JP2006042812A - Method for producing functional macromolecular complex - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for producing a functional macromolecular complex by which various functional molecules can be arranged arbitrarily in nano level, the distance between the functional molecules can be regulated so as to be constant, the combination of kinds of the functional molecules can be easily changed, and the functional molecules can readily be bonded to spacer molecules in an arbitrary solution substantially without damaging the functions of the functional molecules. <P>SOLUTION: The method for producing the functional macromolecular complex involves carrying out a polymerase chain reaction in the presence of a primer pair of two kinds of primers having bondable sites to the functional molecule having a bonding activity to a biopolymer, and a template nucleic acid. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a method for producing a functional polymer complex in which functional molecules of the same species or at least two functional molecules of different species are linked via a nucleic acid spacer.

  When cells are used for evaluation of substances, medical applications, etc., in order to reproduce the original properties of cells, cell functions such as adhesion, proliferation and differentiation in vitro, ie on artificial substrates Therefore, a technique for precisely controlling undifferentiation, cell death and the like is required. The use of cells that have lost their original properties has the disadvantages of, for example, misinterpretation of the action of the drug, and incompatibility of the transplanted cells with the living body. For this reason, attempts have been actively made to variously modify artificial base materials. In that case, the cell adhesion functional molecule which can recognize a cell, especially a cell surface receptor will bear an important function.

  As functional molecules, collagen, elastin, fibronectin, laminin, etc., which are extracellular matrices extracted from the living body, are used to coat artificial substrates and control the cell function by reproducing the environment equivalent to that in the living body. Research has been conducted for a long time (see Non-Patent Documents 1 and 2, for example). In addition, by using a method of coating an artificial substrate using hormones or cytokines as functional molecules, the same environment (concentration, density, etc.) as in the living body is equivalent. Studies have been made to control cell functions by reproducing (environment) (for example, see Non-Patent Document 3).

  However, in the method of controlling cell functions by the extracted extracellular matrix, the types of extracellular matrix that can be purified from the living body are substantially limited, and it is substantially possible to use a small amount of extracellular matrix that is difficult to extract. There are drawbacks such as inability to obtain results with high reproducibility due to the influence of purity and impurities. For this reason, it is difficult to design and reconfigure an environment suitable for each function of each cell on an artificial substrate.

  In order to solve the above drawbacks, to simplify and understand the action of extracellular matrix, and to avoid contamination of pathogens that can occur in the extraction matrix as a transplant material, extracellular matrix such as collagen, fibronectin, laminin, etc. An attempt to control cell function by using synthetic peptide molecules that have the amino acid sequence contained in and that can be recognized by cell surface receptors as functional molecules, and that are coated on an artificial substrate with various changes in density and type. (For example, Non-Patent Documents 4 and 5).

  As one of methods for arranging the functional molecules, a technique (spacer method) in which functional molecules are linked by a polymer spacer having a nanometer length has been reported.

  For example, Dai (W. Dai) et al. Are functional molecules composed of RGD molecules that are functional molecules composed of amino acid sequences in cell surface receptors contained in fibronectin or amino acid sequences in laminin. It has been reported that two YIGSR molecules are bonded to each other via polyethylene glycol chains having an average molecular weight of about 3500 to produce a cell aggregating agent (see, for example, Non-Patent Document 6).

Kawasaki et al. Also converted RGD molecules, which are functional molecules composed of amino acid sequences in cell surface receptors contained in fibronectin, and PHSRN molecules, which are synergistic sequences thereof, into polyethylene glycol having an average molecular weight of 2500-3400. It has been reported that a heteroligand molecule is produced by binding via a chain (see Non-Patent Documents 7 and 8). Similarly, it has been reported that RGD and EILDV, which are functional molecules composed of amino acid sequences in fibronectin, are combined, or PDSGR and YIGSR, which are functional molecules in laminin, are combined to produce a molecule. (For example, see Non-Patent Documents 9 and 10).
PP. Lanza, R.A. Langer, J. et al. Vacanti, Principles of Tissue Engineering, 2nd Ed. , Academic Press (2000) Teruhiko Koide, Toshihiko Hayashi, Extracellular Matrix-Fundamentals and Clinical Studies, Aichi Publishing (2000) Y. Ito, Biomaterials, 20, 2333 (1999) U. Hersel, C.I. Dahmen, H.M. Kessler, Biomaterials, 24, 4385 (2003) H. Shin, S .; Jo, AG. Mikos, Biomaterials, 24, 4353 (2003) W. Dai, J. et al. Belt, WM. Saltzman, Bio / Technology, 12, 797-801 (1994). S. Yamamoto, Y. et al. Kaneda, N .; Okada, S .; Nakagawa, K .; Kubo, S .; Inoue, M .; Maeda, Y .; Yamashiro, K .; Kawasaki, T .; Mayumi, Anti-Cancer Drugs, 5, 424-428 (1994) Y. Suzuki, K .; Hojo, I .; Okazaki, H .; Kamata, M .; Sasaki, M .; Maeda, M .; Nomizu, Y .; Yamamoto, S .; Nakagawa, T .; Mayumi, K. et al. Kawasaki, Chem. Pharm. Bull. 50 (9), 1229-1232 (2002) M.M. Maeda, Y .; Izuno, K.M. Kawasaki, y. Kaneda, y. Mu, y. Tsusumumi, KW. Lem, T .; Mayumi, Biochem. Biophys. Res. Commun. 241, 595-598 (1997) M.M. Maeda, K .; Kawasaki, Y. et al. Mu, H .; Kamada, Y .; Tsutsumi, TJ. Smith, T.M. Mayumi, Biochem. Biophys. Res. Commun. , 248, 485-489 (1998)

  The present invention, in one aspect, arbitrarily arranges various functional molecules at the nano level, controls the distance between the functional molecules in a solution to be constant, controls the types of functional molecules Enabling at least one of easily changing the combination, being able to easily bind the functional molecule to the spacer molecule in any solution without substantially impairing the function of the functional molecule, The object is to provide a method for producing a functional polymer composite. Note that other problems of the present invention are apparent from the description of the present specification and the like.

That is, the gist of the present invention is as follows.
[1] 1) Template nucleic acid;
2) a first containing a binding site for a functional molecule having binding activity with a biopolymer, and a nucleic acid consisting of consecutive nucleotide residues in the base sequence of the sense strand of the template nucleic acid or a derivative thereof A first functional molecule-binding primer in which the functional molecule is bound to the primer of
3) a binding site for a functional molecule having binding activity with a biopolymer, and a nucleic acid consisting of consecutive nucleotide residues in the base sequence of the antisense strand of the template nucleic acid or a derivative thereof, A first molecule in which the functional molecule is bound to a second primer having a nucleotide at the 3 ′ end as a nucleotide present at a position at least 20 nucleotides away from the nucleotide on the template nucleic acid corresponding to the 3 ′ end of the first primer. A method for producing a functional polymer complex having at least two functional molecules, which comprises performing a polymerase chain reaction in the presence of two functional molecule-binding primers. And [2] (A) 1) a template nucleic acid;
2 ′) a binding site for a functional molecule having binding activity with a biopolymer, and a nucleic acid or a derivative thereof comprising a continuous nucleotide residue in the base sequence of the sense strand of the template nucleic acid 1 primer and
3 ′) a binding site for a functional molecule having binding activity with a biopolymer, and a nucleic acid or a derivative thereof consisting of consecutive nucleotide residues in the base sequence of the antisense strand of the template nucleic acid. A polymerase chain in the presence of a second primer having a nucleotide at the 3 ′ end as a nucleotide present at a position at least 20 nucleotides away from the nucleotide on the template nucleic acid corresponding to the 3 ′ end of the first primer. Performing a reaction to synthesize a polymer complex having at least two binding sites for the functional molecule; and (B) a functional molecule in the polymer complex obtained in the step (A). A step of synthesizing a functional polymer complex having at least two functional molecules by bonding
A method for producing a functional polymer complex having at least two functional molecules,
About.

  According to the method for producing a functional polymer composite of the present invention, there is an excellent effect that various functional molecules can be arbitrarily arranged at the nano level. According to the production method, there is an excellent effect that a functional polymer complex in which the positional relationship of functional molecules is controlled to be constant in the nanometer order can be obtained. Therefore, according to the functional polymer composite obtained by the production method of the present invention, the positional relationship of the functional molecules can be defined on the nanometer order, and the functional polymer obtained by the production method of the present invention. The complex is useful for accurately estimating the distance within or between biomolecules.

  In this specification, the amino acid sequence of a peptide may be represented by the one-letter code or the three-letter code of an amino acid in accordance with a conventional biochemical nomenclature. The correspondence between such notations and amino acids is shown below. A or Ala: alanine residue; D or Asp: aspartic acid residue; E or Glu: glutamic acid residue; F or Phe: phenylalanine residue; G or Gly: glycine residue; H or His: histidine residue; Or Ile: isoleucine residue; K or Lys: lysine residue; L or Leu: leucine residue; M or Met: methionine residue; N or Asn: asparagine residue; P or Pro: proline residue; Q or Gln : Glutamine residue; R or Arg: arginine residue; S or Ser: serine residue; T or Thr: threonine residue; V or Val: valine residue; W or Trp: tryptophan residue; Y or Tyr: tyrosine Residue; C or Cys: Cysteine residue.

  In one aspect, the present invention provides a polymerase chain reaction in the presence of a primer pair consisting of two kinds of primers having a binding site for a functional molecule having binding activity with a biopolymer and a template nucleic acid. The present invention relates to a method for producing a functional polymer complex having at least two functional molecules.

  Since the production method of the present invention utilizes the polymerase chain reaction, it has advantages such as the ability to selectively produce a functional polymer complex having the desired base length and easy purification.

