KR102448191B1 - Conjugated bead, process thereof and biological assay system and assay process inlcuidg thereof - Google Patents

Abstract

Disclosed are: a bead conjugate with a linking group having a sufficient length between nanobeads and an oligo-nucleotide that specifically binds to a target nucleic acid molecule; a method for manufacturing the bead conjugate; and an analysis kit and analysis method for detecting the target nucleic acid molecule in a biological sample by using the bead conjugate. By using the bead conjugate, it is possible to specifically and rapidly detect the target nucleic acid molecule present in a biological sample, and the sensitivity of the analysis can be improved.

Description

Bead conjugate, manufacturing method, biological analysis system and analysis method comprising the same {CONJUGATED BEAD, PROCESS THEREOF AND BIOLOGICAL ASSAY SYSTEM AND ASSAY PROCESS INLCUIDG THEREOF}

The present invention relates to nanobeads, and more particularly, a bead complex capable of specifically detecting and purifying nucleic acid molecules present in a biological sample, and a system capable of analyzing nucleic acid molecules present in a biological sample using the same, and It relates to the method of analysis.

As the coronavirus-19 (COVID-19) outbreak in China at the end of 2019 spread worldwide, interest in nucleic acid amplification reactions such as polymerase chain reaction (PCR) that can diagnose infectious diseases at an early stage this is rising Even before COVID-19, PCR was widely used to diagnose infectious diseases caused by cancer, viruses, or bacteria.

For example, when diagnosing infection with Human papillomavirus (HPV), a major pathogen that causes cervical cancer, currently, a cotton swab is inserted into the cervix to collect cells, and then staining Cytodiagnosis to observe the presence or absence is mainly performed. The cytology test has a rather low sensitivity of 50-70%, and to compensate for this, an additional HPV genetic test is performed to determine the presence or absence of HPV in cervical cells. If the HPV gene test is positive, even if the result of the cytology test is negative, there is a high probability of developing cervical cancer.

Currently, there are two main methods used for HPV screening: the pap smear test and the pad-type detection method. The Pap test is a method to check the presence of HPV virus through a PCR reaction by inserting a cotton swab into the cervix to collect cells. The pad-type detection method is a method to check the presence of HPV virus by PCR by collecting HPV virus in secretions by wearing a pad.

Pap tests have the disadvantage that they are invasive and require specialized medical staff to detect them. The pad-type detection method has disadvantages in that it is impossible to shower or wash the vagina 24-72 hours before the test and wear the pad for a long time. Therefore, if it is possible to detect the presence of HPV virus by detecting DNA or RNA present in urine, the disadvantages of the existing test method can be sufficiently compensated and HPV infection can be confirmed within a relatively short time.

In order to perform polymerase chain reaction (PCR), disease-specific nucleic acid molecules must be extracted from a sample. A method of extracting nucleic acid molecules present in a sample is largely divided into a method using a column and a method using a magnetic bead. In the method using a column, cells present in the sample are lysed and collected using a column, and then tested through PCR. In a method using magnetic beads, a desired nucleic acid molecule is captured and purified through charge-charge interaction between the surface charge of the magnetic bead and the nucleic acid molecule in the sample. However, since these methods non-specifically capture various proteins, inhibitors, genomic DNA, and RNA in the sample, an additional purification process is required. In addition, the accuracy is low even when performing a PCR reaction due to various inhibitors present in the sample.

In particular, various inhibitors, proteins, DNA, and RNA exist in the urine, but they are present in very small amounts compared to the total amount of urine. In the currently applied purification method using a column and a purification method using magnetic beads, all substances having a charge in urine are separated. Since foreign substances other than the intended nucleic acid molecule are separated together, the accuracy of the PCR reaction is deteriorated, an additional purification process is required, and there is a problem in that the specificity of the PCR reaction is lowered.

It is an object of the present invention to provide a bead complex capable of specifically isolating and detecting a target nucleic acid molecule present in a biological sample, a method for preparing the same, a kit and an analysis method capable of analyzing a target nucleic acid molecule in a biological sample using the bead complex is intended to provide.

Another object of the present invention is a bead complex capable of rapidly and easily performing a target nucleic acid molecule present in a biological sample, a method for preparing the same, a kit and analysis method capable of analyzing a target nucleic acid molecule in a biological sample using the bead complex is intended to provide.

Another object of the present invention is a bead conjugate capable of improving the detection sensitivity of a target nucleic acid molecule present in a biological sample, a method for preparing the same, and a kit capable of analyzing a target nucleic acid molecule in a biological sample using the bead conjugate and analysis methods.

In one aspect, as a bead conjugate in which one end of an oligo-nucleotide specifically binding to a target nucleic acid molecule is condensed on the surface of a nano-bead, the structure of Formula 1 below between the surface of the nano-bead and the oligo-nucleotide Disclosed is a bead conjugate in which a linking group having a

[Formula 1]

(In Formula 1, R ¹ is a direct bond or a C ₁ -C ₂₀ aliphatic hydrocarbon group; R ² and R ³ are each independently a C ₃ -C ₂₀ divalent aliphatic hydrocarbon linkage group; The asterisk to the left of R ¹ is on the surface of the nanobead. Indicates the site to be connected, and the asterisk to the right of R ³ indicates the site to be connected to the end of the oligo-nucleotide.

For example, R ² and R ³ of Formula 1 may each independently be a C ₃ -C ₂₀ alkylene group.

In one exemplary aspect, the nano-beads may be made of an inorganic material.

The inorganic material may be at least one selected from a non-metallic material selected from the group consisting of iron oxide, silica, glass, or a combination thereof, and a metal material selected from the group consisting of gold, silver, copper, a combination thereof, and an alloy thereof. have.

In another exemplary aspect, the nano-beads may be made of an organic material.

The organic material is a polymer resin selected from the group consisting of polystyrene, polypropylene, polyethylene, poly acrylamide, combinations or copolymers thereof, and fullulane, fullulane acetate, cellulose, hydroxypropylmethylcellulose, agar It may be at least one of polysaccharides selected from the group consisting of rose, chitosan, combinations thereof, and copolymers thereof.

Optionally, the nano-beads may be made of a magnetic material.

The target nucleic acid molecule may be derived from a tumor or a pathogen.

The pathogen may include a pathogenic virus, pathogenic bacterium or pathogenic protozoa.

For example, the target nucleic acid molecule may be derived from human papillomavirus.

In another aspect, as a method for preparing the above-described bead complex, by reacting a nano-bead in which an amino group represented by the following Chemical Formula 2 is connected to the surface, and a carboxylic acid represented by the following Chemical Formula 3, the surface of the nano-bead is formed by the following Chemical Formula transforming into a linker having a structure of 4; A method comprising reacting the nanobead whose surface is modified with a linking group having the structure of Formula 4, and an oligo-nucleotide that specifically binds to a target nucleic acid molecule and whose terminal is modified with an aliphatic thiol of Formula 5 This is initiated.

[Formula 2]

[Formula 3]

[Formula 4]

[Formula 5]

(In Formulas 2 to 5, R ¹ , R ² , R ³ and an asterisk are the same as defined in claim 1, respectively)

In another aspect, an assay kit for detecting a target nucleic acid molecule in a biological sample comprising the above-described bead conjugate is disclosed.

The biological sample may include urine, saliva, sputum, blood and nasopharyngeal smear.

For example, the assay kit may include a nucleic acid amplification kit, an electrophoresis kit, or a next-generation nucleic acid amplification kit comprising a target nucleic acid molecule specifically bound to the oligo-nucleotide constituting the bead conjugate, a nucleic acid polymerase, and a buffer solution for a nucleic acid polymerization reaction. It may be a next generation sequencing kit.

In another aspect, the method comprising: reacting the above-described bead complex with a biological sample; separating the target nucleic acid present in the biological sample and the bead complex to which the oligo-nucleotide is bound; and detecting whether the oligo-nucleotide is bound to the target nucleic acid molecule.

For example, detecting whether the oligo-nucleotide is bound to the target nucleic acid molecule may include performing a nucleic acid amplification reaction using the target nucleic acid molecule bound to the oligo-nucleotide as a template.

Optionally, the step of separating the target nucleic acid molecule and the oligo-nucleotide-coupled bead complex present in the biological sample may be performed using a magnet.

If necessary, before the step of reacting the bead complex with the biological sample, the method may further include transforming a nucleic acid molecule present in the biological sample into a single-stranded nucleic acid molecule.

In another aspect, the method comprising: reacting the above-described bead complex with a biological sample; and separating the target nucleic acid molecule present in the biological sample and the bead complex to which the oligo-nucleotide is bound.

For example, the step of separating the target nucleic acid molecule and the oligo-nucleotide present in the biological sample from the bead complex may be performed using a magnet.

An oligo-nucleotide that specifically binds to a target nucleic acid molecule present in a biological sample is a bead conjugate condensed on the surface of the nanobead through a linker having a predetermined length bound to the surface of the nanobead. Among the nucleic acid molecules present in the biological sample, only the target nucleic acid molecule can specifically bind to the oligo-nucleotide bound to the surface of the nanobead. Accordingly, it is possible to specifically isolate and detect a target nucleic acid molecule in a biological sample.

