MS). If the sample contains a target nucleic acid having a sequence complementary to the sequence of the ligand nucleic acid fixed to the column, the target nucleic acid binds to the ligand nucleic acid and is slowed down in elution. Therefore, it is possible to detect or identify the target nucleic acid based on such elution characteristics. In addition, the ligand nucleic acid distinguishes between base mismatches and binds. For example, the ligand nucleic acid can distinguish between 1-3 base mismatches, preferably 1-2 base mismatches, and particularly 1 base mismatches. Therefore, the method of the present invention can also be used to detect target nucleic acids having base mismatches.

The method of the present invention may include a step of heating the sample before or after the sample is introduced into the column. Since double-stranded nucleic acid cannot bind to a nucleic acid ligand, it is preferable to heat the double-stranded nucleic acid to thermally denature it into a single-stranded nucleic acid form. The heating temperature can be equal to or higher than the melting temperature of the double-stranded nucleic acid, for example, 75 to 100°C.

In another aspect, the present invention relates to a kit for detecting a target nucleic acid in a sample, comprising an affinity chromatography packing. The kit according to the present invention allows easy and convenient detection or identification of a target nucleic acid in a sample. The kit according to the present invention may also include other components useful for detecting a target nucleic acid in addition to the affinity chromatography packing, such as an eluent for affinity chromatography, a control, instructions, etc.

The present invention will now be described in more detail with reference to the following examples and drawings. However, the present invention is not limited to the following examples.

[Example 1] Preparation of the carrier for affinity column and column (1) Carrier for affinity column There are various products for affinity carriers. ChromSpeed ^TM CM103 (Mitsubishi Chemical) is a weakly acidic cation exchange resin of methacrylic acid type, and is used for purification of antibiotics and amino acids. We thought that it might be possible to prepare an affinity column that can obtain precise and pure target products by using a carrier used for purification of biopharmaceuticals, so we decided to use ChromSpeed ^TM CM103 this time.

(2) Affinity column cartridges For the cartridges packed with affinity carriers, Sigma-Aldrich's 0.5 mL Empty PP Rev Tube Kit was used. This is an empty column that allows the preparation of SPE cartridges using any packing material. In addition, since PNA is expensive, there was an issue of preparing cartridges with as little ligand as possible, so Tomoe Manufacturing Co., Ltd.'s Luer type cartridge Type Mini (0.1 mL) was also used in some cases.

(3) Method for immobilizing PNA on the carrier for affinity columns Since the synthesized ligand, PNA, has an _NH2 group on the aromatic ring, peptide bonds cannot be used to immobilize the ligand on the carrier. Therefore, we decided to use a crosslinker that can chemically bond two or more molecules by covalent bonds.

[Example 2] Synthesis of PNA (ligand) for affinity column A PNA for affinity column having the structure shown in Figure 1 was synthesized. The crosslinker used was SMCC having cyclohexane in the spacer portion and having an active ester group and a maleimide group. The maleimide group reacts with SH groups, and the active ester group reacts specifically with _NH2 groups. In order to utilize this feature of SMCC, Fmoc-Cys(Mmt)-OH having an SH group was introduced into the ligand. In addition, in order to avoid steric hindrance between PNA12mer and Fmoc-Cys(Mmt)-OH, Fmoc-Aund(11)-OH(C10) was introduced as a spacer (Figure 1). The base sequence of the PNA portion was GACAATGTAGCT (SEQ ID NO: 1).

Fmoc-PNA12mer-C ₁₀ -Cys was synthesized using the standard Fmoc solid-phase synthesis method. A Biotage fully automated microwave synthesizer, Initiator+Alstra, was used for the synthesis. The support used was Fmoc-NH-SAL-PEG Resin (0.22 mmol/g), whose N-terminus was protected with an Fmoc group. The PNA condensation reagents used were N,N'-diisopropylcarbodiimide (DIC) and ethyl cyanoglyoxylate-2-oxime (Oxyma), as recommended for the automated synthesizer, Initiator+Alstra, in a ratio of 5 equivalents of PNA monomer, 5 equivalents of DIC, and 5 equivalents of Oxyma. The condensation reagents for Fmoc-Cys(Mmt)-OH and Fmoc-Aund(11)-OH were O-(7-aza-1H-benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) and N,N-diisopropylethylamine (DIPEA) in the ratio of Fmoc-Cys(Mmt)-OH 5 equivalents, Fmoc-Aund(11)-OH 5 equivalents, HATU 6.5 equivalents, and DIPEA 2.3 equivalents, to obtain Fmoc-PNA12mer-C ₁₀ -Cys-NH ₂ (1).