  The production method of the present invention has one major feature in that a primer pair consisting of two kinds of primers having a binding site for a functional molecule having binding activity with a biopolymer is used. Therefore, according to the production method of the present invention, when producing a functional polymer complex, functional molecules can be arranged via a spacer composed of a nucleic acid or a derivative thereof. The excellent effect of being able to be made is demonstrated. In addition, according to the production method of the present invention, it is possible to obtain an excellent effect that a functional polymer complex in which the positional relationship between the functional molecules is controlled to be constant in a nanometer order can be obtained.

  In the present specification, the “spacer” refers to a substance that can be bonded via two molecular chains that are arranged between two or more functional molecules and that pair adjacent functional molecules. . According to the production method of the present invention, the spacer is a nucleic acid extended from a primer pair consisting of two kinds of primers having a binding site for a functional molecule having binding activity with a biopolymer or a derivative thereof. Generated.

  The production method of the present invention performs a polymerase chain reaction using a template nucleic acid and a primer pair comprising two kinds of primers having a binding site for a functional molecule having a binding activity between biopolymers. There is one major feature. Therefore, according to the production method of the present invention, it is possible to easily produce a functional polymer complex having a long chain spacer.

  According to the production method of the present invention, a functional polymer complex having a structure in which at least two functional molecules of the same species or different species are linked via a double-stranded spacer can be obtained.

  In the method for producing a functional polymer complex of the present invention, the type and number of functional molecules, the distance between functional molecules, and the like can be selected according to the purpose.

The primer pair is not particularly limited.
1) a first containing a binding site for a functional molecule having binding activity with a biopolymer, and a nucleic acid or a derivative thereof consisting of consecutive nucleotide residues in the base sequence of the sense strand of the template nucleic acid A primer,
2) a binding site for a functional molecule having binding activity with a biopolymer, and a nucleic acid consisting of consecutive nucleotide residues in the base sequence of the antisense strand of the template nucleic acid or a derivative thereof, A second primer having a nucleotide at the 3 ′ end as a nucleotide present at a position at least 20 nucleotides away from the nucleotide on the template nucleic acid corresponding to the 3 ′ end of the first primer;
A primer pair consisting of

  The distance between the first primer and the second primer can be appropriately set according to the distance between the functional molecules in the target functional polymer complex. The distance between the first primer and the second primer is specifically at least 20 nucleotides in length from the nucleotide on the template nucleic acid corresponding to the 3 ′ end of the first primer, more specifically Has a length of 30 nucleotides or more, preferably 40 nucleotides or more, more preferably 50 nucleotides or more from the viewpoint of the efficiency of the polymerase chain reaction and the ease of purification. From the viewpoint of exerting well, it is desirable that the length is 1000 nucleotides or less, preferably 800 nucleotides or less, more preferably 500 nucleotides or less.

  In the present specification, the term “sense strand” indicates one single-stranded nucleic acid of two types of single-stranded nucleic acids in a double-stranded nucleic acid for convenience, and “antisense”. “Strand” is intended to indicate the other single-stranded nucleic acid.

  The length of the primer is not particularly limited, and is, for example, at least 10 nucleotides in length. More specifically, from the viewpoint of accuracy of the annealing position, it is at least 15 nucleotides in length, preferably 18 nucleotides in length. As described above, more preferably, it is 20 nucleotides or more, and from the viewpoint of preventing the yield reduction of primer synthesis, it is 100 nucleotides or less, preferably 60 nucleotides or less, more preferably 30 nucleotides or less. .

  The Tm value (melting temperature) of the primer is not particularly limited, and is preferably in the range of 50 ° C. to 70 ° C. from the viewpoint of suppressing non-specific annealing, and the difference between the Tm values of the two primers is 10 It is desirable that the temperature be 5 ° C. or lower.

  The primer pair may be a primer pair capable of synthesizing a target spacer portion using such a primer pair. In one aspect, the primer pair is a primer composed of a partial base sequence of a template nucleic acid or a primer containing a partial base sequence of the template nucleic acid. In another aspect, the primer may comprise a base sequence that is completely complementary to a part of the base sequence of the template nucleic acid, and a part of the base sequence of the template nucleic acid and one or several nucleotides Primers with different residues may be used.

  In the production method of the present invention, it is preferable to use deoxyribonucleic acid as a raw material of the nucleic acid derivative from the viewpoint of easy synthesis of a long chain exceeding 30 nucleotides. In particular, since a double-stranded DNA strand has a structurally clear structure of 10 base pairs and a length of 3.4 nm, it is preferable to use a double-stranded DNA strand (hereinafter referred to as dsDNA) as a nucleic acid derivative. It may be abbreviated).

  In the present specification, the “functional molecule” refers to a synthetic or natural molecule having binding activity with a biopolymer. Examples of the biopolymer include cells, extracellular matrix, receptors for intercellular adhesion, and proteins, hormones, cytokines (growth factors), antibodies, carbohydrates and the like on the surface of various other biological tissues.

  The binding activity of the functional molecule can be evaluated by, for example, surface plasmon analysis, gel shift assay, phage display method and the like. For example, in the evaluation of binding activity by surface plasmon analysis, a solution containing a functional molecule to be evaluated is fed at a constant flow rate to a chip on which a biopolymer is immobilized, and interaction is performed by an appropriate detection means. This can be done by detecting. Examples of the detection means include optical detection means based on fluorescence intensity, fluorescence deflection, etc .; matrix-assisted laser desorption ionization-time-of-flight mass spectrometer (MALDI-TOF MS), electrospray ionization mass spectrometer (ESI) -MS) and other mass spectrometers; and combinations thereof. Here, when a sensorgram indicating the formation of a complex composed of a functional molecule and a biopolymer is presented, it is an indicator that the functional molecule has binding activity.

  In the production method of the present invention, the “at least two functional molecules” may be functional molecules of the same species or different species.

  The functional molecule used in the production method of the present invention may be chemically derivatized.

  In the present specification, the “receptor” refers to a biopolymer that exists in a cell, specifically recognizes various physiologically active substances, and transmits and expresses the action of the physiologically active substance. Examples of the receptor include receptors present in cells (hereinafter also referred to as intracellular receptors), receptors existing on the plasma membrane (hereinafter also referred to as cell surface receptors), and the like.

  Examples of the extracellular matrix include collagen (eg, procollagen), elastin, fibroin, fibronectin, vitronectin, laminin, entactin, tenascin, agrin, ankaline, thrombospondin, entactin, osteopontin, osteocalcin, fibrinogen, proteoglycan and the like. Can be mentioned. Examples of the proteoglycan include aggrecan, agrin, perlecan, durin, fibrojuline, brevican and the like.

  In the production method of the present invention, a partial structure derived from the extracellular matrix, that is, a cell matrix fragment in vivo or a synthetic peptide corresponding to the fragment may be used as a functional molecule.

  The “in vivo cell matrix fragment” refers to a purified fragment capable of binding to a specific cell surface receptor, obtained after purification by cleaving a part of the extracellular matrix chemically or enzymatically. The cell matrix fragment in the living body is not particularly limited. For example, a purified fragment of type I collagen obtained by CNBr treatment [for example, WD. Staatz, JJ. Walsh, T .; Pexton, SA. Santoro, J .; Biol. Chem. , 265 (9), 4778-4781 (1990), etc.]; a purified fragment of type IV collagen treated with a protease such as CNBr or, if necessary, trypsin, thermolysin, collagenase [eg, JA. Eble, R.M. Golbik, K .; Mann, K .; Kuhn, EMBO J. et al. , 12 (12), 4795-4802 (1993), a fragment having a triple helical structure; EC. Tsilibary, AS. Charonis, J.M. Cell. Biol. , 103 (6), 2467-2473 (1986)], cell adhesion fragments obtained by treating fibronectin with a protease such as trypsin or pepsin [for example, SK. Akiyama, E .; Hasegawa, T .; Hasegawa, KM. Yamada, J .; Biol. Chem. 260 (24), 13256-13260 (1985)].

  The “synthetic peptide corresponding to a fragment of a cell matrix in a living body” means a unit of a minimum amino acid sequence that contains an amino acid sequence contained in an extracellular matrix and that can bind to a specific cell surface receptor. It refers to a chemically synthesized oligopeptide.

  In all peptides that can be used as a functional molecule in the present invention, an additional amino acid residue may be bonded to the N-terminal side and / or the C-terminal side depending on the purpose.

  In addition, the synthetic peptide that can be used as a functional molecule in the present invention includes a non-natural amino acid, a chemical modification group, an L-form amino acid, a D-form amino acid, or both, depending on the purpose. May be.

  The synthetic peptide is not particularly limited. For example, it is commonly contained in fibronectin, vitronectin, laminin, collagen, thrombospondin, etc., and 3 amino acids bind as a unit of the minimum amino acid sequence to which the receptor binds. Arg-Gly-Asp (RGD) sequence and the like.

  The linear synthetic peptide containing the RGD sequence, and the amino acid sequence of the peptide that can be used as a functional molecule in the present invention is not specifically limited. For example, RGD, RGDS, RGDV, RGDT, RGDF, GRGD, GRGDG, GRGDS, GRGDF, GRGDY, GRGDVY, GRGDYPC, GRGDSP, GRGDSG, GRGNP, GRGDSY, GRGDSPK, YRGDS, YRGDG, YRGDG SEQ ID NO: 2), GCGYGRGDSPG (SEQ ID NO: 3), GGGPHSRNGGGGGRGDGG (SEQ ID NO: 4), and the like.

  Moreover, the functional molecule which controlled the binding activity to a receptor by circularizing the peptide containing a RGD sequence can also be utilized for this invention.