In addition, by introducing a linking group having a predetermined length between the nanobead and the oligo-nucleotide, steric hindrance in the molecule is minimized, and non-specific binding between the linking group and the base of the oligo-nucleotide is inhibited, It is possible to implement a specific binding between the nanobead and the linker.

Since only the target nucleic acid molecule is specifically isolated and detected, an additional purification process is not required. Accordingly, the process of purifying the target nucleic acid molecule in the biological sample is simplified, and the target nucleic acid molecule can be isolated quickly and easily.

In addition, since only the target nucleic acid molecule present in the biological sample is specifically isolated and purified, the sensitivity of the analysis can be improved when finally analyzing whether the target nucleic acid molecule is present in the biological sample.

1 is a schematic diagram schematically showing the configuration of a bead assembly according to an exemplary embodiment of the present invention.
2 is a schematic diagram schematically illustrating a process for manufacturing a bead assembly according to an exemplary embodiment of the present invention.
3 is a schematic diagram schematically illustrating a process of isolating a target nucleic acid molecule from a biological sample using a bead binder prepared according to an exemplary embodiment of the present invention.
4 is a graph showing the results of measuring the average particle size of the bead binder synthesized according to an exemplary embodiment of the present invention using the DLS method.
5 is a graph showing the results of measuring the average surface charge of the bead binder synthesized according to an exemplary embodiment of the present invention using a Zetasizer.
6A to 6D are graphs showing PCR analysis results using a bead conjugate synthesized according to an exemplary embodiment of the present invention, respectively.
7 is a photograph showing the result of measuring the detection of a target nucleic acid molecule extracted using a bead conjugate synthesized according to an exemplary embodiment of the present invention using electrophoresis.

In order to find out whether viral disease, bacterial disease, or cancer is present, a biological sample (specimen) such as urine, saliva, sputum, nasopharyngeal smear or blood is obtained and then DNA or RNA inside the biological sample A process for purifying and extracting nucleic acid molecules such as Various inhibitors, DNA, and RNA exist in a biological sample, and the amount of a nucleic acid molecule to be detected is present in a relatively low concentration in the entire biological sample.

The currently used nucleic acid extraction method collects and purifies all charged substances in a biological sample. Next, since the test using a nucleic acid amplification reaction such as a polymerase chain reaction is performed using purified nucleic acid, the accuracy and efficiency are low. In addition, there are disadvantages in that the method of collecting biological samples is invasive and inconvenient, and requires the help of professional medical staff.

The present inventors develop a bead conjugate that can detect and analyze only a specific target nucleic acid molecule in a biological sample, rapidly and easily, and rapidly and accurately detects and analyzes whether a virus or pathogen is infected and/or cancer is developed with high sensitivity did. In order to detect and analyze only a desired specific nucleic acid molecule in a biological sample, an oligo-nucleotide capable of reacting specifically with a desired nucleic acid molecule on the surface of a nanobead is condensed (conjugated) to collect a specific nucleic acid molecule. , purified and detected.

Hereinafter, the present invention will be described with reference to the accompanying drawings, if necessary.

1 is a schematic diagram schematically showing the configuration of a bead assembly according to an exemplary embodiment of the present invention. As shown in FIG. 1, the bead conjugate 100 is condensed on the surface of the nano-bead 112 through the nano-bead 112 and the end of the connector L through a linker (L), and is specific to the target nucleic acid molecule. and an oligo-nucleotide (122) that binds positively. The linking group (L) interposed between the surface of the nano-bead 112 and the oligo-nucleotide 122 may have a structure of Formula 1 below.

[Formula 1]

(In Formula 1, R ¹ is a direct bond or a C ₁ -C ₂₀ aliphatic hydrocarbon group; R ² and R ³ are each independently C ₃ -C ₂₀ A divalent aliphatic hydrocarbon linkage group; The asterisk to the left of R ¹ is the surface of the nanobead indicates a site connected to , and an asterisk to the right of R ³ indicates a site connected to the end of the oligo-nucleotide.

In Formula 1, R ¹ is a direct bond or an aliphatic hydrocarbon group selected from the group consisting of a C ₁ -C ₂₀ alkyl group, a C ₂ -C ₂₀ alkenyl group, a C ₂ -C ₂₀ alkynyl group and a C ₁ -C ₂₀ alkyl ether group. can For example, the aliphatic hydrocarbon group constituting R ¹ of Formula 1 is selected from the group consisting of a C ₁ -C ₁₀ alkyl group, a C ₂ -C ₂₀ alkenyl group, a C ₂ -C ₂₀ alkynyl group, and a C ₁ -C ₂₀ alkyl ether group. may be, but is not limited thereto. In addition, in Formula ₁ , R ² and R ³ are each independently a C ₃ -C ₂₀ alkylene group, for example, a C _3-10 alkylene group or a C _{5-10 alkylene} group, but is not limited thereto. .

The linking group L interposed between the nano-bead 112 and the oligo-nucleotide 122 includes R ² and R ³ , which are two aliphatic hydrocarbon linkages each having at least 3 carbons. By introducing a linker (L) having a predetermined length, the nano-beads 112 constituting the bead complex 100 and the oligo-nucleotides 122 capable of specifically binding to a target nucleic acid molecule are spaced apart with a sufficient distance. do. Accordingly, intra-molecular and/or inter-molecular steric hindrance between the nano-beads 112 and the oligo-nucleotides 122 in the bead complex 100 can be minimized. .

In addition, in the process of synthesizing or manufacturing the bead conjugate 100, an amine group (-NH ₂ ) that is a reactive functional group directly or indirectly connected to the surface of the nano-bead 112 constituting the initial bead (110, see FIG. 2) And, an amide bond can be specifically formed between the carboxylic acid group (-COOH) applied to form the linkage group (L), and it is present in the base constituting the carboxylic acid group and the oligo-nucleotide (122). It can inhibit non-specific binding between reactive amine groups.

One end of the linking group (L) interposed between the nano-bead 112 and the oligo-nucleotide 122 in the bead complex 100 may be connected to the 5' end or the 3' end of the oligo-nucleotide 122 . For example, one end of the linking group (L) may be connected to the 5' end of the oligo-nucleotide (122).

In an exemplary aspect, the nano-beads 112 may be made of an inorganic material. For example, the inorganic material forming the nano-beads 112 is a non-metal material selected from the group consisting of iron oxide, silica, glass, and combinations thereof and/or gold, silver, copper, combinations thereof, and alloys thereof. It may be a metal material selected from the group, but is not limited thereto.

In another exemplary aspect, the nano-beads 112 may be made of an organic material. The organic material may be a polymer material. For example, the organic material forming the nano-beads 112 may be a polymer resin selected from the group consisting of polystyrene, polypropylene, polyethylene, poly acrylamide, combinations thereof, and copolymers thereof and/or furan, flu It may be a polysaccharide material such as lan acetate, cellulose, hydroxypropylmethylcellulose, agarose, chitosan, a combination thereof, or a copolymer thereof, but is not limited thereto.

In another optional aspect, the nano-beads 112 may be a porous material or a paramagnetic material. For example, the nano-beads 110 may be made of a magnetic material. When the nano-bead 112 is made of a paramagnetic material, as will be described later, a target nucleic acid molecule specifically bound to the bead binder 110 can be easily separated and purified using a magnet.

In exemplary aspects, oligo-nucleotides 120 may consist of 20 to 100, eg, 20 to 50, nucleotides. When the oligo-nucleotide 122 consists of 20 to 100 nucleotides, the oligo-nucleotide 122 can hybridize with a target nucleic acid molecule present in a biological sample under stringent conditions. Accordingly, a target nucleic acid molecule specific for the oligo-nucleotide 122 can be separated and purified with high sensitivity.

As used herein, “polynucleotide”, “nucleic acid molecule” or “nucleic acid” are used interchangeably and refer to a polymer of nucleotides of any length, and include DNA (eg, cDNA) and RNA molecules inclusive. include “Nucleotide,” the building block of a nucleic acid molecule, refers to deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or analogs thereof, or incorporated into polymers by DNA or RNA polymerases, or by synthetic reactions. It can be any substrate that can be. Polynucleotides may include modified nucleotides, sugars or bases modified analogs, such as methylated nucleotides and analogs thereof.

A mutation in a nucleotide may not result in a mutation in a protein. Such nucleic acids are functionally equivalent codons or codons encoding the same amino acid (e.g., due to codon degeneracy, there are six codons for arginine (Arg) or serine (Ser)), or a nucleic acid molecule comprising a codon encoding a biologically equivalent amino acid.

Also, mutations in nucleotides may cause changes in the protein itself. Even in the case of a mutation that causes a change in the amino acid of the protein, one exhibiting almost the same activity as the protein of the present invention can be obtained.

To the extent that the nucleic acid molecule or polynucleotide of the present invention has the characteristics, it is clear to those skilled in the art that the peptides and nucleic acid molecules of the present invention are not limited to the amino acid sequence or base sequence described in the sequence listing.