[Example 3] Design of artificial nucleic acid (ligand) for affinity column other than PNA (1) Design of LNA (3) LNA for affinity column having the structure shown in Figure 2 was synthesized. LNA was purchased from Gene Design Co., Ltd. In designing LNA, since mixmers showed stronger enzyme resistance compared to gapmers, a structure was created in which LNA was inserted every 3 bases in the 12mer (in the sequence GacAatGtaGct (SEQ ID NO: 3), capital letters indicate the insertion positions). In addition, in order to adopt the same structure as PNA and to unify the conditions during immobilization, the 3' end was modified with a spacer (C6) and thiol.

(2) Design of S-oligo (4) An S-oligo for affinity column was synthesized with the structure shown in Figure 3. S-oligo with phosphorothioate was purchased from Eurofins. To standardize the insertion conditions, phosphorothioate was inserted every 3 bases as in LNA (in the sequence GacAatGtaGct (SEQ ID NO: 4), capital letters indicate the insertion positions). In addition, the 3' end was modified with a spacer (C3) and a thiol.

[Example 4] Immobilization of ligand PNA (1) on affinity column As described below, the PNA (1) prepared in Example 2 was immobilized on an affinity column ChromSpeed ^™ CM103.

(1) Reagent used: Diisopropylethylamine (DIPEA)
・11-[(9-Fluorenylmethoxycarbonyl)amino]undecanoic acid (Fmoc-Aund(11)-OH)・N,N'-dimethylformamide (DMF)
Dichloromethane (DCM)
・N-methylpyrrolidone (NMP)
・O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU)
・20% piperidine/NMP
・N,N'-disuccinimidyl carbonate (DSC)
6-[(9-fluorenylmethoxycarbonyl)amino]hexanoic acid (Fmoc-Acp(6)-OH)
・^ChromSpeedTM CM103
・Hexamethylenediamine (1,6-diaminohexane)
・4-(N-maleimidomethyl)cyclohexanecarboxylic acid N-succinimidyl ester (SMCC)
・L-cysteine

(2) Synthesis scheme The synthesis scheme is shown in Figure 4. An EYELA personal organic synthesizer (CCS-600) was used to immobilize PNA (1) on ChromSpeed ^TM CM103. To confirm the amount of reactive carboxylic acid per unit volume of ChromSpeed ^TM CM103 using the Fmoc quantification method, Fmoc-Acp(6)-OH was first introduced.

As a preparation for immobilization of PNA on ChromSpeed ^TM CM103 (5, approximately 0.08 meq/ml), 5 (2 mL) soaked in MeOH was replaced with DMF and dried. DSC (0.313 g, 1.2 mmol) was dissolved in DMF (2 mL) and DIPEA (195 μL, 1.2 mmol) was added to the reaction solution, which was then added to 5 and stirred at room temperature for 1 hour. After the reaction was completed, the reaction solution was removed and activation was performed again using the same concentration of DSC reaction solution. After the reaction was completed, the reaction solution was removed and washed with DMF (2 mL) to obtain 6.

Next, 1,6-diaminohexane (0.929 g, 8 mmol) was suspended in NMP (1 mL) and DMF (1 mL), then added to 6 and stirred at room temperature overnight. After the reaction was completed, the reaction solution was removed and washed once with DMF (2 mL) and five times with MeOH (2 mL) to obtain 7.

Next, Fmoc-Acp(6)-OH (0.141 g, 0.4 mmol), HATU (0.1977 g, 0.52 mmol), and DIPEA (90 μL, 0.52 mmol) were dissolved in NMP (1.5 mL) and added to 7, followed by stirring at room temperature for 1 hour. After completion of the reaction, the reaction solution was removed, and the condensation reaction was carried out again at room temperature for 30 minutes using the same concentration of Fmoc-Acp(6)-OH reaction solution. After confirming that the ninhydrin test was negative, the reaction solution was removed, and the mixture was washed twice with DMF (2 mL), twice with DCM (2 mL), and twice with MeOH (2 mL) to obtain 8.