  The cyclic peptide is not particularly limited. For example, GPen * GRGDSPC * A, GAC * RGDC * LGA, AC * RGDGWC * G, cyclo (RGDf (NMe) V), cyclo (RGDfV), cyclo (RGDfK), And cyclo (RGDEv), cyclo (GRGDfL), cyclo (ARGDfV), cyclo (GRGDfV) and the like. In addition, the small letter contained in the amino acid single letter notation which shows a peptide sequence shows D-amino acid. The “Pen” represents penicillamine, and “*” represents a cysteine residue having a disulfide bond in the molecule. The same applies to the following.

  In the production method of the present invention, in addition to the RGD sequence, a minimum amino acid sequence unit derived from various extracellular matrices can be used.

  Although it does not specifically limit as a minimum amino acid sequence derived from fibronectin which is one of the extracellular matrices, For example, sequences, such as NGR, LDV, REDV, EILDV, KQAGDV, are mentioned.

  In addition, the minimum amino acid sequence derived from laminin that is one of the extracellular matrices is not particularly limited, and examples thereof include sequences such as LRGDN, IKVAV, YIGSR, CDPGYIGSR, PDSGR, YFQRYLI, and RNIAEIIKDA (SEQ ID NO: 5). It is done. The minimum amino acid sequence derived from laminin includes, for example, the sequence of peptide having cell adhesion activity described in Noriyoshi Nomizu, Protein Nucleic Acid Enzyme, Vol. 45, No. 15, 2475-2482 (2000) and the like. . Examples of the sequence of the peptide having cell adhesion activity include RQVFQVAYIIIKA (SEQ ID NO: 6) derived from the α1 chain of laminin, RKRLQVQLSIRT (SEQ ID NO: 7) derived from the α1 chain G domain, and the like.

  Similarly, the minimum amino acid sequence derived from type I collagen, which is one of the extracellular matrices, is not particularly limited, and examples thereof include sequences such as DGEA, KDGEA and GPAGKDGEGAQG (SEQ ID NO: 8), GER, and GFOGER. .

  Furthermore, the minimum amino acid sequence derived from elastin that is one of the extracellular matrices is not particularly limited, and examples thereof include sequences such as VAPG, VGVAPG, and VAVAPG.

  Furthermore, examples of the minimum amino acid sequence derived from thrombospondin, which is one of the extracellular matrices, include sequences such as VTXG.

  The minimum amino acid sequence unit can be contained in a synthetic peptide used as a functional molecule.

  In the production method of the present invention, a substance that does not originate from the extracellular matrix but simply binds to a receptor, for example, a substance that is artificially designed and synthesized as a low molecular weight substance, is not functional. It may be used as a molecule.

  The substance that binds to the receptor may contain a part of the structural motif derived from the extracellular matrix, or may not contain the structural motif. Examples of the substance include a ligand molecule for α5β1 integrin, a peptide of C * RRETAWAC *, a ligand molecule for α6β1 integrin, a peptide of VSFSFRHRYSPFAVS (SEQ ID NO: 9), a ligand molecule for αMβ2 integrin, GYRDGAYAGIPILYN (sequence No. 10) and the like.

  Furthermore, in the production method of the present invention, GA. Sulyuok, C.I. Gibson, SL. Goodman, G.M. Holzemann, M .; Wiesner, H.C. Kessler, J. et al. Med. Chem. , 44 (12), 1938-1950 (2001) and the like, and low-molecular substances that bind to receptors can also be used as functional molecules.

  Among receptors, for example, receptors such as syndecan, NG2, and CD44 contain proteoglycans. Therefore, peptides containing amino acid sequences in the receptors and binding to proteoglycans are used as functional molecules. It can also be used.

  Although it does not specifically limit as an amino acid sequence of the peptide couple | bonded with a proteoglycan, For example, consensus sequences, such as BBXB, XBBXBX, and XBBBXXX, are mentioned. “X” represents a hydrophobic amino acid, and “B” represents a basic amino acid. Specific examples of the consensus sequence include, for example, KRSR, FHRRIKA (derived from bone sialoprotein), PRRARV (derived from fibronectin), WQPPPARI, and the like, and YEKPGSPPREVVPRPPGV (SEQ ID NO: 11) (fibronectin) RPSLAKKQRFRHRNRKGYRSQR (SEQ ID NO: 12) (derived from vitronectin), RIQNLLKITNLRIKFVK (SEQ ID NO: 13) (derived from laminin), and the like.

  In the production method of the present invention, for example, U.S. Pat. Hersel, C.I. Dahmen, H.M. Kessler, Biomaterials, 24, 4385-4415 (2003), JA. Hubbell, Bio / Technology, 13, 565-576 (1995), H.C. Shin, S .; Jo, AG. Mikos, Biomaterials, 24, 4353-4364 (2003) and E.I. Koivunen, W.H. Arap, D.A. Rajotte, J. et al. Lahdenranta, R.A. Pasqualini, J. et al. Nuclear Med. , 40 (5), 883-888 (1999) and the like and linear or cyclic synthetic peptides as described in those cited documents can be used as functional molecules.

  The “hormone” refers to a substance that is produced and secreted by endocrine cells of animal tissues, transported to the target cell by the bloodstream, and regulates the activity of the target cell as an exogenous signal. Examples of the hormone include, but are not limited to, insulin and glucagon produced in the pancreas, adrenaline and noradrenaline produced in the adrenal medulla, androgen, estrogen and gestagen produced in the gonad. As in the case of the extracellular matrix, a partial structure or an artificially synthesized substance that has an amino acid sequence contained in a hormone molecule and can be expected to be active may be used.

  The “cytokine” refers to a proteinaceous factor that is released from cells and mediates cell-cell interactions such as immunity, control action of inflammatory reaction, antiviral action, antitumor action, and regulation of cell proliferation / differentiation. The cytokine is not particularly limited. For example, interleukins, interferons, tumor necrosis factor (TNF), lymphotoxin, colony stimulating factor (CSF), bone morphogenetic factor (BMP), epidermal growth factor ( EGF), nerve growth factor (NGF), insulin-like growth factor (IGF), basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF), transforming growth factor (TGF), hepatocyte growth factor ( HGF), vascular endothelial growth factor (VEGF) and the like.

  As for cytokines, as in the case of the extracellular matrix, a part of the structure that is contained in these molecules and can be expected to be active or an artificially synthesized substance may be used. Examples of such substances include NSVNSKIPKACCVPTELSAI (SEQ ID NO: 14) peptide that exhibits the same properties as BMP-2 for bone morphogenetic factor (BMP) [see, for example, Y. Suzuki et al. Biomed. Mater. Res. , 50, 405-409 (2000))]. As for the substance, YXCXXGPXTTWXCXP [X represents a position that can be substituted with several amino acids] for erythropoietin (NC. Wrington, FX. Farrell, R. Chang, AK. Kashapap, FP. Barbone, LS. Mulcahy) , DL. Johnson, RW. Barrett, LK. Jolliffe, WJ. Dower, Science, 273, 458-463 (1996)).

  CD. Reyes, AJ. Garcia, J .; Biomed. Mater. Res. , 65A, 511-523 (2003), a structure in which several peptides including the exemplified amino acid sequences are associated may be used as the functional molecule.

  In the production method of the present invention, an antibody can also be used as a functional molecule. The antibody can be freely selected according to the purpose, and is not particularly limited. Examples thereof include antibodies to various cells, extracellular matrix, other various biological tissue surfaces, lipids and other biopolymers. Specific examples of the antibody include monoclonal antibodies, various anti-integrin antibodies, antibodies against cell surface proteoglycans such as syndecan, NG2, and CD44 / CSPG, anti-cadherin antibodies, and anti-selectin antibodies. When the antibody is used as a functional molecule, the antibody itself may be used, or a fragment obtained by protease treatment such as pepsin or papain may be used.

  In the production method of the present invention, a carbohydrate can also be used as a functional molecule. In the production method of the present invention, any of a monosaccharide, an oligosaccharide which is a condensate composed of several (2 to 10) monosaccharides, and a polysaccharide composed of a large number of monosaccharides can be used as the functional molecule. Good. In addition, oligosaccharides and polysaccharides include very many types due to differences in the types of constituent sugars, differences in the bonding mode between constituent sugars, differences in the degree of polymerization, and the like. Any carbohydrate that has the ability to bind to a polymer or the like can be used as a functional molecule.

  Examples of the carbohydrate include β-galactose, lactose, or a polymerizable lactose monomer that can be polymerized to form an adhesive substrate, such as a monosaccharide that binds to an asialoglycoprotein receptor on hepatocytes; Sialyl Lewis X which is an oligosaccharide containing sialic acid and fucose (see, for example, A. Varki, Proc. Natl. Acad. Sci. USA, 91, 7390-7397 (1994)) and the like; hyaluronic acid, chondroitin sulfate And glycosaminoglycans such as dermatan sulfate, heparan sulfate, heparin, and keratan sulfate.

  In the production method of the present invention, the “molecule that binds to an intercellular adhesion receptor” that can be used as a functional molecule refers to a molecule that binds to an intercellular adhesion receptor present in each extracellular component. . The molecule that binds to the receptor for intercellular adhesion is not particularly limited, and examples thereof include NCAM and cadherin. Furthermore, as a molecule | numerator couple | bonded with the receptor of the said cell adhesion, DITWDQLWDLMK (sequence number: 15) etc. are mentioned as a synthetic peptide among the receptors with respect to selectin, for example. In the production method of the present invention, the use of a molecule that binds to the receptor for cell-cell adhesion as a functional molecule exhibits an excellent effect that a cell-cell interaction can be mimicked.