Meanwhile, the term "amino acid" herein is used in the broadest sense and is intended to include naturally-occurring L-amino acids or residues. One-letter abbreviations and/or three-letter abbreviations commonly used for naturally-occurring amino acids may be used herein.

Amino acids are D-amino acids as well as chemically-modified amino acids, such as amino acid analogs, naturally-occurring amino acids not normally incorporated into proteins, such as norleucine, properties known in the art to characterize amino acids. chemically-synthesized compounds having Included within the definition of amino acids are analogs or mimetics of phenylalanine or proline that allow conformational restriction of the same peptide compound as, for example, native phenylalanine or proline. Such analogs and mimetics may be referred to herein as "functional equivalents" of amino acids. do. Other examples of amino acids are listed in Roberts and Velaccio, The Peptides: Analysis, Synthesis, Biology, Eds. Gross and Meiehofer, Vol. 5, p. 341 (Academic Press, Inc.: N. Y. 1983).

For example, synthetic peptides synthesized by standard solid-phase synthesis techniques are not limited to the amino acids encoded by the gene, and thus allow for a wider variety of substitutions for a given amino acid. Amino acids not encoded by the genetic code may be referred to herein as “amino acid analogs”. For example, amino acid analogs include 2-aminoadipic acid (Aad) for Glu and Asp; 2-aminopimelic acid (Apm) for Glu and Asp; 2-aminobutyric acid (Abu) for Met, Leu and other aliphatic amino acids; 2-aminoheptanoic acid (Ahe) for Met, Leu and other aliphatic amino acids; 2-aminobutyric acid for Gly (Aib); cyclohexylalanine (Cha) for Val, Leu and Ile; Homoarginine (Har) to Arg and Lys; 2,3-diaminopropionic acid (Dap) for Lys, Arg and His; N-ethylglycine (EtGly) for Gly, Pro and Ala; N-ethylglycine (EtGly) for Gly, Pro and Ala; N-ethylasparagine (EtAsn) for Asn and Gin; hydroxylysine (Hyl) to Lys; allohydroxylysine (AHyl) for Lys; 3-(and 4-)hydroxyproline for Pro, Ser and Thr (3Hyp, 4Hyp); allo-isoleucine (AIle) for He, Leu and Val; 4-amidinophenylalanine for Arg; N-methylglycine for Gly, Pro and Ala (MeGly, sarcosine); N-methylisoleucine for Ile (MeIle); norvaline (Nva) for Met and other aliphatic amino acids; norleucine (Nle) for Met and other aliphatic amino acids; ornithine (Orn) for Lys, Arg and His; citrulline (Cit) and methionine sulfoxide (MSO) for Thr, Asn and Gin; and N-methylphenylalanine (MePhe) for Phe, trimethylphenylalanine, halo-(F-, Cl-, Br- or I-)phenylalanine or trifluorylphenylalanine.

As used herein, the term “peptide” includes all proteins, protein fragments, and peptides isolated from naturally occurring ones or synthesized by recombinant techniques or chemically.

In one aspect, peptide variants are provided, such as peptide variants having one or more amino acid substitutions. As used herein, the term “peptide variants” refers to one or more amino acids having substitutions, deletions, additions and/or insertions in the amino acid sequence of a peptide, and It means that it exerts almost the same biological function as a peptide composed of amino acids. The peptide variant should have at least 70%, preferably at least 90%, more preferably at least 95% identity to the original peptide.

Such peptide variants may include amino acid variants known as "conservative". Variants may also include non-conservative changes. In an exemplary aspect, the sequence of the variant polypeptide differs from the original sequence by substitution, deletion, addition, or insertion of 5 or fewer amino acids. Variants may also be altered by deletion or addition of amino acids that have minimal effect on the immunogenicity, secondary structure and hydropathic nature of the peptide.

“Conservative” substitution means that there is no significant change in properties such as secondary structure and hydropathic nature of a polypeptide even when one amino acid is substituted with another amino acid. Amino acid variations are based on the relative similarity of amino acid side chain substituents, such as polarity, charge, solubility, hydrophobicity, hydrophilicity and/or amphipathic nature, etc. can be obtained by In one example, amino acid exchanges in proteins or peptides that do not entirely alter the activity of the molecule are known in the art (H. Neurath, R.L. Hill, The Proteins, Academic Press, New York, 1979). The most common exchanges are amino acid residues Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Thy/Phe, Ala/ Exchange between Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, Asp/Gly.

In consideration of the above-described variation having biological equivalent activity, it is construed that the nucleic acid molecule encoding the peptide and/or protein according to the present invention includes a sequence exhibiting substantial identity to the sequence described in the sequence listing. The substantial identity is at least 61% when the sequence of the present invention and any other sequences are aligned to the maximum extent and the aligned sequence is analyzed using an algorithm commonly used in the art. of homology, more preferably 70% homology, even more preferably 80% homology, and most preferably 90% homology. Alignment methods for sequence comparison are known in the art.

As used herein, "hybridization under stringent conditions" refers to two single-stranded nucleic acid molecules consisting of at least 70%, for example 80% or more, or 90% or more of complementary nucleotides. do.

For example, the oligo-nucleotide 122 that specifically binds to a target nucleic acid molecule may be a probe or a primer. As used herein, "primer" generally refers to a nucleic acid molecule that is complementary to a portion of a template nucleic acid molecule.

In one example, a primer may be complementary to a portion of a strand of a template nucleic acid molecule. The primer may be a strand of nucleic acid for the synthesis of a target nucleic acid molecule in a nucleic acid amplification reaction, in which case the primer may hybridize to the template strand, and the nucleotide may be used at the end of the primer with the help of a polymerase (polymerase). ) can be added. Thus, while the target nucleic acid molecule is being replicated, the polymerase catalyzing the replication of the target nucleic acid molecule initiates replication at the 3' end of the primer attached to the target nucleic acid molecule, thereby synthesizing and replicating the opposite strand of the target nucleic acid molecule .

In an optional aspect, the primer may be fully or partially complementary to the template nucleic acid molecule. A primer may exhibit sequence identity or homology or complementarity to a template nucleic acid. Homology or sequence identity or complementarity between the primer and the template nucleic acid may be based on the length of the primer. For example, when the primer consists of about 20 nucleotides, it may include a base complementary to 10 or more adjacent nucleic acid bases with respect to the template nucleic acid.

On the other hand, the oligo-nucleotide 122 may specifically bind to a target nucleic acid molecule present in a biological sample. The types of target nucleic acid molecules will be described later.

Next, a method for preparing and synthesizing a bead conjugate according to an exemplary embodiment of the present invention will be described with reference to FIG. 2 . As shown in the upper left of Figure 2, by reacting the initial bead 110 with an amine group represented by the following Chemical Formula 2 on the surface of the nano-beads 112 and a carboxylic acid having a structure of the following Chemical Formula 3, the nano-beads A surface of (110) is synthesized by a modified (modified) bead intermediate (110A) with a linking group (first linking group, L ¹ ) having the structure of Formula 4 below.

[Formula 2]

[Formula 3]

[Formula 4]

(In Formulas 2 to 4, R ¹ , R ² , R ³ and an asterisk are the same as defined in Formula 1, respectively)

In an exemplary aspect, the initial bead 110 in which an amine group having the structure of Formula 2 is connected to the surface of the nano-bead 112 may be activated in a buffer solution adjusted to pH 5-6. Buffers capable of activating the initial beads 110 are MES (2- [Nmorpholino] ethane sulfonic acid) buffer, phosphate buffer, sodium acetate, sodium phosphate, and combinations thereof. It may be selected from the group consisting of, but is not limited thereto.

On the other hand, the amine group connected to the surface of the nano-bead 112 constituting the initial bead 110 and the carboxylic acid group constituting the terminal of the aliphatic carboxylic acid having the structure of Formula 2 react to form a stable amide bond. Thus, an appropriate activator may be used. Available activators are EDC(1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride), NHS(N-hydroxysuccinimide), sulfo-NHS(N-hydroxysulfosuccinimide), CMC(1-cyclohexyl-3-(2-morpholinoethyl) ) carbodiimide), DCC (dicyclohexyl carbodiimide), DIC (Diisopropyl carbodiimide), and may be selected from the group consisting of combinations thereof, but is not limited thereto. For example, the activator may be added in a ratio of 10 to 200 molar equivalents, for example, 50 to 150 molar equivalents, based on the amine group connected to the surface of the nano-beads 112 constituting the initial bead 110 .

For example, the reaction between the initial bead 110 in which an amine group is connected to the surface of the nano-bead 112 and the aliphatic carboxylic acid having the structure of Chemical Formula 2 may be performed at room temperature for 12-24 hours, but is not limited thereto. . If necessary, after the reaction between the initial beads 110 and the aliphatic carboxylic acid is completed, a washing process using the above-described buffer may be performed.

Then, the first connecting group (L ¹ ) having the structure of Chemical Formula 4 on the surface of the nano-bead 112 is first modified or modified bead intermediate 110A, and one end of the oligo-nucleotide 122 is of Chemical Formula 5 By reacting the oligo-nucleotide 122 modified with an aliphatic thiol, the oligo-nucleotide 122 is connected to the surface of the nano-bead 112 through the final linking group (L) having the structure of Formula 1 (100) synthesized and manufactured.

