Affinity chromatography based on artificial nucleic acids

Abstract

The present invention relates to an affinity-chromatography filler including a carrier and a nucleic-acid ligand immobilized on the carrier, wherein the nucleic-acid ligand contains at least one type of artificial nucleic acid selected from the group consisting of peptide nucleic acids (PNAs), locked nucleic acids (LNAs), and S-oligos, and the nucleic-acid ligand is immobilized on the carrier by means of a peptide bond, a disulfide bond, or a bond formed via a linker.

Description

[Technical field]

[0001] The present invention relates to a filler for affinity chromatography based on an artificial nucleic acid, and an affinity chromatography column packed with the filler. 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 filler based on an artificial nucleic acid. [Background technology]

[0002] Affinity chromatography is a technique for separating and identifying target molecules that utilizes binding affinity. For example, target molecules are 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 immobilized with a ligand that specifically binds to the target molecule, 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.

[0003] 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 things that cannot be achieved with natural nucleic acids. For example, it is possible to create artificial nucleic acids with high resistance to nucleases and fluorescent emission functions 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-oligo), and morpholino oligonucleotides.

[0004] For example, PNA is a compound with a structure similar to that of nucleic acids, in that the backbone is formed by peptide bonds instead of phosphate bonds. PNA has excellent hybridization affinity with DNA, is not repulsed by negative charges due to phosphate groups, is not affected by salt concentration, and has good biological stability and heat resistance.

[0005] 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. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Orum H., Nielsen PE, Jorgensen M., Larsson C., Stanley C., Koch T. BioTechniques Vol. 19 No. 3, pp. 472-480 (1995) Summary of the Invention [Problem to be solved by the invention]

[0007] An object of the present invention is to prepare an affinity column having excellent nucleic acid affinity and stability, and to utilize the same for detection and identification of nucleic acids. [Means for solving the problem]

[0008] As a result of intensive research to solve the above problems, the present 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 an affinity chromatography packing material whose nucleic acid affinity is not affected by temperature and pH and which has excellent storage stability, and thus completed the present invention.

[0009] For example, the present invention encompasses 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 carrier; 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 packing material for affinity chromatography 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] , further comprising 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 the target nucleic acid based on elution from the affinity chromatography column. The method includes:

[15] The method according to

[14] , wherein the affinity chromatography packing comprises a carrier having a nucleic acid ligand immobilized thereon, the 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 material 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 having a nucleic acid ligand immobilized thereon, the nucleic acid ligand having a sequence complementary to a first sequence of a target nucleic acid, and a second support having a nucleic acid ligand immobilized thereon, the nucleic acid ligand having a sequence complementary to a second sequence of the target nucleic acid.

[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] . Effect of the Invention

[0010] The present invention provides a packing material and a column for affinity chromatography. The packing material and the 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 the 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 the fields of nucleic acid detection and identification, diagnostic agents, molecular biology research, and the like. [Brief description of the drawings]

[0011] [Figure 1] The chemical structure of Fmoc-PNA12mer-C10-Cys-NH2(1) is shown. [Diagram 2] The chemical structure of LNA 12mer (GacAatGtaGct)-C6-SH(3) is shown. [Diagram 3] The chemical structure of S-oligo 12mer (GacAatGtaGct)-C3-SH(4) is shown. [Figure 4]1 shows a synthetic scheme for immobilizing the ligand PNA(1) on an affinity column. [Diagram 5] 1 shows a synthetic scheme for immobilizing the ligand PNA(2) on an affinity column. [Figure 6] 1 shows a synthetic scheme for immobilizing ligands PNA(1) and PNA(2) on an affinity column. [Figure 7] 1 shows a synthetic scheme for immobilizing the ligand LNA (3) on an affinity column. [Figure 8] 1 shows a synthetic scheme for immobilizing ligand S-oligo (4) on an affinity column. [Figure 9] An outline of an affinity column evaluation method is given below. [Figure 10] FIG. 1 shows conceptual diagrams of variations in the form of immobilizing a ligand on an affinity column. [Figure 11] 1 is a graph showing the evaluation results of an affinity column using the ligand PNA(1). [Figure 12] 1 is a graph showing the evaluation results of an affinity column using the ligand PNA(2). [Figure 13] 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, respectively. [Figure 14] 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. [Figure 15] 1 is a graph showing the outline and results of evaluation of an affinity column for DNA having base mismatches. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] 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.