  In the production method of the present invention, the functional molecule is optimal for each cell from an extracellular matrix, a hormone, a cytokine (growth factor), an antibody, a carbohydrate, a molecule that binds to a receptor for intercellular adhesion, and the like. Two or more things can be selected. From the viewpoint of practically producing a functional polymer complex in which the arrangement of functional molecules is controlled in the order of nanometers, fragments of functional molecules derived from nature, synthetic peptide molecules, saccharides, and artificially synthesized substances ( It is preferable to use a low molecular weight substance) or a complex thereof. In addition, when a functional molecule consists of a peptide, it is preferable that the chain length of this peptide is about 3-150 amino acids. Moreover, when a functional molecule consists of sugar chains, the chain length of the sugar chains is preferably about 1 to 100 residues of monosaccharides.

  In the method for producing a functional polymer complex of the present invention, a functional molecule can be bound to a primer or a spacer by a covalent bond or a non-covalent bond via a binding site on the primer or the spacer.

  The functional molecule and the primer or spacer are preferably bonded to each other by introducing an appropriate functional group into each of the functional molecule and the primer or spacer.

  Hereinafter, the functional group to be introduced into the functional molecule is referred to as “primer or spacer binding functional group”, and the functional group to be introduced into the primer or spacer is referred to as “functional molecule binding functional group”. The site where the functional group for binding a functional molecule introduced on the primer or spacer is also referred to as “binding site”.

  The primer or spacer binding functional group and the functional molecule binding functional group are not particularly limited, and the primer or spacer binding functional group and the functional molecule binding functional group bind to each other correspondingly. Thus, it can be appropriately selected.

  The reaction for binding the functional molecule and the primer or spacer can be appropriately selected.

  In addition, the binding mode of the functional group for functional molecule binding contained in the primer or spacer and the functional group for primer or spacer binding contained in the functional molecule is such that the primer, spacer, functional molecule, etc. are decomposed during the reaction. If the introduction reaction does not occur non-specifically between unintended sites, both covalent bonds and non-covalent bonds can be used.

  As the introduction reaction by covalent bond, amide bond forming reaction (condensation of amino group and carboxyl group using a condensing agent such as carbodiimide, or carboxyl group active ester previously activated by amino group and N-hydroxysuccinimide or the like) And a Michael addition reaction (a method of reacting an α, β-unsaturated carbonyl group such as a maleimide group, an acrylic ester group, an acrylamide group, and a vinyl sulfone group with a thiol group), a thioether Formation reaction (reaction of halogenated alkyl group such as haloacetyl group, epoxy group, aziridine group, etc. with thiol), Diels-Alder cycloaddition reaction (reaction of conjugated dienes such as cyclopentadiene and olefins such as quinone) Schiff base type) Examples of the reaction include a method of reacting an aldehyde group such as glyoxylic acid and an amino group such as oxyamino group, and the Schiff base group formed may be reduced with an appropriate reducing agent), a thiazolidine ring formation reaction ( And a cysteine group in which an amino group and a thiol group remain, and an aldehyde group). Furthermore, examples of the amide bond forming reaction include a method in which an amide bond is formed by reacting a thioester group, an amino group, and cysteine in which a thiol group remains.

  Examples of non-covalent bond-introducing reactions include complex formation reactions (eg, His-Tag formation reaction utilizing ternary complex formation of trinitriloacetic acid-nickel complex and oligohistidine, gold-thiol bond reaction, etc.), biological Recognition reactions (for example, sugar chain-lectin reaction, avidin-biotin reaction, antigen-antibody reaction, etc.) and the like.

  The introduction reaction can be performed by setting a combination of a functional group for functional molecule binding and a functional group for primer or spacer binding to each other in the above reaction mode. Such a combination can be appropriately selected by referring to the above. The introduction reaction described above may be initiated by other factors such as light, heat, vibration, time, and deprotection.

  As a functional group for primer or spacer binding, a reactive amino acid residue or the like originally contained in a functional molecule, a reactive site contained in a sugar, or the like may be used. A group may be intentionally introduced and used. As a method for introducing a functional group for primer or spacer binding into a functional molecule, a bifunctional reagent containing a functional group for primer or spacer and containing a functional group for binding to a functional molecule in the same molecule is used. And a method of chemically modifying functional molecules.

  One method of chemically modifying a functional molecule containing a peptide is to synthesize a functional molecule and then bind a bifunctional reagent using the reactivity of amino acid residues, etc. For example, Toru Komano and Ken Shimura Assist, Kenzo Nakamura, Michinori Nakamura, Nobuyuki Yamazaki, Motonori Ohno, Yuichi Kanaoka, Fumio Sakiyama, Hiroshi Maeda, "Chemical Modification of Proteins <above>" Biochemical Experimental Method 12, Society of Science Publishing Center (1981), etc. Known methods are used. Further, at the stage of chemically synthesizing a functional molecule, for example, MW. Pennington, BM. Dunn Ed. "Peptide Synthesis Protocols", Methods in Molecular Biology, Vol. 35, Humana Press (1994), etc., and can be freely introduced by using a known method. The same can be said for functional molecules including saccharides. For example, known methods described in “Glycotechnology” edited by the Glycoengineering Association, Industrial Research Council, Biotechnology Information Center (1992), etc. are used. be able to.

  As a method for introducing functional groups for functional molecule binding into nucleic acids or their derivatives in a position-specific manner, for example, “Polymer Chemistry and Functional Design of Nucleic Acids” Polymer Research Method 6, Academic Publishing Center (1996) Ito, E .; Fukusaki, J. et al. Mol. Catal. B: Enzymatic, 28, 155-166 (2004) and the methods described in the references described in the document can be used.

  Examples of the nucleic acid or derivative thereof into which a functional group for functional molecule binding is introduced include, for example, a chemical in which a functional group for functional molecule binding is introduced into a main chain or a nucleobase containing a phosphate site, ribose or deoxyribose, etc. And chemically modified products in which a functional group for functional molecule binding is introduced at the 5 ′ end or 3 ′ end. If the position in the primer or spacer of the functional group for functional molecule binding can be specified as described above, the functional group binding functional group is not limited to the above examples.

  A linker may be included between the primer or spacer and the functional group for functional molecule binding and / or between the functional molecule and the functional group for primer or spacer binding. According to the linker, the distance between the primer or spacer and the functional molecule can be kept slightly separated, and introduced with reduced steric hindrance in the reaction between the primer or spacer and the functional molecule. The function of rapidly advancing the reaction or the function of not inhibiting the binding reaction between the cell surface receptor and the functional molecule when the functional polymer complex having the nanostructure of the present invention is brought into contact with the cell. It exhibits the excellent effect of being able to. The linker is not particularly limited as long as it has a length of about 0.1 to 5 nm. For example, an alkyl chain, a peptide chain, an oxyethylene chain, an oxypropylene chain, and the like are mixed. The structure may be different. The linker is not limited to the above structure, and an optimum linker is selected depending on the purpose. The linker may be introduced into the primer or spacer, the functional molecule, or both as part of the functional group for functional molecule binding, the primer or the spacer binding functional group, for example.

  The “template nucleic acid” is a nucleic acid having a base sequence complementary to the primer, and preferably a double-stranded nucleic acid composed of complementary strands. For example, RNA, DNA Etc.

  The template nucleic acid is not particularly limited, and may be, for example, a nucleic acid extracted or purified from a biological material such as human tissue, mammalian cell, eukaryotic microorganism, or bacteria, and is artificially synthesized. It may be a nucleic acid.

  Since the polymerase chain reaction in the production method of the present invention includes a nucleic acid elongation reaction, it is preferably performed in the presence of a nucleic acid synthase. The “nucleic acid synthase” refers to an enzyme having an action of extending the chain length of nucleic acid using deoxyribonucleotide triphosphate as a substrate. The nucleic acid synthase is not particularly limited, and examples thereof include Taq DNA polymerase (derived from Thermus aquaticus), Tfl DNA polymerase (derived from Thermus flavus), Tth DNA polymerase (derived from Thermus thermophilus HB-8 strain), Tli. Examples thereof include heat-resistant DNA polymerases such as DNA polymerase (derived from Thermococcus litoralis) and Pfu DNA polymerase (derived from Pyrococcus furiossis).

  In the production method of the present invention, the stability of the functional polymer complex itself can be increased, for example, under conditions of contact with biological components, as long as the nucleic acid synthesizing enzyme can sufficiently perform a nucleic acid elongation reaction. A chemically modified nucleotide from the viewpoint of allowing the functional polymer complex to be quantitatively analyzed by enzyme immunoassay or a fluorescent label, labeled with fine particles and observed with an electron microscope, etc. A nucleic acid elongation reaction with a nucleic acid synthase may be performed in the presence of an analog.

Examples of the chemically modified nucleotide analog include deoxyribonucleotide triphosphates containing various radioisotopes (for example, [α- ³² P], [α- ³⁵ S], [ ³ H] deoxycytidine triphosphate. Acid) bromodeoxyuridine triphosphate, digoxigenin labeled deoxyuridine triphosphate, fluorescein labeled deoxyuridine triphosphate, biotin labeled deoxyuridine triphosphate, biotin labeled deoxycytidine triphosphate and the like.