[Formula 5]

(In Formula 5, R ³ and an asterisk are the same as defined in Formula 1, respectively)

The reaction between the bead intermediate (110A) and the modified oligo-nucleotide (120) may be carried out at room temperature for 12-24 hours, for example, it may be carried out at a pH of 7-8. If necessary, a reducing agent for activating the terminal thiol group of the modified oligo-nucleotide 120 before the reaction may be used. For example, the reducing agent for activating the thiol group linked to one end of the oligo-nucleotide 122 is tris (2-carboxyethyl) phosphine (TCEP), tris (3-hydroxypropyl) phosphine (tris (3-hydroxypropyl) phosphine: THPP), dithiothreitol (DTT), 2-mercaptoethanol (ME), 2-mercaptoethylamine (MEA), combinations thereof and these It may be selected from the group consisting of a salt (eg, hydrochloride), but is not limited thereto.

If necessary, the modified oligo-nucleotide 120 not bound to the surface of the intermediate bead 110A may be removed.

As shown in Figure 2 and Chemical Formula 1, the finally prepared and synthesized bead conjugate 100 has a linker (L) interposed between the nano-bead 112 and the oligo-nucleotide 122 is at least 3 carbon atoms, respectively. and a divalent aliphatic hydrocarbon linking group including (R ² , R ³ ). Accordingly, the nano-beads 112 and the oligo-nucleotides 122 are not directly connected, and are spaced apart from each other by a predetermined distance. Intramolecular and/or intermolecular steric hindrance between the nano-beads 112 and the oligo-nucleotides 122 constituting the bead conjugate 100 of the present invention can be prevented and minimized.

On the other hand, in the present invention, by reacting the initial bead 110 having an amine group connected to the surface of the nano-bead 112 with a carboxylic acid having the structure of Formula 3, the first connecting group (L ¹ ) having an amide bond intermediate bead ( 110A) is synthesized, and the oligo-nucleotide 120 modified with an aliphatic thiol having the structure of Formula 5 is reacted with the oligo-nucleotide 120 at the end of the oligo-nucleotide 122 for the bead intermediate 110A, and finally the nano-bead 112 and the oligo - A bead conjugate 100 with a linker (L) having a predetermined length between the nucleotides 122 is synthesized.

That is, the amine group, which is a reactive functional group connected to the surface of the nano-bead 112, does not directly react with the carboxylic acid group formed at the end of the oligo-nucleotide, and the amine group connected to the surface of the nano-bead 112 is primarily a structure of Formula 2 Forms an amide bond with the carboxylic acid group of a carboxylic acid having Accordingly, an amide bond may be specifically formed between the amine group connected to the surface of the nanobead 112 and the carboxylic acid group of the carboxylic acid having the structure of Formula 2 .

According to the present invention, the base constituting the oligo-nucleotide 122 is not directly connected to the nano-bead 112 . The base constituting the oligo-nucleotide 122 constituting the bead conjugate 100 is inhibited from steric hindrance due to direct binding to the nano-bead 112 . Accordingly, since the oligo-nucleotide 122 efficiently hybridizes with the target nucleic acid molecule, the presence or absence of the target nucleic acid molecule in the biological sample can be detected and detected by applying the bead conjugate 100 of the present invention.

On the other hand, when the oligo-nucleotide is condensed on the surface of the nano-bead, intramolecular and/or intermolecular steric hindrance may be caused between the nano-bead and the oligo-nucleotide. In addition, for example, in the case of condensation (conjugation) to have an amide bond between a carboxylic acid group connected to the surface of the nanobead and an amine group modified at one end of the oligo-nucleotide, the carboxylic acid group connected to the surface of the nanobead is In addition to reacting with the amine group located at one end of the oligo-nucleotide, it also reacts with reactive primary amine groups present in adenine, cytosine, and guanine among the bases constituting the oligo-nucleotide to form an amide bond. to form That is, the amide bond between the nanobead and the oligo-nucleotide is formed non-specifically.

By the non-specific amide bond between the nanobead and the base constituting the oligo-nucleotide, steric hindrance is induced in the base constituting the oligo-nucleotide, and the oligo-nucleotide including the steric hindered base is a target nucleic acid Molecular and specific hybridization does not occur efficiently. Accordingly, when inducing a direct amide bond between the nanobead and the oligo-nucleotide, there is a limit in detecting and detecting the presence of a target nucleic acid molecule in a biological sample due to the modification of the oligo-nucleotide.

Subsequently, a method for analyzing the presence of a target nucleic acid molecule in a biological sample and a method for purifying a target nucleic acid molecule in a biological sample using the bead conjugate according to the present invention will be described. 3 is a schematic diagram schematically illustrating a process of isolating a target nucleic acid molecule from a biological sample using a bead binder prepared according to an exemplary embodiment of the present invention. As shown in FIG. 3 , the biological sample is reacted with the above-described bead complex 100 , and the target nucleic acid molecule and the oligo-nucleotide 122 present in the biological sample are separated by separating the bead complex 100 , which is a biological sample. Target nucleic acid molecules present within can be selectively isolated and purified.

For example, biological samples may include, but are not limited to, urine, saliva, sputum, blood, and nasopharyngeal smears. When the nucleic acid molecule present in the biological sample is in the form of DNA, the nucleic acid molecule in the biological sample may be transformed into a single-stranded nucleic acid molecule before the biological sample and the bead complex 100 react. For example, the transformation into a single-stranded nucleic acid molecule may be performed by applying heat to the biological sample, in which case the heat treatment may be performed at 70 to 100° C. for 2 to 10 minutes, but is not limited thereto.

In addition to the target nucleic acid molecule, various non-target nucleic acid molecules may be present in a biological sample. An oligo-nucleotide 122 capable of hybridizing with a target nucleic acid molecule is coupled to the outside of the bead conjugate 100 according to the present invention. Therefore, when the bead complex 100 according to the present invention is reacted with a biological sample in which a target nucleic acid molecule is present, only the target nucleic acid molecule present in the biological sample comprises an oligo-nucleotide (100) constituting the bead complex 100 according to the present invention. 122) specifically. On the other hand, non-target nucleic acid molecules present in the biological sample do not bind to the bead assembly 100 according to the present invention, and remain as free nucleic acid molecules in the biological sample.

Then, only the target nucleic acid molecule specifically bound to the bead conjugate 100 according to the present invention from among the biological sample may be separated and purified. For example, when the nano-bead 112 is made of a magnetic material, the bead assembly 100 may be separated from the non-target nucleic acid molecule remaining in the sample using a magnet. Accordingly, from among the nucleic acid molecules present in the biological sample, only the target nucleic acid molecule specifically bound to the bead conjugate 100 according to the present invention can be separated and purified with sensitivity.

According to the above-described embodiment, only the target nucleic acid molecule bound to the bead complex may be specifically purified and separated, and then the presence or absence of the target nucleic acid molecule in the biological sample may be analyzed.

On the other hand, the target nucleic acid molecule may specifically bind to the oligo-nucleotide 112 located outside the bead complex 100 isolated and purified from the biological sample. In this state, when a nucleic acid amplification reaction is performed using the target nucleic acid molecule as a template and the oligo-nucleotide 122 as a primer, nucleotides complementary to the target nucleic acid molecule are synthesized at the end of the oligo-nucleotide 122 . do. Accordingly, a plurality of nucleic acid molecules corresponding to the target nucleic acid molecule is amplified, and the presence of the amplification product is checked, so that the presence or absence of the target nucleic acid molecule in the biological sample can be detected and analyzed.

For example, the target nucleic acid molecule to which the oligo-nucleotide 122 specifically binds may be derived from a pathogen or cancer or tumor, and may be in the form of DNA or RNA.

A nucleic acid molecule derived from a pathogen may be a nucleic acid molecule that is a factor or marker indicating whether or not the pathogen is infected. For example, a nucleic acid molecule derived from a pathogen may include a nucleic acid molecule derived from a pathogenic virus, pathogenic bacterium, or pathogenic protozoa. In one example, the target nucleic acid molecule may be a nucleic acid molecule encoding a protein or peptide comprising a pathogenic antigen or a fragment thereof, a variant or derivative thereof.

A pathogenic antigen may be derived from a pathogenic organism, such as a bacterium, virus or protozoa, that elicits an immunological response in an individual, in particular a mammalian individual, in particular a human. In one example, the pathogenic antigen may be a surface antigen or a portion thereof located on the surface of a bacterial, viral or protozoan organism.