Next, the amount of Fmoc was quantified using an absorption spectrometer, and the amount of Fmoc-Acp(6)-OH introduced was calculated. 20% piperidine/DMF (400 μL) was added to a well-dried solid support (5-10 mg), stirred at room temperature for 30 minutes, and the solution was diluted 50-fold with DMF to prepare a sample. The absorbance at 301 nm was measured, and the amount of Fmoc groups was calculated using the molar extinction coefficient ε (301 nm) of 7800 M ^-1 cm ^-1. The amount of Fmoc-Acp(6)-OH introduced in this case was 0.20838 mmol/g.

The Fmoc deprotection reaction was performed with 20% piperidine/DMF to obtain 9.

Next, SMCC (17.1 mg, 0.051 mmol) was dissolved in DMF (3 mL), added to 9 (48.9 mg), and stirred at room temperature for 1 hour. After the reaction was completed, the reaction solution was removed and washed with DMF (2 mL) to remove excess Sulfo-SMCC. After that, washing with DMF was repeated, and the absorbance (280 nm) was measured every 1 mL. Washing was continued until the absorbance was completely gone, and 10 was obtained.

Next, the ligand Fmoc-PNA12mer-C ₁₀ -Cys-NH ₂ (1, 1.94 mg, 0.51 μmol) was dissolved in DMF (500 μL) and HFIP (100 μL) and added to 10, and the mixture was stirred at room temperature overnight. After the reaction was completed, the reaction solution was removed and washed with DMF (2 mL) to remove excess Fmoc-PNA12mer-C ₁₀ -Cys-NH ₂ (1). The mixture was then repeatedly washed with DMF, and the absorbance (260 nm) was measured every 1 mL. The washing was continued until the absorbance was completely gone, yielding 11. Fmoc quantification using an absorption spectrophotometer revealed that the amount of Fmoc-PNA12mer-C10-Cys-NH ₂ (1) introduced into 10 was 0.01532 mmol/g.

Next, a capping reaction was carried out to inactivate unreacted SMCC. L-cysteine (61.8 mg, 0.51 mmol) was dissolved in NMP (10 mL) and added to 11, followed by stirring at room temperature for 3 hours. After completion of the reaction, the reaction solution was removed and washed with DMF (5 mL) to remove excess L-cysteine.

Finally, 20% piperidine/DMF was added and the mixture was stirred for 5 minutes to remove the Fmoc group of Fmoc-PNA12mer-C ₁₀ -Cys-NH ₂ (1) to obtain 12.

[Example 5] Immobilization of ligand PNA (2) on affinity column PNA (2) was immobilized on affinity column ChromSpeed ^™ CM103 as described below.

Using a procedure similar to that of Example 2, PNA (2) was synthesized having the base sequence GAAACCCAGCAG (sequence number 2) instead of the base sequence GACAATGTAGCCT (sequence number 1) of PNA (1).

The immobilization of PNA (2) onto ChromSpeed ^™ CM103 was carried out using an EYELA personal organic synthesizer, similar to the immobilization of PNA (1) in Example 4 (Figure 5). That is, the synthesis of 10 was carried out in the same manner as in Example 4, except that the ligand was changed to FMOC-PNA12MER-C ₁₀ -Cys-NH ₂ (2).

The ligand Fmoc-PNA12mer-C ₁₀ -Cys-NH ₂ (2, 1.93 mg, 0.51 μmol) was dissolved in DMF (600 μL) and HFIP (200 μL) and added to 10, and the mixture was stirred at room temperature overnight. After the reaction was completed, the reaction solution was removed and washed with DMF (2 mL) to remove excess Fmoc-PNA12mer-C ₁₀ -Cys-NH ₂ (2). The mixture was then repeatedly washed with DMF, and the absorbance (260 nm) was measured every 1 mL. The washing was continued until the absorbance was completely gone, yielding 13. Fmoc quantification using an absorption spectrophotometer revealed that the amount of Fmoc-PNA12mer-C ₁₀ -Cys-NH ₂ (2) introduced into 10 was 0.0113 mmol/g.

Next, a capping reaction was carried out to inactivate unreacted SMCC. L-cysteine (6.18 mg, 0.051 mmol) was dissolved in NMP (10 mL) and added to 13, and the mixture was stirred at room temperature for 3 hours. After the reaction was completed, the reaction solution was removed and washed with DMF (5 mL) to remove excess L-cysteine.