  In the production method of the present invention, a product obtained by a nucleic acid elongation reaction using a nucleic acid synthase may be chemically modified. Such chemical modifications include, for example, chromophores such as fluorescein derivatives, rhodamine derivatives and Alexa, and, for example, Yasuyuki Kurihara, Tsuneari Takeuchi, Yoichi Matsuda, “The latest protocol of non-RI experiments—the principle and practice of fluorescence: gene analysis Chromophores described in separate experimental medicine, Yodosha (1999), etc .; chemical modifications in which antigenic sites such as biotin, bromodeoxyuridine and digoxigenin are introduced

The manufacturing method of the present invention, in one embodiment,
1) a template nucleic acid;
2) a first containing a binding site for a functional molecule having binding activity with a biopolymer, and a nucleic acid consisting of consecutive nucleotide residues in the base sequence of the sense strand of the template nucleic acid or a derivative thereof A first functional molecule-binding primer in which the functional molecule is bound to the primer of
3) a binding site for a functional molecule having binding activity with a biopolymer, and a nucleic acid consisting of consecutive nucleotide residues in the base sequence of the antisense strand of the template nucleic acid or a derivative thereof, A first molecule in which the functional molecule is bound to a second primer having a nucleotide at the 3 ′ end as a nucleotide present at a position at least 20 nucleotides away from the nucleotide on the template nucleic acid corresponding to the 3 ′ end of the first primer. In this method, the polymerase chain reaction is carried out in the presence of two functional molecule binding primers (referred to as “Production Method 1”).

  According to the production method 1, since the first functional molecule-binding primer and the second functional molecule-binding primer are used as a primer pair, the base sequence of the primer pair and the template nucleic acid According to the base sequence, an excellent effect is obtained that a functional polymer complex in which at least two functional molecules are arranged at a certain distance can be obtained.

  In addition, in the said manufacturing method 1, you may perform the process which couple | bonds a functional molecule with a 1st primer and a 2nd primer prior to the said process (a). In this step, as described above, the functional molecule and the primer precursor can be bound by forming a covalent bond or a non-covalent bond. Moreover, you may include the process of processing a functional molecule and / or a nucleic acid derivative (for example, chemical modification, deprotection, etc.) as needed.

  The functional polymer complex obtained by such a production method has a molecular structure similar to that of a nucleic acid, and binds to a biopolymer at any one end of the main chain or at any midpoint of the main chain. It is a polymer substance into which a functional molecule having activity is introduced. The “nucleic acid” refers to deoxyribonucleic acid, ribonucleic acid and the like in which the main chain is formed through a phosphodiester bond.

  In the step (a), in the polymerase chain reaction, a template nucleic acid and the primer pair are paired under appropriate conditions, and a catalytic reaction is performed in the presence of a nucleic acid synthase using deoxyribonucleotide triphosphate as a substrate. .

  The polymerase chain reaction is performed, for example, in an appropriate buffer (for example, 50 mM potassium chloride, 10 mM Tris buffer (pH 8.4), 1.5 mM magnesium chloride, 0.01 wt% gelatin) in the presence of an appropriate DNA polymerase. , Template nucleic acid (genomic DNA of organisms such as bacteria) 0.05 μg, 4 types of deoxyribonucleotide triphosphates (dATP, dGTP, dTTP and dCTP) 0.2 mM each, 2 types of primers 0.5 μM each 50 μL of the reaction solution prepared as described above was performed under conditions such as 25 cycles of heat denaturation at 94 ° C. for 1 minute, annealing at 58 ° C. for 1 minute, and extension reaction at 72 ° C. for 1 minute. It can be done. The volume of the reaction solution, the concentration of magnesium chloride, the amount of template nucleic acid, the amount of primer, etc., for example, the expression level of the product, the purity of the product, etc. can be determined by conventional agarose gel electrophoresis, polyacrylamide gel electrophoresis, etc. It can be set as appropriate by confirming the amount, purity and the like.

  In the production method 1, the obtained reactive organism is purified by a conventional nucleic acid purification means such as gel filtration, agarose gel electrophoresis, purification using polyacrylamide gel electrophoresis, or magnetic bead method. Thus, the intended functional polymer complex can be obtained.

In the production method 1, the polymerase chain reaction is, in one aspect,
(A1) Two types of functional molecule-binding primers are each complementarily extended with respect to the chain nucleic acid derived from the template nucleic acid or the other primer to generate an extended functional site-containing polymer complex. The process of
(A2) a step of denaturing the elongated functional site-containing polymer complex produced in step (a1) to produce a single-stranded construct, and (a3) at least two kinds of one produced in step (a2) Pairing the chain constructs,
Can be subdivided into With respect to the product in the step (a1) or (a2), a necessary amount is taken out, and only the necessary amount of the product is subjected to a desired subsequent step, thereby easily changing the combination of functional molecules. Is also possible.

  The above-mentioned “denaturation” means that the nucleic acid has a molecular structure (tertiary structure, higher order of nucleic acid) inherently possessed by the nucleic acid, for example, due to an increase in temperature of the solution, an increase or decrease in pH, or the presence of an appropriate drug. This means that the structure cannot be maintained. Specifically, the denaturation refers to, for example, breaking the double helix structure of DNA and dissociating it into two single-stranded polynucleotides.

  In the step (a2), the denaturation is preferably performed by a method of increasing the temperature of the nucleic acid solution, from the viewpoint of preventing the decomposition of the generated extended functional site-containing polymer.

  In the step (a3), the pairing can be performed by conventional conditions and techniques such as annealing and hybridization.

  According to the production method of the present invention, when a primer having a specific base sequence is used, a functional polymer complex having a desired chain length can be obtained depending on the position where the template nucleic acid is paired. Moreover, according to the production method of the present invention, when a plurality of types of base sequence primers are used, a mixture of a plurality of functional polymer complexes having different chain lengths can be obtained.

  In the present specification, the production method 1 is also referred to as a pre-denaturing method. Since this pre-denaturing method is a production method using a primer to which a functional molecule is bound in advance, any combination of functional molecules to be introduced into the functional polymer complex can be freely selected by arbitrarily selecting a primer combination. Excellent effect that can be selected.

In another embodiment, the production method of the present invention is,
(A) 1) a template nucleic acid;
2 ′) a binding site for a functional molecule having binding activity with a biopolymer, and a nucleic acid or a derivative thereof comprising a continuous nucleotide residue in the base sequence of the sense strand of the template nucleic acid 1 primer and
3 ′) a binding site for a functional molecule having binding activity with a biopolymer, and a nucleic acid or a derivative thereof consisting of consecutive nucleotide residues in the base sequence of the antisense strand of the template nucleic acid. A polymerase chain in the presence of a second primer having a nucleotide at the 3 ′ end as a nucleotide present at a position at least 20 nucleotides away from the nucleotide on the template nucleic acid corresponding to the 3 ′ end of the first primer. Performing a reaction to synthesize a polymer complex having at least two binding sites for the functional molecule; and (B) a functional molecule in the polymer complex obtained in the step (A). A step of synthesizing a functional polymer complex having at least two functional molecules by bonding
(Also referred to as “manufacturing method 2”).

  In the production method 2, first, a polymer composite having a polymerase chain reaction in the presence of a template nucleic acid, a first primer, and a second primer and having at least two binding sites for the functional molecule. The body is synthesized [referred to as “step (A)”].

  According to the production method 2, the polymer complex is synthesized prior to the binding of the functional molecule. Therefore, if the polymer complex is synthesized in advance, only the necessary amount in the polymer complex can be obtained in any kind. Alternatively, it exhibits an excellent effect that it can be used for introducing functional molecules in an amount.

  In addition, the polymerase chain reaction in the said process (A) can be performed similarly to the case of the said manufacturing method 1.

  Next, in the production method 2, a functional molecule is bonded to the polymer complex obtained in the step (A) to obtain a target functional polymer complex (referred to as “step (B)”). .

  In the step (B), the binding between the polymer complex and the functional molecule can be performed by the same method as described above.

  In the production method 2, the product of step (B) is purified by a conventional nucleic acid purification means such as a gel filtration method, a purification method using agarose gel electrophoresis or polyacrylamide gel electrophoresis, or a magnetic bead method. As a result, the intended functional polymer composite can be obtained.

In the production method 2, the polymerase chain reaction in the step (A) is, in one aspect,
(A1) a step in which two types of primers each extend complementarily to a chain extension product derived from the template nucleic acid or the other primer to form an extended polymer complex,
(A2) a step of denaturing the extended polymer complex produced in step (A1) to produce a single-stranded construct, and (A3) at least two types of single-stranded constructs produced in step (A2) The process of combining,
Can be subdivided into

  In the step (A2), the extension polymer complex can be modified by the same method as in the production method 1.

  In the step (A3), at least two kinds of single-stranded constructs can be paired by the same method as in step (a3) in the case of the production method 1.

  In the present specification, the production method 2 is also referred to as a post-modification method. Since this post-denaturation method is a production method in which a functional molecule is introduced into a double-stranded nucleic acid spacer prepared in advance, if a polymer complex having at least two binding sites is synthesized in advance, Only the required amount among them can be used for introduction of any kind or amount of functional molecules.

  The production method of the present invention may be either the pre-modification method or the post-modification method. From the viewpoint of easy introduction of the functional molecule into the nucleic acid spacer and production efficiency of the functional polymer complex, the pre-denaturation method is preferred.

  Since the functional polymer complex obtained by the production method of the present invention has a nucleic acid spacer as a spacer, the interval between the functional molecules is regulated to an arbitrary distance, and the molecular weight of the spacer portion is substantially single. It exhibits the excellent property of having a certain nanostructure. The functional polymer composite obtained by the production method of the present invention has a substantially distributed distance between the functional molecules compared to, for example, a conventionally used substance having a spacer such as polyethylene glycol. Excellent effect that can be made uniform.

  In the functional polymer complex obtained by the production method of the present invention, the preferred position for introducing the functional molecule in the nucleic acid spacer consisting of dsDNA can be selected according to the purpose and is not particularly limited. 5 'end (there are 2 places in total if there are no halfway branches, etc.) and 3' end (2 places in total, if there are no halfway branches, etc.) and dsDNA spacer end sites and nucleic acids An arbitrary position in the middle of the dsDNA spacer such as a base site can be mentioned. When describing the case of containing two functional molecules, molecules having functional molecules bound to two 5 ′ ends of the dsDNA strand, or molecules having functional molecules bound to two 3 ′ ends, respectively, A combined molecule (for example, a molecule in which a functional molecule is bonded to each of the 5 ′ end and the 3 ′ end) can be mentioned. When three or more functional molecules are introduced, a vacant end can be used.