In one example, the pathogenic antigen may be a peptide or protein antigen derived from a pathogen associated with an infectious disease. More specifically, the pathogenic antigen is Acinetobacter baumannii, Anaplasma genus, Anaplasma phagocytophilum, Ancylostoma braziliense, Ancylostoma duodena Arcanobacterium haemolyticum, Ascaris lumbricoides, Aspergillus genus, Astroviridae, Babesia genus, Bacillus anthracis, Bacillus cereus ( Bacillus cereus), Bartonella henselae, BK virus, Blastocystis hominis, Blastomyces dermatitidis, Bordetella pertussis, Borrelia burgdor Berry (Borrelia burgdorferi), Borrelia genus, Borrelia spp, Brucella genus, Brugia malayi, Bunyaviridae family, Buryaviridae family (Burkholderia cepacia) and other Burkholderia species, Burkholderia mallei, Burkholderia pseudomalei, Caliciviridae family, Campylobacter genus, Candida albicans albicans), Candida spp, Chlamydia trachomatis, Chlamydophila pneumoniae, Chlamydophila psittaci (Ch lamydophila psittaci), CJD prion, Clonorchis sinensis, Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium perfringens, Clostridium spp, Clostridium tetani, Coccidioides spp, coronaviruses, Coronaviruses, Corynebacterium diphtheria, Coxiella burnetii, Crimean-Congo hemorrhagic fever virus, Cryptococcus neoformans, Cryptosporidium genus, Cytomegalovirus (CMV), Dengue viruses (DENgue viruses) -1, DEN-2, DEN-3 and DEN-4), Dientamoeba fragilis, Ebolavirus (EBOV), Echinococcus genus, Ehrlicia chaffeensis, E. Ehrlichia ewingii, Ehrlichia genus, Entamoeba histolytica, Enterococcus genus, Enterovirus genus, Enteroviruses, mainly Coxsackie A virus ( Coxsackie A virus) and Enterovirus 71 (EV71), Epidermophyton spp, Epstein-Barr Virus (EBV), Esche richia coli) O157:H7, O111 and O104:H4, Fasciola hepatica and Fasciola gigantica, FFI prions, Filarioidea superfamily, Flaviviruses, Francisella tularensis), Fusobacterium genus, Geotrichum candidum, Giardia intestinalis, Gnathostoma spp, GSS prion, Guanarito virus ), Haemophilus ducreyi, Haemophilus influenza, Helicobacter pylori, Henipavirus (Hendra virus Nipah virus), hepatitis A virus, hepatitis B virus (HBV), hepatitis C Virus (HCV), hepatitis D virus, hepatitis E virus, herpes simplex virus 1 and 2 (HSV-1 and HSV-2), Histoplasma capsulatum, HIV (human immunodeficiency virus) , Hortaea werneckii, human bocavirus (HBoV), human herpesvirus 6 (HHV-6) and human herpesvirus 7 (HHV-7), human metapneumovirus ( Human metapneumovirus (hMPV), Human papillomavirus (HPV), Human parainfluenza viruses (HPIV), influenza virus, Japanese encephalitis virus, JC Virus, Junin virus, Kingella kingae, Klebsiella granulomatis, Kuru prion, Lassa virus, Legionella pneumophila ), Leishmania genus, Leptospira genus, Listeria monocytogenes, Lymphocytic choriomeningitis virus (LCMV), Machupo virus, Mala Malassezia spp, Marburg virus, Measles virus, Metagonimus yokagawai, Microsporidia phylum, Molluscum contagiosum virus ) (MCV), Mumps virus, Mycobacterium leprae and Mycobacterium lepromatosis, Mycobacterium tuberculosis, Mycobacterium urcerans ( Mycobacterium ulcerans, Mycoplasma pneumoniae, Naegleria fowleri, Necator americanus, Neisseria gonorrhoeae, Neisseria meningitides, Nocardia asteroids, Nocardia asteroids species (Nocardia spp), Onchocerca volvulus, Orientia tsutsugamushi, Orientia tsutsugamushi Tomyxovirus family (Orthomyxoviridae family, influenza), Brazil Paracoccidioides brasiliensis, Lung fluke species (Paragonimus spp), Pulmonary dystoma (Paragonimus westermani), Parvovirus B19, Pasteurella genus , Plasmodium genus, Pneumocystis jirovecii, Poliovirus, Rabies virus, Respiratory syncytial virus (RSV), Rhinovirus, rhinovirus, Rickettsia akari, Rickettsia genus, typhus Rickettsia prowazekii, Rockettsia rickettsia, Rickettsia fever, Rickettsia typhi virus, Rickettsia typhi virus Rotavirus, Rubella virus, Sabia virus, Salmonella genus, Sarcoptes scabiei, SARS coronavirus, Schistosoma genus, Severe fever thrombocytopenia Severe fever with thrombocytopenia syndrome virus (SFTSV), Shigella genus, Sin Nombre virus, Hantavirus, Sporothrix schenckii, Staphylococcus ( Staphylococcus genus), Staphylococcus genus, Streptococcus agalactiae, Streptococcus pneumoniae (St reptococcus pneumoniae), Streptococcus pyogenes, Strongyloides stercoralis, Taenia genus, Taenia solium, tickborne encephalitis virus, TBEV) (Toxocara canis) or cat roundworm (Toxocara cati), Toxoplasma gondii, Treponema pallidum, Trichinella spiralis, Trichomonas vaginalis, Trichophyton spp. , Trichuris trichiura, Trypanosoma brucei, Trypanosoma cruzi, Ureaplasma urealyticum, Varicella zoster virus (VZV), varicella zoster Virus (VZV), Variola major or Variola minor, vCJD prion, Venezuelan equine encephalitis virus, Vibrio cholera, West Nile virus ), Western equine encephalitis virus, Wuchereria bancrofti, Yellow fever virus, Yersinia enterocolitica, Yersinia pestis, and It may be derived from Yersinia pseudotuberculosis, but is not limited thereto.

The target nucleic acid molecule to which the oligo-nucleotide 122 specifically binds may be from cancer or a tumor. In an exemplary aspect, the target nucleic acid molecule derived from cancer or tumor may be a nucleic acid molecule specific for cancer or tumor, or a nucleic acid molecule that is a factor or marker indicating whether or not cancer or tumor develops. In one example, cancers or tumors include, but are not limited to, stomach cancer, lung cancer, liver cancer, colorectal cancer, small intestine cancer, skin cancer, pancreatic cancer, and prostate cancer.

In an optional aspect, the target nucleic acid molecule derived from cancer or tumor may be a nucleic acid molecule encoding a protein or peptide that is a tumor antigen or fragment thereof, variant or derivative thereof.

In one example, the target nucleic acid molecule derived from cancer or tumor is 5T4, 707-AP, 9D7, AFP, AlbZIP HPG1, alpha-5-beta-1-integrin, alpha-5-beta-6-integrin, alpha-actinin -4/m, alpha-methylacyl-Coenzyme A racemase, ART-4, ARTC1/m, B7H4, BAGE-1, BCL-2, bcr/abl, beta-catenin/m, BING-4, BRCA1/m , BRCA2/m, CA 15-3/CA 27-29, CA 19-9, CA72-4, CA125, calreticulin, CAMEL, CASP-8/m, cathepsin B, Ca Depsin L, CD19, CD20, CD22, CD25, CDE30, CD33, CD4, CD52, CD55, CD56, CD80, CDC27/m, CDK4/m, CDKN2A/m, CEA, CLCA2, CML28, CML66, COA-1/m , coactosin-like protein, collage XXIII, COX-2, CT-9/BRD6, Cten, cyclin B1, cyclin D1, cyp-B, CYPB1, DAM-10, DAM-6, DEKCAN, EFTUD2/ m, EGFR, ELF2/m, EMMPRIN, EpCam, EphA2, EphA3, ErbB3, ETV6-AML1, EZH2, FGF-5, FN, Frau-1, G250, GAGE-1, GAGE-2, GAGE-3, GAGE- 4, GAGE-5, GAGE-6, GAGE7b, GAGE-8, GDEP, GnT-V, gp100, GPC3, GPNMB/m, HAGE, HAST-2, Hepsin, Her2/neu, HERV-K-MEL, HLA- A*0201-R17I, HLA-A11/m, HLA-A2/m, HNE, Homeobox NKX3.1, HOM-TES-14/SCP-1, HOM-TES-85, HPV-L1, HPV-E6 , HPV-E7, HSP70-2M, HST-2, hTERT, iCE, IGF-1R, IL-13Ra2, IL-2R, IL-5, immature laminin receptor, kallikrein in)-2, Crane-4, Ki67, KIAA0205, KIAA0205/m, KK-LC-1, K-Ras/m, LAGE-A1, LDLR-FUT, MAGE-A1, MAGE-A2, MAGE-A3, MAGE -A4, MAGE-A6, MAGE-A9, MAGE-A10, MAGE-A12, MAGE-B1, MAGE-B2, MAGE-B3, MAGE-B4, MAGE-B5, MAGE-B6, MAGE-B10, MAGE-B16 , MAGE-B17, MAGE-C1, MAGE-C2, MAGE-C3, MAGE-D1, MAGE-D2, MAGE-D4, MAGE-E1, MAGE-E2, MAGE-F1, MAGE-H1, MAGEL2, mammaglobin A, MART-1/Melan-A, MART-2, MART-2/m, matrix protein 22, MC1R, M-CSF, ME1/m, mesothelin, MG50/PXDN, MMP11, MN/CA IX-antigen, MRP-3, MUC-1, MUC-2, MUM-1/m, MUM-2/m, MUM-3/m, myosin class I/m, NA88-A, N -Acetylglucosaminyltransferase-V, neo-PAP, neo-PAP/m, NFYC/m, NGEP, NMP22, NPM/ALK, N-Ras/m, NSE, NY-ESO-1, NY-ESO- B, OA1, OFA-iLRP, OGT, OGT/m, OS-9, OS-9/m, osteocalcin, osteopontin, p15, p190 minor bcr-abl, p53, p53/m, PAGE-4, PAI- 1, PAI-2, PAP, PART-1, PATE, PDEF, Pim-1-kinase, Pin-1, Pml/PARalpha, POTE, PRAME, PRDX5/m, Prosteine, Proteinase-3, PSA , PSCA, PSGR, PSM, PSMA, PTPRK/m, RAGE-1, RBAF600/m, RHAMM/CD168, RU1, RU2, S-100, SAGE, SART-1, SART-2, SART-3, SCC, SIRT2 /m, Sp17, S SX-1, SSX-2/HOM-MEL-40, SSX-4, STAMP-1, STEAP-1, Survivin, Survivin-2B, SYT-SSX-1, SYT-SSX-2, TA- 90, TAG-72, TARP, TEL-AML1, TGFbeta, TGFbetaRII, TGM-4, TPI/m, TRAG-3, TRG, TRP-1, TRP-2/6b, TRP/INT2, TRP-p8 , a nucleotide sequence encoding tyrosinase, UPA, VEGFR1, VEGFR-2/FLK-1, WT1 and the immunoglobulin genotype of lymphocytes or the T cell receptor genotype of lymphocytes, fragments thereof, variants or derivatives thereof, or It may consist of a transcript sequence thereof, but is not limited thereto.