[0013] 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. Artificial nucleic acids are nucleic acids whose physical properties, such as hydrogen bond patterns, higher-order structures, 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. [ka]

[0014] 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, so that it is completely fixed to 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 when forming a double strand with RNA is greatly reduced. This enables LNA to form an unprecedentedly stable double strand. [ka]

[0015] In general, the ability of artificial nucleic acids to form double strands is 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 a target RNA showed that the Tm value increased by +4 to +6 degrees per LNA modification (Obika S et al., Tetrahedron Lett, 39:5401-5404, 1998).

[0016] 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). [ka]

[0017] 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.

[0018] Another type of artificial nucleic acid is the morpholino oligonucleotide, which is an oligonucleotide that has a morpholine ring instead of the ribose or deoxyribose of nucleic acids (RNA, DNA).

[0019] The nucleic acid ligand contains at least one artificial nucleic acid, and may be entirely made up of an artificial nucleic acid, or may contain an artificial nucleic acid only in part. When an artificial nucleic acid is only partially contained, for example, an artificial nucleic acid may be contained every few bases (2, 3, 4, 5, etc.), or an artificial nucleic acid may be contained in the region at the end opposite to the end of the nucleic acid ligand immobilized on the carrier.

[0020] 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 (target sequence) of the target nucleic acid. In this specification, the term "sequence" means a base sequence unless otherwise specified.

[0021] 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.

[0022] In another embodiment, the affinity chromatography packing of the present invention comprises a first support having a nucleic acid ligand immobilized thereon, the nucleic acid ligand having a sequence complementary to a first sequence, and a second support having a nucleic acid ligand immobilized thereon, the nucleic acid ligand having a sequence complementary to a second sequence.

[0023] 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).

[0024] "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 between 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.

[0025] The length of the nucleic acid ligand is selected so as to have an appropriate affinity for the target nucleic acid to be isolated or detected in affinity chromatography. In consideration of the retention of affinity and ease of synthesis, the length of the nucleic acid ligand is generally 8 to 30 mers, preferably 10 to 25 mers, and more preferably 10 to 23 mers.

[0026] The design of the nucleic acid ligand, i.e., the overall length, the type and arrangement of the artificial nucleic acid contained therein, etc., can be appropriately determined by those skilled in the art, taking into consideration the type and length of the target nucleic acid, the purpose, the type of elution solution used, etc. Furthermore, the preparation of the nucleic acid ligand can also be performed by a conventional method, for example, using a synthesis device capable of synthesizing artificial nucleic acids.

[0027] The carrier for immobilizing the ligand may be a 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, a carrier in the form of porous particles, gel, 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.

[0028] The ligand can be immobilized on the support by a peptide bond, a disulfide bond, or a bond via a linker. When a peptide bond is used, the reactive terminal of the support is a carboxylic acid, and then the peptide bond can be obtained by converting the reactive terminal of the support into an active ester using a peptide condensing agent such as HATU, and then reacting with a primary amino group on the ligand. In addition, when the reactive terminal of the support is a primary amino group, it is also possible to obtain a peptide bond by converting the reactive terminal of the support into an active ester and then reacting with the carboxylic acid on the ligand. In the case of a disulfide bond, it is possible to use a support and a ligand whose reactive terminals are both SH groups, and then bond them via an oxidation reaction.

[0029] When the ligand is immobilized on the support via a linker, the linker can be, for example, a heterocrosslinker, an epoxy resin curing agent, a homocrosslinker, or the like. As heterocrosslinkers, 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, like heterocrosslinkers, can also be used as reagents for crosslinking amino groups or sulfhydryl groups, and are commercially available, for example, jER Cure (registered trademark) (Mitsubishi Chemical). 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, and specific examples include DSG (disuccinimidyl glutarate), DMP (dimethyl pimelimidate), and BMOE (bismaleimidoethane).As another homo-crosslinker, 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) and BM(PEG)3 (1,11-bismaleimido-triethyleneglycol) can be used.