  In order to introduce a functional molecule by a pre-denaturing method, a functional group for functional molecule binding is introduced into a single-stranded DNA (hereinafter also referred to as “ssDNA”) as a raw material of a nucleic acid spacer at the stage of chemical synthesis of DNA. As described above, various functional group-binding functional groups can be selected and are not particularly limited. From the viewpoint of exhibiting reaction selectivity, it is preferable to introduce a thiol group or an amino group.

  In the production method of the present invention, the method for introducing a functional group for binding a functional molecule in ssDNA, the type of site and functional group, the method for introducing a functional group for binding a nucleic acid spacer in a functional molecule, the site and type are appropriately determined depending on the purpose. Can be selected. In the production method of the present invention, the nucleic acid spacer structure is not limited to a simple double helix, but various complex structures such as a branched structure, a three-dimensional structure, a two-dimensional structure, and a three-dimensional structure. The functional polymer composite of the present invention having a linked structure or the like can be produced.

  The functional polymer composite obtained by the production method of the present invention may be used alone, or two or more functional polymer composites may be mixed and used. The optimal use form is selected for each cell.

  The functional polymer complex obtained by the production method of the present invention may be sterilized as necessary. The sterilization method is not particularly limited, and can be appropriately selected depending on the application. Examples of the sterilization method include autoclave sterilization, ethylene oxide gas sterilization, γ-ray sterilization, electron beam sterilization, and filter sterilization.

EXAMPLES Hereinafter, although an Example etc. demonstrate this invention concretely, this invention is not limited at all by them. The measurement methods or evaluation methods used in the following examples and the like are collectively shown.

(Analysis of peptides by reversed-phase high-performance liquid chromatography (HPLC))
Analysis of peptides by reverse phase HPLC was performed under the following conditions. A trade name: TSKgel ODS-80TM (manufactured by Tosoh Corporation) was used as the column. The eluent flow rate during elution was set to 1.0 mL / min, and the detection wavelength was set to 210 nm. The peptides were eluted with a 20 minute linear gradient with 5-30% by weight acetonitrile. The linear gradient includes a 5 wt% aqueous acetonitrile solution containing 0.05 wt% trifluoroacetic acid (hereinafter also referred to as “TFA”) and a 30 wt% aqueous acetonitrile solution containing 0.05 wt% TFA. Using.

(Peptide purification by reverse phase HPLC)
Peptide purification by reverse phase HPLC was performed under the following conditions. As the column, trade name: PREP-ODS (manufactured by Shimadzu Corporation) was used. The eluent flow rate during elution was set to 10 mL / min, and the detection wavelength was set to 210 nm. Peptides were eluted with a linear gradient of 8 minutes with 10 wt% to 20 wt% acetonitrile. For the linear gradient, a 10 wt% acetonitrile aqueous solution containing 0.05 wt% trifluoroacetic acid and a 20 wt% acetonitrile aqueous solution containing 0.05 wt% TFA were used.

(Measurement of mass spectrum)
Using a matrix-assisted laser desorption / ionization time-of-flight mass spectrometer [trade name: Voyager-DE STR (manufactured by Applied Biosystems)], using α-cyan-4-hydroxycinnamic acid (CHCA) as a matrix, mass spectrum Was measured.

(Analysis of ssDNA (conjugate) introduced with functional molecule by reverse phase HPLC)
Analysis of the conjugate by reverse phase HPLC was performed under the following conditions. The trade name: OligoDNA RP (manufactured by Tosoh Corporation) was used as the column. The flow rate was set to 1.0 mL / min, and the detection wavelength was set to 260 nm. Elute with 0.1 M aqueous ammonium acetate containing 5% conjugate of acetonitrile for 5 minutes, then 5-30%
Elute with a 50 minute linear gradient with% acetonitrile. For the linear gradient, a 0.1 M aqueous ammonium acetate solution containing 5% by weight of acetonitrile and a 0.1M aqueous ammonium acetate solution containing 30% by weight of acetonitrile were used.

(SsDNA concentration measurement)
25 μL of ssDNA solution was added to 975 μL of 10 mM Tris-HCl buffer (pH 7.0) for dilution. About the obtained dilution, the light absorbency of 260 nm was measured with the spectrophotometer which carried out base correction | amendment only with 10 mM tris hydrochloric acid buffer. The value obtained by multiplying the absorbance measured at this time by 40 was defined as the OD value of the ssDNA solution. The weight (μg) per OD of the ssDNA used in this example can be calculated from the calculated OD value. By using the molecular weight of ssDNA, the OD value can be converted into a molar concentration.

(Measurement of concentration of functional polymer complex having DNA spacer)
The concentration of the functional polymer complex shown in the following examples was determined using a trade name: PicoGreen DNA Quantification Kit (Pierce). A calibration curve was prepared using a λDNA standard solution with a known concentration. All the measured values are average values of three measurements.

  The following example illustrates one embodiment of the present invention. That is, as a functional molecule, two functional molecules, a KDGEA peptide (functional molecule derived from type I collagen) and a GRGDS peptide (functional molecule derived from fibronectin), are linked between the two functional molecules. As the structure of the nucleic acid spacer, dsDNA having a double helix structure in which complementary base pairs are formed and two functional molecules are introduced at the 5 ′ end present in two places in the dsDNA.

  The length of the DNA spacer was 40 nm (120 mer as DNA), 80 nm (240 mer as DNA) and 160 nm (480 mer as DNA). The structure (schematic diagram) of the functional polymer designed according to the above concept is shown below.

  Functional polymer complexes having DNA spacer lengths of 40 nm, 80 nm, and 160 nm are referred to as KDGEA-SP40-GRGDS, KDGEA-SP80-GRGDS, and KDGEA-SP160-GRGDS, respectively.

  In the following examples, functional polymer composites were prepared by a pre-modification method. First, a conjugate in which a functional molecule was bound to ssDNA was synthesized. A maleimide group was selected as the functional group for binding the nucleic acid spacer. Those introduced at the N-terminus of each of the KDGEA peptide molecule and the GRGDS peptide molecule (referred to as maleimidated KDGEA and maleimidated GRGDS, respectively) were used. The ssDNA used was one having a thiol group introduced at the 5 'end as a functional group for functional molecule binding. More specifically, the structure is a synthetic ssDNA in which the OH site of 6-mercapto-1-hexanol is bound to the 5'-terminal phosphate group. Immediately after synthesis, the thiol moiety at the 5 'end is protected by a protecting group having a thiol group and a disulfide bond. This is deprotected to form a thiol group, and maleimidated KDGEA or maleimidated GRGDS is introduced to form a conjugate with ssDNA.

Examples 1-3
KDGEA-SP40-GRGDS with 40 nm (120 mer) dsDNA as spacer, KDGEA-SP80-GRGDS with 80 nm (240 mer) dsDNA, and KDGEA-SP160-GRGDS with 160 nm (480 mer) dsDNA Was prepared as follows. An outline of the synthesis process is shown in FIG.

(Synthesis of maleimidated KDGEA)
Synthesis of maleimidated KDGEA was started from a resin (manufactured by Shimadzu Corporation, trade name: Preloaded HMP resin) in which Fmoc-Ala (wherein “Fmoc” is an abbreviation of 9-fluorenylmethoxycarbonyl) was previously introduced. Deprotection of the Fmoc group was performed using a 20 wt% piperidine (Applied Biosystems) / dimethylformamide (hereinafter also referred to as DMF) solution. In addition, the condensation reaction for extending the peptide chain comprises 10 equivalents of Fmoc-aa (aa represents an appropriately protected amino acid) with respect to the amount of amino acids in the resin, and 10 equivalents of HBTU [ Made by Shimadzu Corporation, HBTU is 2- (1-H-benzotriazol-1-yl-1,1,3,3-tetramethyluronium hexafluorophosphate), 10 equivalents of HOBt [Peptide Research Institute, HOBt is an abbreviation for 1-hydroxybenzotriazole) and 20 equivalents of DIEA (manufactured by Applied Biosystems, DIEA is an abbreviation for Diisopropylenelamine) in DMF, sequentially corresponding to the amino acid sequence. In Performed on the resin by adding to the resin. The N-terminal Fmoc group was deprotected with 20 wt% piperidine / DMF. In the obtained product, 5 equivalents of GMBS (manufactured by Dojin Chemical Co., Ltd., GMBS is an abbreviation of N- (4-maleidobutyryloxy) succinimide) and 10 equivalents of DIEA with respect to the produced free N-terminal amino group. Were dissolved in DMF and added. The mixture was allowed to react at room temperature for 4 hours, and then another equivalent of GMBS was added to the obtained product and reacted at room temperature for 1 hour. The resin holding the resulting product was washed with DMF, and then the resin was washed with methanol and diethyl ether. The washed resin was dried under reduced pressure. For cleavage of the maleimide group-containing peptide from the resin, 90% by volume TFA (manufactured by Peptide Institute, Inc.) / 5% by volume thioanisole (manufactured by Aldrich) / 3% by volume H ₂ O / 2% by volume anisole (V / V / V / V) solution was used. The solution was added to the resin and the resulting mixture was stirred for 2 hours at room temperature. Thereafter, the resin was filtered off and the filtrate was concentrated under reduced pressure. Peptide was precipitated by adding cooled diethyl ether to the residue, and the solid was separated by filtration and dried under reduced pressure. The resulting peptide was purified by purification HPLC under the conditions described above. The isolated peptide was confirmed to be a single peak (retention time 10.4 minutes) by analytical HPLC. Further, when the molecular weight of the peptide was measured by mass spectrum, m / z = 684.60 [M + H] ⁺ , which was in good agreement with the calculated value. Freeze drying gave solid maleimidated KDGEA.