Accordingly, the present invention also relates to an assay kit for detecting a target nucleic acid molecule in a biological sample using the composition for analysis including the above-described bead conjugate 100 .

For example, the assay kit may be a nucleic acid amplification kit comprising a target nucleic acid molecule specifically bound to the oligo-nucleotide 122 constituting the bead complex 100, a nucleic acid polymerase, and a buffer for a nucleic acid amplification reaction. In addition to the bead conjugate 100 and nucleic acid polymerase, the buffer for the nucleic acid amplification reaction is deoxyribonucleotide triphosphate (dNTP) and/or nucleotide triphosphate (NTP), and the presence of an amplification product according to the nucleic acid amplification reaction. Fluorescent so that it can be detected. It may further include functional additives such as probes and polymerase stabilizers labeled with substances and/or radioactive isotopes.

Polymerase stabilizers include bovine serum albumin (BSA), cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, glycosaminoglycan, pullulan, alginic acid, carrageenan, aribinogalactan, hemicellulose, It may include a polysaccharide selected from the group consisting of dextran, chitosan, glycol chitosan, starch, and combinations thereof. The content of the polymerase stabilizer in the buffer for the nucleic acid amplification reaction may be included in a concentration of 0.01 to 10% (w/v), for example, 0.5 to 5% (w/v) or 0.5 to 3% (w/v). have. When the content of the polymerase stabilizer satisfies the above-mentioned range, the nucleic acid amplification reaction can be performed efficiently.

As used herein, the term "polymerase" generally refers to a substance that catalyzes a polymerization reaction. Polymerases can be used to extend nucleic acid primers paired with a template strand by introduction of nucleotides or nucleotide analogues. The polymerase can add new strands of DNA by adding new nucleotides corresponding to the template strand one at a time through the creation of phosphodiester bonds, thereby extending the 3' end of the existing nucleotide chain.

The nucleic acid polymerase is not particularly limited as long as the amplification reaction of the target nucleic acid molecule can be performed using the polymerase. More specifically, polymerases include DNA polymerases, RNA polymerases, thermostable polymerases, wild-type polymerases, and modified polymerases. More specifically, the polymerase is "Klenow fragment" of E. coli DNA polymerase I, bacteriophage T7 DNA polymerase, bacteriophage T4 DNA polymerase, 029 (phi29) DNA polymerase agent, Taq polymerase, Tth Polymerase, Tli Polymerase, Pfu Polymerase, Pwo Polymerase, VENT Polymerase, DEEPVENT Polymerase, EXTaq Polymerase, LA-Taq Polymerase, Sso Polymerase, Poc Polymerase, Pab Polymerase, Mth Polymerase, ES4 Polymerase, Tru Polymerase, Tac Polymerase, Tne Polymerase, Tma Polymerase, Tea Polymerase, Tih Polymerase, Tfi Polymerase, Platinum Taq Polymerase, Tbr Polymerase, Tfl Polymerase, Pfutubo Polymerase, Pyrobest Polymerase enzymes, Pwo polymerase, KOD polymerase, Bst polymerase, Sac polymerase, polymerase having 3' to 5' exonuclease activity, and variants and derivatives thereof. In one example, the polymerase is a thermostable DNA polymerase that can be obtained from various bacterial species, including Thermus aquaticus (Taq), Thermus thermophilus (Tth), Thermus filiformis , Thermis flavus , Thermococcus literalis and/or Pyrococcus furiosus (Pfu). However, the present invention is not limited thereto.

For example, a polymerase, dNTPs and/or NTPs in a buffer for nucleic acid amplification reaction may be commercialized in the form of a premix. In one example, the nucleic acid polymerase and the dNTP (and/or NTP) are 5-40% (w/v), for example 10-33% (w/v) or 15-25% (w) in a buffer for a nucleic acid amplification reaction. /v) may be included.

Buffers for nucleic acid amplification reactions may contain dNTP mixtures (dATP, dCTP, dGTP, dTTP) and/or NTP mixtures (ATP, CTP, GTP, TTP), other nucleic acid amplification reaction additives and nucleic acid polymerase cofactors. When performing a nucleic acid amplification reaction, it may be desirable to supply the reaction vessel with components necessary for the reaction in excess. The excess amount of the components required for the nucleic acid amplification reaction means an amount such that the amplification reaction is not substantially limited to the concentration of the component. A cofactor, such as Mg ²⁺ , dNTP may be supplied to the reaction solution to such an extent that a desired amplification reaction can be achieved.

For example, in a nucleic acid amplification reaction, annealing is performed under stringent conditions that enhance specific binding between the nucleotide sequence of the target nucleic acid molecule and the primer sequence. The term annealing or priming refers to the apposition of an oligonucleotide or nucleic acid to a template nucleic acid molecule, whereby a polymerase polymerizes the nucleotides to form a complementary nucleic acid molecule in the template nucleic acid molecule or fragment thereof. Stringent conditions for annealing are sequence-dependent and vary according to environmental variables. In an exemplary aspect, the bead conjugate 100 according to the present invention in a buffer for a nucleic acid amplification reaction is 5 to 40% (w/v), for example, 5 to 30% (w/v) or 5 to 20% (w/v) v) may be included.

If necessary, the buffer for the nucleic acid amplification reaction may further include an additive for the nucleic acid amplification reaction. For example, the additive for the nucleic acid amplification reaction may be selected from the group consisting of mannitol, polyethylene glycol (eg, PEG 10,000), trehalose, betaine, and combinations thereof. In this case, the additive for the nucleic acid amplification reaction in the buffer for the nucleic acid amplification reaction is at a concentration of 0.5 to 30% (w/v), for example, 0.5 to 20% (w/v) or 1 to 15% (w/v). may be added.

The buffer for the nucleic acid amplification reaction may include an appropriate buffer for the nucleic acid amplification reaction. The type of the buffer is not particularly limited, but organic acids, glycine, histidine, glutamate, succinate, phosphate, acetate, citrate, tris (eg, tris-EDTA), HEPES (Hydroxyethyl Piperazine Ethane Sulfonic acid), amino acids and these It can be selected from the group consisting of a combination of.

The amplified target nucleic acid molecule may be labeled with a detectable labeling substance. In one exemplary aspect, the labeling material may be a material that emits fluorescence, phosphorescence, chemiluminescence, or radioactivity, but is not limited thereto. For example, the labeling material may be fluorescein, phycoerythrin, rhodamine, lissamine Cy-5 or Cy-3.

When amplifying a target nucleic acid molecule, Cy-5 or Cy-3 is labeled at the 5'-end and/or 3' end of the oligo-nucleotide 122, and a nucleic acid amplification reaction (eg, real-time polymerase chain reaction) ), the target nucleic acid may be labeled with a detectable fluorescent labeling material. In addition, when labeling with a radioactive material is performed in a nucleic acid amplification reaction (eg, real-time polymerase chain reaction), radioactive isotopes such as P ³² and/or S ³⁵ are added to the buffer for the nucleic acid amplification reaction according to the present invention. In addition, as the amplification product is synthesized, radioactivity may be incorporated into the amplification product so that the amplification product may be radioactively labeled.