Finally, 20% piperidine/DMF was added and the mixture was stirred for 5 minutes to remove the Fmoc group of Fmoc-PNA12mer-C ₁₀ -Cys-NH ₂ (2) to obtain 14.

Example 6 Immobilization of Ligands PNA(1) and PNA(2) on Affinity Column As described below, a mixture of PNA(1) and PNA(2) (1:1) was immobilized on an affinity column ChromSpeed ^™ CM103.

The immobilization of a mixture (1:1) of PNA (1) and PNA (2) to ChromSpeed ^™ CM103 was carried out using an EYELA personal organic synthesizer, similar to the immobilization of PNA (1) in Example 4 (Figure 6). That is, the synthesis of 10 was carried out in the same manner as in Example 4, except that the ligand was changed to a mixture of Fmoc-PNA12mer-C ₁₀ -Cys-NH ₂ (1 and 2).

The ligands Fmoc-PNA12mer-C ₁₀ -Cys-NH ₂ (1, 1.62 mg, 0.85 μmol: 2, 1.61 mg, 0.85 μmol) were mixed and dissolved in DMF (600 μL) and HFIP (200 μL), and the mixture was added to 10 and stirred at room temperature overnight. After the reaction was completed, the reaction solution was removed and washed with DMF (2 mL) to remove excess Fmoc-PNA12mer-C ₁₀ -Cys-NH _2. The DMF washing was then repeated, and the absorbance (260 nm) was measured for every 1 mL. Washing was continued until the absorbance was completely gone, yielding 15.

Next, a capping reaction was carried out to inactivate unreacted SMCC. L-cysteine (10.4 mg, 0.085 mmol) was dissolved in NMP (10 mL) and added to 15, and the mixture was stirred at room temperature for 3 hours. After the reaction was completed, the reaction solution was removed and washed with DMF (5 mL) to remove excess L-cysteine.

Finally, 20% piperidine/DMF was added and the mixture was stirred for 5 minutes to remove the Fmoc group from Fmoc-PNA12mer-C ₁₀ -Cys-NH ₂ (1 and 2) to obtain 16.

Example 7 Immobilization of Ligand LNA (3) on Affinity Column LNA (3) was immobilized on affinity column ChromSpeed ^™ CM103 as described below.

Immobilization of LNA (3) onto ChromSpeed ^™ CM103 was carried out in the same manner as immobilization of PNA (1) in Example 4 ( FIG. 7 ). That is, the synthesis of 10 was carried out as described above, but the ligand was changed to LNA 12mer L(GacAatGtaGct: SEQ ID NO: 3)-C ₆ -SH (3).

LNA (3) was dissolved in DMSO and TE buffer, added to 10, and stirred overnight at room temperature. After the reaction was completed, the reaction solution was removed and washed with DMSO (2 mL) to remove excess LNA (3). After that, washing with DMSO was repeated, and the absorbance (260 nm) was measured for every 1 mL. Washing was continued until the absorbance completely disappeared, obtaining 17. The absorbance (260 nm) of each fraction (1 ml) of the washing solution was measured using a spectrophotometer, and washing was repeated until LNA (3) completely disappeared.

Next, a capping reaction was carried out to inactivate unreacted SMCC. Fmoc-L-cysteine was dissolved in DMSO, added to 17 and stirred at room temperature for 10 minutes (reaction (i)). After the reaction was completed, the reaction solution was removed, and the mixture was washed twice with DMSO and twice with DCM to obtain 18. Finally, the Fmoc group of LNA (3) may be removed by adding 20% piperidine/NMP and stirring for 5 minutes (reaction (ii)).

Next, Fmoc quantification was performed using a spectrophotometer, and the amount of LNA (3) introduced was found to be 0.01474 mmol/g.

[Example 8] Immobilization of ligand S-oligo (4) on affinity column S-oligo (4) was immobilized on affinity column ChromSpeed ^™ CM103 as described below.

Immobilization of S-oligo (4) onto ChromSpeed ^™ CM103 was carried out in the same manner as immobilization of PNA (1) in Example 4 ( FIG. 8 ). That is, the synthesis of 11 was carried out as described above, but the ligand was changed to S-oligo 12mer H-(GacAatGtaGct: SEQ ID NO: 4)-C ₃ -SH (4).