(Synthesis of maleimidated GRGDS)
Using a resin in which Fmoc-Ser was previously introduced (manufactured by Shimadzu Corporation, trade name: Preloaded HMP resin), the synthesis was performed in the same manner as in the case of the maleimidated KDGEA. The resulting crude peptide was purified by purification HPLC under the conditions described above. The isolated peptide was confirmed to be a single peak (retention time 8.6 minutes) by analytical HPLC. Further, when the molecular weight of the peptide was measured by mass spectrum, m / z = 656.61 [M + H] ⁺ , which was in good agreement with the calculated value. Lyophilization gave a solid maleimidated GRGDS.

(Deprotection of ssDNA containing thiol group)
There are two types of ssDNA with protected thiol groups as raw materials for KDGEA-SP40-GRGDS: PS-S-SP40A and PS-S-SP40B. In FIG. 1, PS-S-SP40A and PS-S-SP40B are parts represented by base sequences, the left ssDNA is PS-S-SP40A, and the right ssDNA is PS-S-SP40B. is there. Other raw materials are also shown.

  PS-S-SP40A was dissolved in TE buffer (10 mM Tris-HCl buffer, pH 8.0 containing 1 mM EDTA) so as to be 100 nmol / mL. 0.2 mL of 0.08 M dithiothreitol solution (prepared by dissolving in 0.25 M phosphate buffer (pH 8.0)) is mixed with 0.2 mL of the obtained solution, and then the resulting mixture is mixed. Was stirred at room temperature for 16 hours. PS-S-SP40A in which the thiol group is deprotected is obtained by purification using a NAP-5 column manufactured by Pharmacia Biotech equilibrated with an eluent [0.1 M phosphate buffer (pH 6.0)]. It was. About the obtained product, the density | concentration of HS-SP40A was determined by measuring the light absorbency of 260 nm. It carried out similarly about the case of KDGEA-SP80-GRGDS and KDGEA-SP160-GRGDS, and obtained HS-SP80A, HS-SP80B, HS-SP160A, and HS-SP160B. The concentrations of the obtained HS-SP40B, HS-SP80A, HS-SP80B, HS-SP160A and HS-SP160B were determined in the same manner. The results are shown in Table 1.

(Preparation of conjugate)
0.8 mL of an HS-SP40A solution (concentration: 17.0 nmol / mL) and 0.8 mL of a solution obtained by dissolving 25 equivalents of maleimidated KDGEA in 0.1 M phosphate buffer (pH 7.0) with respect to HS-SP40A; Were mixed and incubated at 4 ° C. for 24 hours to react. The resulting reaction solution was lyophilized. The obtained product was dissolved in 10 mM Tris-HCl buffer (pH 7.0) containing 0.5 mL of 1 mM EDTA. The resulting solution was subjected to gel filtration using NAP-5 equilibrated with an eluent to purify KGDEA-S-SP40A, which is a conjugate of maleimidated KDGEA and HS-SP40A. In addition, 10 mM Tris-HCl buffer (pH 7.0) containing 1 mM EDTA was used as an eluent. About the obtained product, the light absorbency in 260 nm was measured and the density | concentration was determined using the data of HS-SP40A (Table 1). Similarly, GRGDS-S-SP40B, which is a conjugate of maleimidated GRGDS and HS-SP40B, KGDEA-S-SP80A, which is a conjugate of maleimidated KDGEA and HS-SP80A, and maleimide GRGDS and HS-SP80B GRGDS-S-SP80B which is a conjugate, KDGEA-S-SP160A which is a conjugate of maleimide KDGEA and HS-SP160A, and GRGDS-S-SP160B which is a conjugate of maleimide GRGDS and HS-SP160B were obtained. . The results are shown in Table 2.

  All conjugates were confirmed to be single peaks by HPLC.

(Preparation of functional polymer composite)
Using the KDGEA-S-SP40A and GRGDS-S-SP40B as a primer pair for PCR, using Escherichia coli 16s rRNA as a template nucleic acid, and a functional polymer complex containing a 120-mer DNA spacer as follows: Was prepared.

  Distilled water was added to 25 μL of the primer pair (each 50 pmol) and trade name: Premix Ex Taq (manufactured by Takara Bio Inc.) and 5 μL of the E. coli suspension to obtain a reaction solution having a total volume of 50 μL. The Escherichia coli suspension is a suspension prepared by dissolving 1 colony of Escherichia coli IFO 3972 grown on an agar medium in 100 μL of distilled water. Using the obtained reaction solution, 24 cycles of reaction (polymerase chain reaction) were performed with 94 ° C. for 0.5 minute, 55 ° C. for 0.5 minute, and 72 ° C. for 1 minute as one cycle. The obtained reaction product was subjected to a spin column (trade name: QIA PCR Purification Kit, manufactured by Qiagen) and purified to obtain KDGEA-SP40-GRGDS. The obtained KDGEA-SP40-GRGDS was subjected to polyacrylamide gel electrophoresis, then visualized and detected. As a result, it was confirmed that the obtained KDGEA-SP40-GRGDS contained 120-mer dsDNA. As a result, it was found that the spacer site composed of dsDNA could be synthesized with the designed length. In the same manner as above, for KDGEA-SP80-GRGDS, KDGEA-S-SP80A and GRGDS-S-SP80B are used as a primer pair, and for KDGEA-SP160-GRGDS, KDGEA-S-SP160A and GRGDS-S-SP160B. Were used as primer pairs to prepare a functional polymer complex. The molecular weights of KDGEA-SP80-GRGDS and KDGEA-SP160-GRGDS were confirmed by polyacrylamide gel electrophoresis. As a result, it was confirmed by comparison of the relative mobility with the molecular weight marker that the dsDNA part was a 240-480 mer, respectively. As a result, it was found that the spacer site composed of each dsDNA could be synthesized with the designed length. Table 3 shows the results of quantifying the concentration using the product name: PicoGreen.

(Evaluation of the ability of the functional polymer complex to control cell functions)
The ability to control cell function was assessed by comparing the ability to differentiate immature osteoblasts from mice (MC3T3-E1 cells) into mature osteoblasts. MC3T3-E1 cells were cultured in α-MEM (Gibco, α-MEM is an abbreviation for Alpha-Minimum Essential Medium) supplemented with 10% by weight fetal bovine serum (FBS). Thereafter, MC3T3-E1 cells were recovered by trypsin treatment when 90% confluent. The α-MEM to which 10 mM β-glycerophosphoric acid (manufactured by Sigma), 50 μg / mL ascorbic acid (manufactured by Sigma) and 10% by weight FBS (manufactured by Sigma) were added, was adjusted to 19000 / mL. MC3T3-E1 cells were redispersed. Thereafter, the MC3T3-E1 cells were seeded at 2 mL per well in a 12-well plate in which KDGEA-SP40-GRGDS, KDGEA-SP80-GRGDS, or KDGEA-SP160-GRGDS was immobilized by the method described below. The cell density at this time was 5000 cells / cm ² . The MC3T3-E1 cells were cultured in humid air containing 5% CO ₂ at 37 ° C. The medium was changed every 3 days. Eleven days later, alkaline phosphatase (ALP) activity of MC3T3-E1 cells was measured. Using the ALP activity as an index of the degree of differentiation of MC3T3-E1 cells, the ability of the functional polymer complex to control cell functions was evaluated.

The cultured cells were collected by trypsin treatment and redispersed in 0.5 mL of PBS. Using the obtained dispersion, the number of cells was counted with a Birkerchilk hemocytometer. Thereafter, ALP activity of the cells was measured as an index of differentiation. ALP activity was measured by a p-nitrophenyl phosphate (pNPP) method using a substrate solution [trade name: FAST p-NITROPHENYL PHOSPHATE TABLE SET (manufactured by Sigma) was used]. Here, utilizing the fact that pNPP, which is a substrate, becomes p-nitrophenol and exhibits yellow color by ALP, the enzyme activity value was shown by the amount of p-nitrophenol generated in a certain time. The amount of p-nitrophenol produced was obtained by dissolving p-nitrophenol in PBS to prepare a calibration curve. In addition, Triton ^™ X-100 (manufactured by Sigma), which is a surfactant, was used for lysis of the cell membrane. A 96-well plate was charged with 100 μL of cell solution or calibration curve solution, 50 μL of 0.3 wt% Triton ^™ X-100, and 100 μL of substrate solution, and the resulting mixture was incubated at 37 ° C. for 1 hour for reaction. It was. To the obtained reaction product, 50 μL of 3N aqueous sodium hydroxide solution was added to stop the enzyme reaction. Thereafter, the absorbance of the obtained product was measured at 450 nm with a microplate reader (Denios, manufactured by Deccan). The ALP activity per cell (p-nitrophenol production rate per cell (fmol / min / cell)) was calculated.

Test example 1
(Preparation of poly-L-lysine surface)
Poly-L-lysine (PLL) hydrochloride (manufactured by Peptide Institute, average molecular weight 8000 or more) was dissolved in phosphate buffered saline (PBS) so as to be 10 mg / mL. The subsequent operations were performed using instruments and buffers that were all sterilized in the safety cabinet. The obtained solution was sterilized by filtration through a filter having an inner diameter of 0.2 μm and further diluted 10-fold with PBS to obtain a 1 mg / mL PLL solution. The resulting PLL solution was added at 0.5 mL per well to a Nuku Delta-treated 12-well plate, and then the plate was sealed. By incubating the plate at 37 ° C. for 2 hours, PLL was adsorbed on the plate. Thereafter, the PLL solution was aspirated from the plate and removed from the well. Furthermore, 1 mL of PBS per well was added to the plate and allowed to stand for 3 minutes, and aspirating washing operation was performed 3 times to prepare a PLL surface.