For labeling, various methods commonly practiced in the art to which the present invention pertains may be used. For example, nick translation methods, random priming methods (Multiprime DNA labeling systems booklet, "Amersham" (1989)) and kynation methods (Maxam & Gilbert, Methods in Enzymology, 65:499 (1986)) can be done through The label is a signal detectable by fluorescence, radioactivity, phosphorescence, chromometry, gravimetric measurement, X-ray diffraction or absorption, magnetic, enzymatic activity, mass analysis, binding affinity, hybridization radiofrequency, nano-crystal. to provide

With respect to a nucleic acid amplification reaction, for example, polymerase chain reaction (PCR), heat denaturation, annealing and polymerization for separation of the target nucleic acid molecule and the oligo-nucleotide 122 double chain are performed. It can be appropriately adjusted and controlled according to the composition and length of the oligo-nucleotide 122 and the target nucleic acid molecule specifically binding thereto.

The nucleic acid amplification reaction is not particularly limited as long as it can amplify a target nucleic acid molecule that may be present in a biological sample. For example, the nucleic acid amplification reaction is a polymerase chain reaction (PCR), a reverse transcription-polymerase chain reaction (RT-PCR), a real-time polymerase chain reaction (Real-Time PCR) , Reverse Transcription, Complementary DNA Synthesis, repair chain reaction, multiplex PCR, transcription-mediated amplification (TMA), self-maintaining sequence replication ( self-sustained sequence replication), selective amplification of target polynucleotide sequences, consensus sequence primed polymerase chain reaction (CP-PCR), optionally priming polymerase chain Arbitrarily primed polymerase chain reaction (AP-PCR, Loop-Mediated Isothermal Amplification, LAMP), Real-time Nucleic Acid Sequence-Based Amplification (NASBA), Strand Displacement Amplification , SDA), Multiple Displacement Amplification (MDA), Rolling Circle Amplification (RCA), Ligase Chain Reaction (LCR), Helicase Dependent Amplification (HDA), Branch-extension amplification (Ram) ification-extension Amplification Method (RAM), a method selected from the group consisting of an in vitro transcription-based amplification system (TAS), and combinations thereof, but is not limited thereto.

In an optional embodiment, the assay kit for detecting binding of the oligo-nucleotides 122 constituting the bead complex 100 to the target nucleic acid molecule present in the biological sample is an electrophoresis kit or next generation sequencing (next generation). sequencing, NGS) kit, but is not limited thereto.

Hereinafter, the present invention will be described through exemplary embodiments, but the present invention is not limited to the technical ideas described in the following examples.

Synthesis Example 1: Synthesis of a bead complex

Nanobeads (silica beads, Bioneer) with amine functional groups connected to the surface were dispersed in 1 ml of 100 mM MES buffer (pH 5.5) at a concentration of 100 mg/ml. Using the carbodiimide reaction of the amine group of the nanobead surface with the carboxyl group of 6-maleimidohexanic acid, EDC and NHS were first transformed into a linking group having an amide bond on the surface of the nanobead, 100 times the mole of the amino group connected to the nanobead surface The equivalent amount was dissolved in 4 ml of MES buffer. After that, the solution was added dropwise to the solution in which the nano-beads were dispersed and reacted at room temperature for 12-24 hours. After the reaction was completed, it was washed 3 times using MES buffer and stored in 100 mM tris buffer, pH 7.4. In order to connect the primer with the maleimide group on the surface of the intermediate nanobead on which the primary linker modification reaction has been completed, a primer whose 5' end is modified with a 1-hexyl thiol group (a sequence specific to the human papillomavirus (HPV) L1 region, purchased from Cosmogene Tech, Korea) ) was used. A primer whose 5' end is modified with a 1-hexyl thiol group (5'-TTITIACIKKIGTIGAYACIAC - 3', SEQ ID NO: 1, I is ideoxyl thyimine, GP5+ primer or Thiol GP5+ primer) was mixed with Tris (2-carboxylethyl) at a concentration of 1 μm. It was reduced in pH 7.4 buffer in which phosphine hydrochloride (TCEP) was dissolved for 2 hours. 10 mg of the nanobeads first modified to have a maleimide group and 10 nM of a primer with a thiol group were reacted at room temperature for 12-24 hours in a pH 7.4 tris buffer. After the reaction was completed, the nano-beads were washed 3 times using tris buffer and stored in tris buffer at pH 8.0 at a concentration of 10 mg/ml. Hereinafter, the bead conjugate prepared in Synthesis Example 1 is referred to as Thiol GP5+_NH ₂ beads.

Synthesis Example 2: Synthesis of a bead complex

In place of the GP5+ primer, a primer whose 5' end was modified with a 1-hexyl thiol group (5'-GAAAIAYIAAITGYAIIWCRWAYTCYTC-3', SEQ ID NO: 2, hereinafter, GP6+ primer or Thiol GP6+ primer) Synthesis Example except that it was used The procedure of 1 was repeated to prepare a bead complex. Hereinafter, the bead conjugate prepared in Synthesis Example 2 is referred to as Thiol GP6+_NH ₂ beads.

Comparative Synthesis Example 1: Amino Group-Coupled Nano Beads

Nanobeads with amine groups connected to the surface were used as they were without surface modification and connection with a primer. Hereinafter, the nano-beads of Comparative Synthesis Example 1 are referred to as NH ₂ beads.

Comparative Synthesis Example 2: Amide Bonded Nanobeads

Surface-modified nanobeads were used with an amino group connected to the surface of the nanobead and a linker having an amide bond on the surface of the nanobead using a carboiimide reaction of carboxyalic acid of 6-maleimidohxexanoic acid. Hereinafter, the nanobeads prepared in Comparative Synthesis Example 2 are referred to as NH ₂ -Maleimide beads.

Comparative Synthesis Example 3: Hydroxy-Group Silica Beads

Nanobeads (silica beads, bioneers) having a hydroxyl group on the surface were used as they were without surface modification and connection with a primer. Hereinafter, the beads prepared in Comparative Synthesis Example 3 are referred to as COOH beads.

Comparative Synthesis Example 4: Synthesis of amide bead conjugate

Nanobeads (COOH magnetic beads, Bioneer) having carboxylic acid present on the surface and oligo-nucleotides substituted with amine groups at the 3' end were combined using carbodiimide reaction. Nanobeads (silica beads, Bioneer) connected to the surface of carboxylic acid were dispersed in 1 ml of 100 mM MES buffer (pH 5.5) at a concentration of 100 mg/ml. In order to connect the primer with the carboxyl group on the surface of the nanobead, a primer having an amine group at the 5' end was used (a sequence specific to the human papillomavirus (HPV) L1 region, GP5+ primer, purchased from Cosmogintech Korea). The procedure of Synthesis Example 1 was repeated to prepare a bead complex. The bead conjugate prepared in Comparative Synthesis Example 4 is referred to as NH ₂ GP5+_COOH beads.

Comparative Synthesis Example 5: Preparation of a bead complex

Instead of the GP5+ primer, the procedure of Synthesis Example 1 was repeated, except that a GP6+ primer whose 5' end was modified with an amino group was used to prepare a bead complex. Hereinafter, the bead conjugate prepared in Comparative Synthesis Example 5 is referred to as NH ₂ GP6+_COOH beads.

Comparative Synthesis Example 6: Silica Beads

Nanobeads (silica beads, Bioneer) that were not modified with functional groups on the surface were used as they were without surface modification and connection with a primer. The beads prepared in Comparative Synthesis Example 6 are referred to as silica beads.

Example 1: Measurement of Average Particle Size of Bead Aggregates

The average particle size of the bead binders finally prepared in Synthesis Example 1 and Synthesis Example 2, respectively, and the bead binders prepared in Comparative Synthesis Examples 1-5, respectively, were measured using a dynamic light scattering (DLS) method. The results of measuring the average particle size of the bead binder and silica beads are shown in FIG. 4 . As shown in Figure 4, according to Synthesis Example 1 and Synthesis Example 2, it was confirmed that the size of the bead conjugate to which the primer was conjugated through the amide bond and the sulfide bond on the surface of the nanobead was greatly increased.

Example 2: Measurement of average surface charge of a bead complex

The average surface charge of the bead conjugates finally prepared in Synthesis Example 1 and Synthesis Example 2, respectively, and the bead conjugates prepared in Comparative Synthesis Examples 1-5, respectively, were measured using a Zetasizer. The measurement result is shown in FIG. From the measurement results, it was confirmed that the average surface charge was changed as the linking group on the surface of the nanobead was changed.

Example 3: Evaluation of extraction efficiency of target nucleic acid molecules using clinical specimens

In order to confirm the possibility of separating the nucleic acid molecules of the bead conjugates prepared in Synthesis Example 1 and Synthesis Example 2, respectively, and the bead conjugates prepared in Comparative Synthesis Examples 3 to 6, cell free DNA (cfDNA) was collected. and recovery efficiency. In order to confirm that the bead binder prepared in Synthesis Example 1 and Synthesis Example 2 specifically binds to a specific cfDNA, a vaginal secretion (cervix swab) sample with a positive history of HPV and sexually transmitted disease (STD) prepared. The extraction experiment was carried out by diluting the sample prepared in negative urine. Considering the optimal extraction pH (8-9) for cfDNA of the bead complex, mix pH 8.5 with 1M tris buffer and 200 mM EDTA (ethylenediamine-N, N, N', N'-tetraacetic acid) with urine in a 1:9 ratio. mixed Extraction was performed using the Kingfisher magmax protocol using the Kingfisher equipment using the Kingfisher Magmax 96 Cell-free DNA isolation kit.