S-oligo (4) was dissolved in DMSO and TE buffer, added to 10, and stirred overnight at room temperature. After the reaction was completed, the reaction solution was removed and washed with DMSO (2 mL) to remove excess S-oligo (4). After that, washing with DMSO was repeated, and the absorbance (260 nm) was measured for every 1 mL. Washing was continued until the absorbance completely disappeared, obtaining 19. The absorbance (260 nm) of each fraction (1 ml) of the washing solution was measured using a spectrophotometer, and washing was repeated until S-oligo (4) completely disappeared.

Next, a capping reaction was carried out to inactivate unreacted SMCC. Fmoc-L-cysteine was dissolved in DMSO, added to 19, and stirred at room temperature for 10 minutes (reaction (i)). After the reaction was completed, the reaction solution was removed and the mixture was washed 15 times with DMF to remove excess L-cysteine, yielding 20. Finally, the Fmoc group of S-oligo (4) may be removed by adding 20% piperidine/NMP and stirring for 5 minutes (reaction (ii)).

Next, Fmoc quantification was performed using a spectrophotometer, and the amount of S-oligo (4) introduced was found to be 0.00618 mmol/g.

[Example 9] Preparation of DNA oligos The target single-stranded DNA sequences to be captured are shown in Table 1. Standard oligos purchased from Eurofins Genomics K.K. were used.

The oligos were dried and the concentrations were adjusted based on the molar yield. After adjustment, all were stored in a refrigerator.
A: 150 μL of TE solution (10 mM Tris-HCl, 1 mM EDTA) was added to adjust the concentration to 100 pmol/μL.
・B: 200 μL of TE solution was added to adjust the concentration to 100 pmol/μL.
・C: 150 μL of TE solution was added to adjust the concentration to 100 pmol/μL.
-D: 150 μL of TE solution was added to adjust the concentration to 100 pmol/μL.
E: 381 μL of TE solution was added to adjust the concentration to 100 pmol/μL.
・AB: A (25 μL) and B (25 μL) were mixed and heated in hot water for 5 minutes, then the temperature was gradually reduced and the mixture was allowed to cool to produce 50 pmol/μL double-stranded DNA.
・AB-C: Prepared by mixing AB (1 μL) and C (9 μL).
・AB-D: Prepared by mixing AB (1 μL) and D (9 μL).
Each was further diluted 50-fold and 1000-fold to adjust to 2 pmol/μL and 100 fmol/μL.

[Example 10] Affinity column evaluation experiment-1
(1) Basic procedure: column washing and initialization Using a disposable syringe (10 mL), 10 mL of distilled water was poured through the top of the cartridge to remove DMSO from the cartridge. Next, to completely remove the DMSO present at the top of the cartridge, the cartridge was turned upside down and washed in the same manner. After confirming that there was no absorption due to DMSO at UV 260 nm, 12 mL of 1xSSC was poured through the cartridge to initialize it.

・Sample application Using a pipette (for 2 μL), 2 μl of the sample adjusted to a concentration of 100 fmol/μl was injected from the top of the cartridge, and the sample was allowed to soak into the cartridge by gravity. At this time, 100 fmol/μl (2 μl) of the sample was measured using a Nanodrop3300, and the RFU value was set to 100%, and the cumulative RFU value obtained from each fraction was calculated as a percentage.

- Fraction collection method
1xSSC was used as the mobile phase, and 200 µl of the solution obtained by gravity flow was collected.

- Method for confirming the recovered DNA Each fraction was measured using Nanodrop3300 to determine its RFU value. The cumulative RFU value obtained from each fraction was calculated. The horizontal axis was plotted as the fraction number and the vertical axis was plotted as the DNA recovery rate (%) to visualize the DNA elution status.

(2) Confirmation of retention of single-stranded DNA In order to confirm whether single-stranded DNA is retained in the prepared affinity column, an affinity column evaluation experiment was performed as shown in FIG. 9. First, the fluorescent dye TAMRA (TMR) was bound to the end of the target double-stranded DNA, and the fluorescent dye FAM was bound to the end of the single-stranded DNA to be removed. Specifically, Oligo C prepared in Example 9 was used as a single-stranded DNA sample (SSDNA), Oligo AB was used as a double-stranded DNA sample (DSDNA), and Oligo AB-C was used as a mixed sample of single-stranded DNA and double-stranded DNA (SSDNA, DSDNA). The affinity column used contained a carrier to which PNA (1) prepared in Example 4 was immobilized as a ligand, as shown in FIG. 10 (a).