[0030] One of the preferred linkers is SMCC, which is a crosslinker having an active ester group and a maleimide group. For example, the ligand can be immobilized on the support via SMCC by reacting an NH group bound to the support with the active ester group of SMCC and reacting an SH group introduced into the ligand with the maleimide group of SMCC.

[0031] One or more linkers can be immobilized on the surface of the support. One ligand can be immobilized on a support to which one linker is immobilized. On the other hand, when multiple linkers are immobilized on the surface of the support, 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 the ligand is bound to a part of the multiple linkers, it is preferable to bind a capping agent to the linker to which the ligand is not bound. Examples of such capping agents include 1,4-diaminobutane (amino group), H-Arg-NH2·2HCl (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 immobilized on the surface of the support, 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.

[0032] A spacer may be introduced into the nucleic acid ligand and / or the 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.

[0033] The affinity chromatography packing material according to the present invention can bind 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. In addition, it can be used repeatedly, and has the advantage of being low cost.

[0034] In another aspect, the present invention relates to an affinity chromatography column packed with the above affinity chromatography packing. The affinity chromatography column is not particularly limited as long as it is a column commonly 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.

[0035] The affinity chromatography column according to the present invention may further include a heating means. The heating means may be one that is integrated with the column, or may be one that is removable from the column. The heating means may be one that heats the entire column, or one that heats 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.

[0036] 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 the target nucleic acid based on elution from the affinity chromatography column. The present invention relates to a method comprising the steps of:

[0037] 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.

[0038] The sample is not particularly limited as long as it is a sample for detecting or identifying a target nucleic acid. Examples include biological fluid samples (blood, serum, plasma, urine, etc.), tissue or cell samples, and environmental samples (soil, river, 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 it is also possible to use heparin, sodium citrate, or other anticoagulants known or commonly used in the field.

[0039] 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.

[0040] 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 solution.

[0041] Subsequently, 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). When the sample contains a target nucleic acid having a sequence complementary to the sequence of the ligand nucleic acid immobilized on 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 according to the present invention can also be used to detect a target nucleic acid having a base mismatch.

[0042] The method according to 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. 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.

[0043] 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, such as an eluent for affinity chromatography, a control, instructions, etc., in addition to the affinity chromatography packing. EXAMPLES

[0044] The present invention will be described in more detail below with reference to examples and drawings, although the present invention is not limited to the following examples.

[0045] [Example 1] Preparation of affinity column carrier and column (1) Affinity column carrier There are various affinity carriers available. TM CM103 (Mitsubishi Chemical) is a methacrylic acid-based weakly acidic cation exchange resin, and is used for the purification of antibiotics and amino acids. We thought that by using a carrier for the purification of biopharmaceuticals, we could create an affinity column that would allow us to obtain precise and pure target products. TM We decided to use CM103.

[0046] (2) Affinity column cartridge The cartridge to be filled with the affinity support was a 0.5 mL Empty PP Rev Tube Kit from Sigma-Aldrich. This is an empty column that allows the preparation of SPE cartridges using any packing material. In addition, since PNA is expensive, it was an issue to prepare the cartridge with as little ligand as possible, so the Luer type cartridge Type Mini (0.1 mL) from Tomoe Manufacturing Co., Ltd. was also used in some cases.

[0047] (3) Method for immobilizing PNA on a support for an affinity column 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 together by covalent bonds.

[0048] [Example 2] Synthesis of PNA (ligand) for affinity column A PNA for affinity columns with the structure shown in Figure 1 was synthesized. The crosslinker used was SMCC, which has cyclohexane in the spacer portion and has 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. To utilize this feature of SMCC, Fmoc-Cys(Mmt)-OH, which has an SH group, was introduced into the ligand. In addition, 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 (sequence number 1).

[0049] Fmoc-PNA12mer-C 10 -Cys was synthesized using the usual Fmoc solid-phase synthesis method. A Biotage fully automatic 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 is 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 automatic 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), with 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-NH2(1) was obtained.