(1) Evaluation of cfDNA extraction efficiency using PCR

To check whether HPV cfDNA and STD cfDNA were amplified among the cfDNA extracted above, Taq polymerase, primer, probe, dNTP, and EvaGreen were added to the extraction solution, and real time PCR (RT-PCR) was performed. For HPV PCR, 12 μl of a mixture of Invitrogen's platinum Ⅱ Hot start DNA Taq polymerase, dNTP, and MgCl ₂ , 5 μl of primer containing our GP5 & GP6 targeting sequence, 0.8 μl of TE buffer, 1.2 μl of EvaGreen, urine extract 5 μl was used. To perform the nucleic acid amplification reaction, each sample was treated at 95°C for 10 minutes, followed by 45 nucleic acid amplification cycles of 20 seconds at 95°C, 30 seconds at 50°C, and 40 seconds at 72°C, and finally 60 Treated at ~95°C for 5 seconds.

In the case of STD PCR, the company's Ezplex○R STD PCR Kit (in vitro approval No. 18-828 classification number [grade]: N05030.01[3]) was used. 10 μl of STD RQ mixture, 6 μl of Primer mix, and 4 μl of urine extract were used. In order to perform the nucleic acid amplification reaction, each sample was sequentially treated for 2 minutes at 25°C, 2 minutes at 50°C, and 10 minutes at 95°C, followed by a nucleic acid amplification cycle of 20 seconds at 95°C and 1 minute at 60°C. 40 times were performed. The measurement result of nucleic acid amplification according to this example is shown in FIG. 6 .

As shown in FIG. 6 , the bead conjugate to which a primer specific to the HPV L1 region is bound according to Synthesis Examples 1 and 2 is the COOH bead of Comparative Synthesis Example 3 that is not bound with a specific primer for HPV cfDNA or It was confirmed that the Cq value was earlier than that of the silica beads of Comparative Synthesis Example 6. Conversely, in the case of STD cfDNA, it was confirmed that the COOH beads of Comparative Synthesis Example 1 or the silica beads of Comparative Synthesis Example 6 had a higher Cq value than the composite beads of Synthesis Examples 1 and 2. On the other hand, compared to the bead conjugate in which an amide bond was formed between an amino group and a carboxylic acid group without a linking group in Comparative Synthesis Examples 4-5, the bead conjugate synthesized in Synthesis Examples 1 and 2 produced the intended HPV nucleic acid more rapidly and efficiently. amplified. From these results, it was confirmed that the bead conjugates of Synthesis Examples 1 and 2 were more specifically bound to HPV cfDNA than the beads or bead conjugates of Comparative Synthesis Examples.

(2) Evaluation of cfDNA extraction efficiency using electrophoresis

In order to confirm HPV cfDNA and STD cfDNA from the cfDNA extracted above, 4 μl of the extracted solution was loaded and electrophoresis was performed at 150V for 20 minutes. The electrophoretic evaluation result is shown in FIG.

In the case of HPV, 12 μl of a mixture of Invitrogen's platinum Ⅱ Hot start DNA Taq polymerase, dNTP, and MgCl ₂ , 5 μl of primer containing our GP5 & GP6 targeting sequence, 2 μl of TE buffer, and 5 μl of urine extract were used. Real-time PCR was performed. After real-time PCR, the effective nucleic acid in the sample was amplified, and then electrophoresis (150V, 20 minutes) was performed on an agarose gel. To perform a real-time nucleic acid amplification reaction (PCR), each sample was treated at 95°C for 5 minutes, followed by 45 nucleic acid amplification cycles of 20 seconds at 95°C, 30 seconds at 50°C, and 40 seconds at 72°C, and , and finally at 60 °C for 5 minutes and sequentially at 10 °C.

For STD PCR, the company's Ezplex○R STD PCR Kit (in vitro permission No. 18-828 classification number [grade]: N05030.01[3]) was used. 8 μl of STD RQ mixture, 2 μl of Primer mix, 2 μl of internal control, and 4 μl of urine extract were used. In order to perform the nucleic acid amplification reaction, treatment was performed at 5 ° C. for 2 minutes, 94 ° C. for 10 minutes, and nucleic acid amplification cycles of 94 ° C. for 20 seconds, 62 ° C. for 80 seconds, and 72 ° C. for 1 minute were performed 40 times, Finally, it was sequentially treated at 72°C for 5 minutes and at 4°C.

As shown in FIG. 7 , the band of HPV was clearly shown in the bead conjugate synthesized in Synthesis Example 1 and Synthesis Example 2, and the STD band was clearly shown compared to the silica bead of Comparative Synthesis Example 6. From these results, it was confirmed that the bead conjugates prepared in Synthesis Example 1 and Synthesis Example 2 respectively bind to HPV cfDNA more specifically than the beads or bead conjugates prepared in Comparative Synthesis Example.

In the above, the present invention has been described based on exemplary embodiments and examples of the present invention, but the present invention is not limited to the technical ideas described in the above embodiments and examples. Rather, those skilled in the art to which the present invention pertains can easily propose various modifications and changes based on the above-described embodiments and examples. However, it is clear from the appended claims that all such modifications and changes are within the scope of the present invention.

100: bead combination
110: initial bead
112: nano beads
114: bead intermediate
120: modified oligo-nucleotide
122: oligo-nucleotide
L, L ¹ : linker

<110> SML Genetree Co., Ltd. <120> CONJUGATED BEAD, PROCESS THEREOF AND BIOLOGICAL ASSAY SYSTEM AND ASSAY PROCESS INLCUIDG THEREOF <130> 2990-1 <150> KR 2021/0105130 <151> 2021-08-10 <160> 2 <170> KoPatentIn 3.0 <210> 1 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Primer for amplifying HPV <400> 1 ttttackkgt gayaciac 18 <210> 2 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Primer for amplifying HPV <400> 2 gaaaayaatg yawcrwaytc ytc 23

Claims (20)

A bead conjugate in which one end of an oligo-nucleotide specifically binding to a target nucleic acid molecule is condensed on the surface of a nano-bead, between the surface of the nano-bead and the oligo-nucleotide, a linking group having the structure of Formula 1 As a method of preparing an interposed bead complex,
Transforming the surface of the nano-bead into a linker having a structure of the following Chemical Formula 4 by reacting the nano-beads having an amino group represented by the following Chemical Formula 2 to the surface thereof with a carboxylic acid represented by the following Chemical Formula 3;
Reacting the nano-beads whose surface is modified with a linking group having the structure of Formula 4, and an oligo-nucleotide that specifically binds to a target nucleic acid molecule and whose 5' end is modified with an aliphatic thiol of Formula 5 A method for preparing a bead conjugate to
[Formula 1]

[Formula 2]

[Formula 3]

[Formula 4]

[Formula 5]

(In Formulas 1 to 5, R ¹ is a direct bond, or an aliphatic hydrocarbon selected from a C ₁ -C ₁₀ alkyl group, a C ₂ -C ₂₀ alkenyl group, a C ₂ -C ₂₀ alkynyl group, and a C ₁ -C ₂₀ alkyl ether group. R ² and R ³ are each independently a C ₃ -C ₁₀ alkylene group; The asterisk on the left of R ¹ indicates a site connected to the nanobead surface, and the asterisk on the right of R ³ is at the 5' end of the oligo-nucleotide Indicates the area to be connected.
The method of claim 1,
The target nucleic acid molecule is a method for preparing a bead conjugate derived from human papillomavirus.
The method of claim 1,
The nano-bead is a method of manufacturing a bead assembly made of an inorganic material.
4. The method of claim 3,
The inorganic material is a bead which is at least one of a non-metal material selected from the group consisting of iron oxide, silica, glass, or a combination thereof, and a metal material selected from the group consisting of gold, silver, copper, combinations thereof, and alloys thereof. A method for preparing a binder.
The method of claim 1,
The nano-bead is a method of manufacturing a bead assembly made of an organic material.
6. The method of claim 5,
The organic material is a polymer resin selected from the group consisting of polystyrene, polypropylene, polyethylene, poly acrylamide, combinations or copolymers thereof, and fullulane, fullulane acetate, cellulose, hydroxypropylmethylcellulose, agar A method for producing a bead complex, which is at least one of polysaccharides selected from the group consisting of rose, chitosan, combinations thereof, and copolymers thereof.
The method of claim 1,
The nano-bead is a method of manufacturing a bead assembly made of a magnetic material.
The method of claim 1,
The target nucleic acid molecule is a method for producing a bead conjugate derived from a tumor or pathogen.
9. The method of claim 8,
The pathogen is a method for producing a bead conjugate comprising a pathogenic virus, pathogenic bacteria or pathogenic protozoa. delete delete delete delete delete delete delete delete delete delete delete

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