Following the procedure in (1) above, the sample was applied to the affinity column, each fraction was collected, and the RFU value was calculated. The RFU values of each fluorescent dye wavelength before the sample was passed through the cartridge were compared with the RFU values of the filtrate after passing through the cartridge using a fluorometer.

The results are shown in Figure 11. Single-stranded DNA appeared from fraction 10 onwards (Figure 11, A and C), and double-stranded DNA was 100% recovered in fractions 1 to 4 (Figure 11, B and C). These results show that PNA as a ligand recognizes single-stranded DNA with a complementary sequence and retains it in the affinity column, whereas double-stranded DNA is not recognized by PNA even if it has a complementary sequence.

[Example 11] Affinity column evaluation experiment-2
Affinity columns containing PNAs with different sequences as ligands were evaluated. Specifically, as shown in Fig. 10(b), an affinity column containing a carrier on which PNA (2) prepared in Example 5 was immobilized as a ligand was used, and the sample was applied to the affinity column in the same manner as in Example 10, and each fraction was collected and the RFU value was calculated.

The results are shown in Figure 12. Single-stranded DNA appeared from fraction 8 onwards (Figure 12, A and C), while double-stranded DNA was recovered in fractions 1 to 3 (Figure 12, B and C). From these results, it was found that, as in Example 10, PNA as a ligand recognizes single-stranded DNA with a complementary sequence and retains it in the affinity column, whereas double-stranded DNA is not recognized by PNA even if it has a complementary sequence.

[Example 12] Affinity column evaluation experiment-3
Affinity columns containing two types of carriers were evaluated. Specifically, as shown in (c) of FIG. 10, affinity columns containing a carrier on which PNA (1) prepared in Example 4 was immobilized as a ligand and a carrier on which PNA (2) prepared in Example 5 was immobilized were used, and samples were applied to the affinity columns in the same manner as in Example 10, and each fraction was collected and the RFU value was determined. Note that PNA (1) and PNA (2) have sequences complementary to different sequences of the target DNA.

The results are shown in Figure 13. Single-stranded DNA appeared from fraction 12 onwards (Figure 13, A and C), while double-stranded DNA was mainly recovered in fractions 1 to 3 (Figure 13, B and C). Comparing these results with those of Examples 10 and 11 (Figures 11 and 12), it was found that an affinity column containing two types of carriers, each with two types of ligands immobilized thereon, was able to separate single-stranded DNA and double-stranded DNA with better separation ability than the use of a single type of ligand.

[Example 13] Affinity column evaluation experiment-4
An affinity column containing two kinds of ligands was evaluated. Specifically, as shown in (d) of FIG. 10, an affinity column containing a carrier on which PNA (1) prepared in Example 4 and PNA (2) prepared in Example 5 were immobilized as ligands was used, and the sample was applied to the affinity column in the same manner as in Example 10, and each fraction was collected and the RFU value was calculated. Note that PNA (1) and PNA (2) have sequences complementary to different sequences of the target DNA.

The results are shown in Figure 14. Single-stranded DNA appeared from fraction 13 onwards (Figure 14, A and C), while double-stranded DNA was mainly recovered in fractions 1 to 4 (Figure 14, B and C). Comparing these results with those of Examples 10 and 11 (Figures 11 and 12), it was found that an affinity column containing a carrier to which two types of ligands were immobilised was able to separate single-stranded DNA and double-stranded DNA with better separation ability than the case in which one type of ligand was used.

[Example 14] Affinity column evaluation experiment-5
In order to confirm whether the affinity column prepared above can discriminate between base mismatches, the following affinity column evaluation experiment was carried out.

Oligo D prepared in Example 9 was used as the single-stranded DNA sample (SSDNA). This oligo D differs from oligo C evaluated in Example 10 by three bases, and as shown in Figure 15A, PNA (1) has a sequence complementary to the region containing the base mismatch. Using an affinity column containing a carrier on which PNA (1) prepared in Example 4 was immobilized as a ligand, the sample was applied to the affinity column in the same manner as in Example 10, and each fraction was collected and the RFU value was calculated. The results are shown in Figure 15B.