[0050] [Example 3] Design of artificial nucleic acids (ligands) for affinity columns other than PNA (1) Design of LNA(3) An LNA for affinity columns 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 a 12mer (in the sequence GacAatGtaGct (SEQ ID NO: 3), capital letters indicate the insertion positions). In addition, in order to adopt a structure similar to that of PNA and to standardize the conditions for immobilization, a spacer (C6) and thiol were modified at the 3' end.

[0051] (2) Design of S-oligo (4) An S-oligo for affinity column having the structure shown in Figure 3 was synthesized. The S-oligo having 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.

[0052] [Example 4] Immobilization of ligand PNA (1) on affinity column As described below, the affinity column ChromSpeed TM The PNA (1) prepared in Example 2 was immobilized on CM103.

[0053] (1) Reagents 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) ChromSpeed TM CM103 Hexamethylenediamine (1,6-diaminohexane) ·4-(N-Maleimidomethyl)cyclohexanecarboxylic acid N-succinimidyl ester (SMCC) L-Cysteine

[0054] (2) Synthesis scheme The synthesis scheme is shown in Figure 4. TM The immobilization of PNA (1) onto CM103 was performed using an EYELA personal organic synthesizer (CCS-600). TM To confirm the amount of carboxylic acid capable of reacting per unit volume of CM103, Fmoc-Acp(6)-OH was first introduced.

[0055] ChromSpeed TM As a preliminary preparation for PNA immobilization on 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 then 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.

[0056] 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 completion of the reaction, the reaction solution was removed and washed once with DMF (2 mL) and five times with MeOH (2 mL) to obtain 7.

[0057] 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, and the mixture was stirred at room temperature for 1 hour. After the reaction was completed, 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.

[0058] Next, Fmoc was quantified using an absorption spectrophotometer, 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 molar extinction coefficient ε (301 nm) was calculated to be 7800 M -1 cm -1 The amount of Fmoc groups was calculated as 0.20838 mmol / g.

[0059] The Fmoc deprotection reaction was carried out with 20% piperidine / DMF to give 9.

[0060] Next, SMCC (17.1 mg, 0.051 mmol) was dissolved in DMF (3 mL) and 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.

[0061] Next, the ligand Fmoc-PNA12mer-C 10-Cys-NH2 (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), and the excess Fmoc-PNA12mer-C 10 -Cys-NH2(1) was removed. After that, washing with DMF was repeated and the absorbance (260 nm) was measured for every 1 mL. Washing was continued until the absorbance completely disappeared, and 11 was obtained. Fmoc quantification was performed using an absorption spectrophotometer, and the amount of Fmoc-PNA12mer-C10-Cys-NH2(1) introduced into 10 was 0.01532 mmol / g.

[0062] 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, 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.

[0063] Finally, add 20% piperidine / DMF and stir for 5 minutes to prepare Fmoc-PNA12mer-C. 10 The Fmoc group of -Cys-NH2(1) was removed to give 12.

[0064] [Example 5] Immobilization of ligand PNA (2) on affinity column As described below, the affinity column ChromSpeed TM PNA(2) was immobilized on CM103.

[0065] Using the same procedure as in Example 2, PNA (2) having the base sequence GAAACCCAGCAG (SEQ ID NO: 2) instead of the base sequence GACAATGTAGCCT (SEQ ID NO: 1) of PNA (1) was synthesized.

[0066] ChromSpeed TMThe immobilization of PNA (2) on CM103 was carried out using an EYELA personal organic synthesizer in the same manner as the immobilization of PNA (1) in Example 4 ( FIG. 5 ). That is, the synthesis of 10 was carried out in the same manner as in Example 4, and the ligand was FMOC-PNA12MER-C. 10 -Cys-NH2(2).

[0067] Ligand Fmoc-PNA12mer-C 10 -Cys-NH2 (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), and the excess Fmoc-PNA12mer-C 10 -Cys-NH2(2) was removed. After that, washing with DMF was repeated and the absorbance (260 nm) was measured for every 1 mL. Washing was continued until the absorbance was completely gone, and 13 was obtained. Fmoc quantification using an absorption spectrophotometer revealed that the amount of Fmoc-PNA12mer-C in 10 was 13. 10 The amount of -Cys-NH2(2) introduced was 0.0113 mmol / g.

[0068] 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.