(Immobilization of functional polymer complex on poly-L-lysine surface by polyion complex formation)
All manipulations were performed using sterilized instruments and buffers in a safety cabinet. The functional polymer composites prepared in Examples 1 to 3, ie, 10 mM containing 2 mM EDTA so that KDGEA-SP40-GRGDS, KDGEA-SP80-GRGDS, and KDGEA-SP160-GRGDS each have a target density. Dilute with Tris-HCl buffer (pH 7.4). The resulting dilution was sterilized by filtration through a 0.2 μm inner diameter filter. The sterilized product was added to 0.5 mL per well to a 12-well plate on which a PLL surface was formed. Here, KDGEA-SP40-GRGDS, KDGEA-SP80-GRGDS, and KDGEA-SP160-GRGDS were each prepared to be contained in 0.5 mL of the product. In the case of a 12-well plate, when 0.5 mL of liquid is added to one well, the area covered with the liquid is about 4.5 cm ² . Therefore, the density when the functional polymer complex in this case is all bonded to the PLL surface is determined to be 100 fmol / cm ² . Thereafter, it was adsorbed at 4 ° C. for 12 hours or more. The functional polymer complex solution after adsorption was collected. Subsequently, the dsDNA concentration of the obtained functional polymer complex solution was measured by a trade name: PicoGreen, and it was confirmed how much the added functional polymer complex was immobilized on the PLL surface. The results are shown in Table 4.

  As a result, it was found that the functional polymer complex was immobilized on the PLL surface.

Test example 2
(Evaluation of the ability of the functional polymer complex to control cell functions)
The ability to control the function of the cells on the PLL surface on which KDGEA-SP40-GRGDS, KDGEA-SP80-GRGDS or KDGEA-SP160-GRGDS prepared in Examples 1 and 2 was immobilized was the same as described above. Evaluation was made by measuring. Table 5 shows the results of culturing MC3T3-E1 cells and measuring ALP activity per cell on day 11.

  As a result, it was found that immature osteoblasts derived from mice (MC3T3-E1 cells) were differentiated into mature osteoblasts by KDGEA-SP40-GRGDS, KDGEA-SP80-GRGDS and KDGEA-SP160-GRGDS. .

Comparative Example 1
(Preparation of functional polymer having polyethylene glycol spacer)
A functional polymer containing a KDGEA molecule and a GRGDS molecule as a ligand molecule and having an average molecular weight of 3000 polyethylene glycol (hereinafter sometimes abbreviated as PEG) as a spacer was synthesized. Fmoc-NH-PEG-NHS (manufactured by NEKTAR, molecular weight 3400) having an amino group protected by an Fmoc group and a carboxyl group activated by N-hydroxysuccinimide at the other end was used as a PEG spacer binding site. Except S. Yamamoto, Y. et al. Kaneda, N .; Okada, S .; Nakagawa, K .; Kubo, S .; Inoue, M .; Maeda, Y .; Yamashiro, K .; Kawasaki, T .; Mayumi, Anti-Cancer Drugs, 5, 424-428 (1994) and Y.M. Suzuki, K .; Hojo, I .; Okazaki, H .; Kamata, M .; Sasaki, M .; Maeda, M .; Nomizu, Y .; Yamamoto, S .; Nakagawa, T .; Mayumi, K. et al. Kawasaki, Chem. Pharm. Bull. 50 (9), 1229-1232 (2002) was applied to synthesize KDGEA-PEG-GRGDS.

(Immobilization of functional polymer with polyethylene glycol spacer on dish surface)
S. Yamamoto, Y. et al. Kaneda, N .; Okada, S .; Nakagawa, K .; Kubo, S .; Inoue, M .; Maeda, Y .; Yamashiro, K .; Kawasaki, T .; Mayumi, Anti-Cancer Drugs, 5, 424-428 (1994) and Y.M. Suzuki, K .; Hojo, I .; Okazaki, H .; Kamata, M .; Sasaki, M .; Maeda, M .; Nomizu, Y .; Yamamoto, S .; Nakagawa, T .; Mayumi, K. et al. Kawasaki, Chem. Pharm. Bull. 50 (9), 1229-1232 (2002), KDGEA-PEG-GRGDS was dissolved in pure water so as to be 9 nmol / mL. 0.5 mL of the resulting solution was added to each well of a 12-well plate (Nunc, delta treatment). The plate was left as it was in a clean bench, and the solution was air-dried. To the wells of the plate, 2 mL of 3% by weight bovine serum albumin / DMEM (Gibco, DMEM is an abbreviation for Dulbecco's Modified Eagle's Medium) was added per well and allowed to stand at room temperature for 1 hour. Thereafter, the solution was aspirated. A series of washing operations in which 2 mL of 0.1 wt% bovine serum albumin (BSA) / DMEM solution was added per well of the plate and aspirated were performed three times to prepare a surface on which the functional polymer was immobilized. .

(Cell function control by the surface immobilizing functional polymer with polyethylene glycol spacer)
Using the 12-well plate prepared as described above, the cell function was evaluated simultaneously under exactly the same conditions as in the case of using the functional polymer complex of Example 1. As a result, when the culture was continued for 11 days, MC3T3-E1 cells were detached from the bottom of the plate. The ALP activity per cell was 28 fmol / min / cell, which was extremely low. This result means that the cell function could not be controlled on the surface of the plate obtained in Comparative Example 1.

  Using the functional polymer complex obtained by the production method of the present invention, the distance between specific sites in a biopolymer such as a protein or carbohydrate on the surface of a cell, extracellular matrix, or other biological tissue, or Estimating the distance between multiple identical or different proteins, carbohydrates and other biomolecules is useful for understanding the structure of cells, extracellular matrix, and other biological tissues, and for the interaction between these biomolecules. This is useful for estimating the presence or absence. In addition, by simultaneously binding to a plurality of specific sites on the cell surface and simultaneously giving a signal to the cell and observing the reaction of the cell, the function of those biomolecules is examined in terms of cell physiology. Very useful. The functional polymer complex obtained by the production method of the present invention greatly contributes to the fields of medical, diagnostic, and production of useful substances such as pharmaceuticals, for example, by the development of the above-mentioned studies.

FIG. 1 shows KDGEA-SP40-GRGDS with 40 nm (120-mer) dsDNA, KDGEA-SP80-GRGDS with 80 nm (240-mer) dsDNA and KDGEA- with 160 nm (480-mer) dsDNA as spacers. It is the schematic which shows the structure of SP160-GRGDS, and the structure of the conjugate used for preparation of those functional polymer composites.

  SEQ ID NO: 1 shows the sequence of the ligand.

  SEQ ID NO: 2 shows the sequence of the ligand.

  SEQ ID NO: 3 shows the sequence of the ligand.

  SEQ ID NO: 4 shows the sequence of the ligand.

  SEQ ID NO: 5 shows the sequence of the ligand.

  SEQ ID NO: 6 shows the sequence of the ligand.

  SEQ ID NO: 7 shows the sequence of the ligand.

  SEQ ID NO: 8 shows the sequence of the ligand.

  SEQ ID NO: 9 shows the sequence of the ligand.

  SEQ ID NO: 10 shows the sequence of the ligand.

  SEQ ID NO: 11 shows the sequence of the ligand.

  SEQ ID NO: 12 shows the sequence of the ligand.

  SEQ ID NO: 13 shows the sequence of the ligand.

  SEQ ID NO: 14 shows the sequence of the ligand.

  SEQ ID NO: 15 shows the sequence of the ligand.

Claims (2)

1) a template nucleic acid;
2) a first containing a binding site for a functional molecule having binding activity with a biopolymer, and a nucleic acid consisting of consecutive nucleotide residues in the base sequence of the sense strand of the template nucleic acid or a derivative thereof A first functional molecule-binding primer in which the functional molecule is bound to the primer of
3) a binding site for a functional molecule having binding activity with a biopolymer, and a nucleic acid consisting of consecutive nucleotide residues in the base sequence of the antisense strand of the template nucleic acid or a derivative thereof, A first molecule in which the functional molecule is bound to a second primer having a nucleotide at the 3 ′ end as a nucleotide present at a position at least 20 nucleotides away from the nucleotide on the template nucleic acid corresponding to the 3 ′ end of the first primer. A method for producing a functional polymer complex having at least two functional molecules, which comprises performing a polymerase chain reaction in the presence of two functional molecule-binding primers. (A) 1) a template nucleic acid;
2 ′) a binding site for a functional molecule having binding activity with a biopolymer, and a nucleic acid or a derivative thereof comprising a continuous nucleotide residue in the base sequence of the sense strand of the template nucleic acid 1 primer and
3 ′) a binding site for a functional molecule having binding activity with a biopolymer, and a nucleic acid or a derivative thereof consisting of consecutive nucleotide residues in the base sequence of the antisense strand of the template nucleic acid. A polymerase chain in the presence of a second primer having a nucleotide at the 3 ′ end as a nucleotide present at a position at least 20 nucleotides away from the nucleotide on the template nucleic acid corresponding to the 3 ′ end of the first primer. Performing a reaction to synthesize a polymer complex having at least two binding sites for the functional molecule; and (B) a functional molecule in the polymer complex obtained in the step (A). A step of synthesizing a functional polymer complex having at least two functional molecules by bonding
A method for producing a functional polymer complex having at least two functional molecules.

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