In Figure 15B, the sample was eluted in multiple fractions, and single-stranded DNA could not be separated. From the results in Figure 12A and Figure 15B, it can be seen that PNA as a ligand recognizes base mismatches, binds to single-stranded DNA, and can separate it.

[Example 15] Selection of a capping agent capable of further improving the affinity of a ligand It is possible to cap the sites on the carrier other than the site where the ligand is immobilized. Stationary phases were prepared using several types of capping agents, and the effects on purification using an affinity column were compared.

Five types of functional groups were considered as candidates for the capping agent: amino group, carboxyl group, methyl group, guanidino group, and phenyl group. In addition, a total of six types of capping agents containing one amino group and one carboxyl group were compared.

As a result, it was found that the column using the positively charged 1,4-diaminobutane (amino group) and H-Arg- _NH2・2HCl (guanidino group) as coating agents had the highest ability to retain single-stranded DNA, followed in order by the negatively charged 5-aminovaleric acid (carboxyl group), the uncharged amylamine (methyl group) and 4-phenylbutylamine (phenyl group), and Fmoc-Lys-OH・HCl (amino group・carboxyl group) with a ±0 charge.

Therefore, it has been found that when isolating or detecting single-stranded DNA, it is preferable to coat areas of the carrier other than the ligand fixing area with a coating agent such as those described above.

Claims (14)

A filler for affinity chromatography comprising a carrier and a nucleic acid ligand immobilized on the carrier, the filler comprising a carrier on which a nucleic acid ligand having a sequence complementary to a first sequence and a nucleic acid ligand having a sequence complementary to a second sequence are immobilized;
The nucleic acid ligand comprises at least one artificial nucleic acid selected from the group consisting of peptide nucleic acid (PNA), locked nucleic acid (LNA) and S-oligo;
A packing material for affinity chromatography, wherein the nucleic acid ligand is immobilized on the carrier by a peptide bond, a disulfide bond or a bond via a linker. A filler for affinity chromatography comprising a carrier and a nucleic acid ligand immobilized on the carrier, the filler comprising a first carrier having a nucleic acid ligand immobilized thereon, the nucleic acid ligand having a sequence complementary to a first sequence, and a second carrier having a nucleic acid ligand having a sequence complementary to a second sequence,
The nucleic acid ligand comprises at least one artificial nucleic acid selected from the group consisting of peptide nucleic acid (PNA), locked nucleic acid (LNA) and S-oligo;
A packing material for affinity chromatography, wherein the nucleic acid ligand is immobilized on the carrier by a peptide bond, a disulfide bond or a bond via a linker. The affinity chromatography packing material according to claim 1 or 2, wherein the nucleic acid ligand comprises PNA. The affinity chromatography packing material according to any one of claims 1 to 3, wherein the nucleic acid ligand is an 8-30 mer. The affinity chromatography packing material according to any one of claims 1 to 4, wherein the first sequence and the second sequence have sequences complementary to different regions of the target nucleic acid. The affinity chromatography packing material according to any one of claims 1 to 4, wherein the first sequence and the second sequence have sequences that differ by 1 to 3 bases. The affinity chromatography packing material according to any one of claims 1 to 6, wherein the linker is selected from the group consisting of SMCC, EMCS, LC-SMCC, SM(PEG)2, SIA, Sulfo-SANPAH, MBS, and an epoxy resin curing agent. A plurality of linkers are immobilized on the surface of the support,
8. The affinity chromatography packing material according to claim 1, wherein the nucleic acid ligand is bound to some of the plurality of linkers. The affinity chromatography packing material according to claim 8, wherein a capping agent is bound to a linker among the plurality of linkers to which the nucleic acid ligand is not bound. The affinity chromatography packing material according to any one of claims 1 to 9, wherein the carrier is selected from the group consisting of resins, porous particles, gels, and magnetic beads. An affinity chromatography column packed with the affinity chromatography packing material according to any one of claims 1 to 10. The affinity chromatography column according to claim 11, which is provided with a heating means. 1. A method for detecting or identifying a target nucleic acid in a sample, comprising:
A step of introducing a sample into an affinity chromatography column packed with the affinity chromatography packing material according to any one of claims 1 to 10;
detecting or identifying a target nucleic acid based on elution from the affinity chromatography column. A kit for detecting a target nucleic acid in a sample, comprising the affinity chromatography packing material according to any one of claims 1 to 10.