[0069] Finally, add 20% piperidine / DMF and stir for 5 minutes to prepare Fmoc-PNA12mer-C. 10 The Fmoc group of -Cys-NH2(2) was removed to give 14.

[0070] [Example 6] Immobilization of ligands PNA(1) and PNA(2) on affinity columns As described below, the affinity column ChromSpeed TM A mixture (1:1) of PNA(1) and PNA(2) was immobilized on CM103.

[0071] ChromSpeed TM The immobilization of a mixture (1:1) of PNA(1) and PNA(2) to CM103 was carried out using an EYELA personal organic synthesizer, similar to the immobilization of PNA(1) in Example 4 (FIG. 6). That is, the synthesis of 10 was carried out in the same manner as in Example 4, and the ligand was Fmoc-PNA12mer-C. 10 -Cys-NH2 (1 and 2) mixture.

[0072] Each Fmoc-PNA12mer-C ligand 10 -Cys-NH2 (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 added to 10, which was stirred overnight at room temperature. After the reaction was completed, the reaction solution was removed and washed with DMF (2 mL), and excess Fmoc-PNA12mer-C 10 -Cys-NH2 was removed. Then, washing with DMF was repeated and the absorbance (260 nm) was measured every 1 mL. Washing was continued until the absorbance was completely eliminated, and 15 was obtained.

[0073] 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.

[0074] Finally, add 20% piperidine / DMF and stir for 5 minutes to prepare Fmoc-PNA12mer-C. 10 The Fmoc group of -Cys-NH2 (1 and 2) was removed to give 16.

[0075] [Example 7] Immobilization of ligand LNA (3) on affinity column As described below, the affinity column ChromSpeed TM LNA(3) was immobilized on CM103.

[0076] ChromSpeed TM Immobilization of LNA (3) to 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)-C6-SH (3).

[0077] 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). Then, 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.

[0078] 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)).

[0079] Next, Fmoc quantification was carried out using a spectrophotometer, and the amount of LNA (3) introduced was found to be 0.01474 mmol / g.

[0080] [Example 8] Immobilization of ligand S-oligo (4) on affinity column As described below, the affinity column ChromSpeed TM S-oligo (4) was immobilized on CM103.

[0081] ChromSpeed TMImmobilization of S-oligo (4) on CM103 was carried out in the same manner as immobilization of PNA (1) in Example 4 ( FIG. 8 ). That is, synthesis of 11 was carried out as described above, but the ligand was changed to S-oligo 12mer H-(GacAatGtaGct: SEQ ID NO: 4)-C3-SH(4).

[0082] S-oligo (4) was dissolved in DMSO and TE buffer, added to 10, and stirred overnight at room temperature. After completion of the reaction, the reaction solution was removed and washed with DMSO (2 mL) to remove excess S-oligo (4). Then, washing with DMSO was repeated, and the absorbance (260 nm) was measured for every 1 mL. Washing was continued until the absorbance completely disappeared, to obtain 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.

[0083] 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 completion of the reaction, 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)).

[0084] Next, Fmoc quantification was carried out using a spectrophotometer, and the amount of S-oligo (4) introduced was found to be 0.00618 mmol / g.

[0085] [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, Inc. were used. [Table 1]

[0086] 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 lowered and the mixture was cooled to produce 50 pmol / μL double-stranded DNA. · AB-C: Prepared by mixing AB (1μL) and C (9μL). · AB-D: Prepared by mixing AB (1μL) and D (9μL). Each was further diluted 50-fold and 1000-fold to adjust the concentrations to 2 pmol / μL and 100 fmol / μL.

[0087] [Example 10] Affinity column evaluation experiment-1 (1) Basic Procedure Column cleaning and initialization Using a disposable syringe (10 mL), 10 mL of distilled water was poured from the top of the cartridge to remove DMSO from the cartridge. Next, in order 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 to initialize the cartridge.

[0088] · Applying samples Using a pipette (for 2 μL), 2 μl of a 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.

[0089] Fraction collection method 1xSSC was used as the mobile phase, and 200 µl of the solution obtained by gravity flow was collected.

[0090] How to confirm the DNA recovered Each fraction was measured using a Nanodrop 3300 to determine its RFU value. The cumulative RFU value obtained from each fraction was calculated. The horizontal axis represents the fraction number, and the vertical axis represents the DNA recovery rate (%), and the DNA elution status was visualized.

[0091] (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).

[0092] The sample was applied to the affinity column according to the procedure described in (1) above, and 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 each filtrate after passing through the cartridge using a fluorometer.

[0093] 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 demonstrated that PNA as a ligand recognized single-stranded DNA having a complementary sequence and retained it in the affinity column, whereas double-stranded DNA was not recognized by PNA even if it had a complementary sequence.

[0094] [Example 11] Affinity column evaluation experiment-2 Affinity columns containing PNAs with different sequences as ligands were evaluated. Specifically, as shown in (b) of FIG. 10, 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.

[0095] The results are shown in Figure 12. Single-stranded DNA appeared from fraction 8 onwards (Figure 12, A and C), and 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 recognized single-stranded DNA having a complementary sequence and retained it in the affinity column, whereas double-stranded DNA was not recognized by PNA even if it had a complementary sequence.

[0096] [Example 12] Affinity column evaluation experiment-3 An affinity column containing two types of carriers was evaluated. Specifically, as shown in (c) of Fig. 10, an affinity column 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 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 determined. Note that PNA(1) and PNA(2) have sequences complementary to different sequences of the target DNA.

[0097] The results are shown in Figure 13. Single-stranded DNA appeared in fraction 12 and after (A and C in Figure 13), and double-stranded DNA was mainly recovered in fractions 1 to 3 (B and C in Figure 13). Comparing these results with those in Examples 10 and 11 (Figures 11 and 12), it was found that the affinity column containing two types of carriers each having two types of ligands immobilized thereon can separate single-stranded DNA and double-stranded DNA with better separation ability than the case where one type of ligand is used.

[0098] [Example 13] Affinity column evaluation experiment-4 An affinity column containing two types of ligands was evaluated. Specifically, as shown in (d) of Figure 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 determined. Note that PNA(1) and PNA(2) have sequences complementary to different sequences of the target DNA.

[0099] The results are shown in Figure 14. Single-stranded DNA appeared in fraction 13 and after (Figure 14, A and C), and 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 the affinity column containing the carrier on which two types of ligands were immobilized can separate single-stranded DNA and double-stranded DNA with better separation ability than the case where one type of ligand is used.

[0100] [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.

[0101] Oligo D prepared in Example 9 was used as a single-stranded DNA sample (SSDNA). This oligo D differs from oligo C evaluated in Example 10 by three bases, and as shown in FIG. 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 determined. The results are shown in FIG. 15B.

[0102] 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.

[0103] [Example 15] Selection of a capping agent capable of better improving the affinity of a ligand It is possible to cap the sites on the carrier other than where the ligand is immobilized. We prepared stationary phases using several types of capping agents and compared their effects on purification using an affinity column.

[0104] 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 having one amino group and one carboxyl group were compared. [Table 2]

[0105] 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.

[0106] Therefore, it was found that, when isolating or detecting single-stranded DNA, it is preferable to coat the areas of the carrier other than the ligand fixing area with the above-mentioned coating agent.

Claims

1. 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.

2. 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.

3. The affinity chromatography packing material according to claim 1 or 2, wherein the nucleic acid ligand comprises a PNA.

4. The affinity chromatography packing material according to any one of claims 1 to 3, wherein the nucleic acid ligand is an 8-30 mer.

5. 5. The affinity chromatography packing material according to claim 1, wherein the first sequence and the second sequence have sequences complementary to different regions of a target nucleic acid.

6. 5. The affinity chromatography packing material according to claim 1, wherein the first sequence and the second sequence differ by 1 to 3 bases.

7. 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.

8. 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.

9. 9. 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.

10. The affinity chromatography packing material according to any one of claims 1 to 9, wherein the support is selected from the group consisting of a resin, a porous particle, a gel, and a magnetic bead.

11. 11. A column for affinity chromatography, packed with the affinity chromatography packing material according to any one of claims 1 to 10.

12. 12. An affinity chromatography column according to claim 11, comprising a heating means.

13. 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 the target nucleic acid based on elution from the affinity chromatography column. A method comprising:

14. 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.

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