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    • 专利摘要:
    The present invention comprises the steps of reacting a nucleic acid specific metal complex with a nucleic acid to produce a metal complex-nucleic acid conjugate, removing unconjugated metal complexes and / or unconjugated by-products and metal complex-nucleic acid conjugates An improved method for direct and selective metallization of nucleic acids via in-situ nanometer sized metal particles, comprising reacting with a reducing agent to produce nanometer sized metal particle-nucleic acid complexes. . The nanometer sized metal particle-nucleic acid composites can be used, for example, to form nanometer sized wires in electronic networks and circuits that allow for high density arrangements.
    公开号:KR20020040650A
    申请号:KR1020010073583
    申请日:2001-11-24
    公开日:2002-05-30
    发明作者:포르트빌리암；베쎌스유리나；하르낙올리버；야스다아키오
    申请人:소니 인터내셔널(유로파) 게엠베하；
    IPC主号:
    专利说明:

    Selective metallisation of nucleic acids via metal nanoparticles produced in-situ}
    [15] The present invention provides direct and selective metallization of nucleic acids via in-situ nanometer sized metal particles that can be used to form nanometer sized wires in electronic networks and circuits that allow for high density arrangements. To an improved method.
    [16] The electronics industry is showing ongoing efforts to obtain high density wiring and circuits. One of the main issues in achieving this goal is to make the industrial wire as small as possible. One approach known in the prior art is to metallize a nucleic acid, which once acts as an electrically conductive wire.
    [17] "Metalization" of a nucleic acid also refers to a method of directly linking a position within a nucleic acid, in particular between the N-7 atoms (G and A) of a purine nucleotide and a metal atom. This reaction has been widely studied because of its association with anticancer agents, mainly platinum (II) or platinum (IV) complexes. Other metal complexes exhibiting this behavior include complexes of Pd, Ru, Au and Rh. The complex requires one or more "unstable" ligands as "leaving groups" to bind in this way.
    [18] In addition, nucleic acid binders have been widely studied as anticancer agents. Non-covalent binders include "intercalator" and "groove binder". Covalently bound formulations are commonly referred to as "alkylating agents". Many examples of molecules having complex functions with each class of agents are known. Specific base pair combinations and selectivity to sequences or other “recognition positions” are highly coordinated (eg drug targeting).
    [19] WO 99/04440, published January 28, 1999, describes a three step process for metallization of DNA. In the first step, silver ions (Ag + ) are biased along the DNA through Ag + / Na + ion exchange and complex formation between Ag + and DNA nucleotide bases. The silver ion / DNA complex is then reduced using a basic hydroquinone solution to form nanometer-sized silver particles bound to the DNA backbone. Subsequently, nanometer sized silver particles are "developed" under low light conditions similar to standard photographic procedures using an acidic solution of hydroquinone and Ag + . This process produces a silver wire with a width of about 100 nm with a differential resistance of about 10 MΩ.
    [20] However, silver wires having a width of 100 nm and in particular having a differential resistance of about 10 MΩ produced in accordance with the method described in WO 99/04440 do not meet the demands of the industry in terms of high density wiring and high density circuits.
    [21] The metallization process described in WO 99/04440 is similar to the process for detecting DNA fractions by silver staining. This process is known as a result of nonspecific staining of DNA fractions and does not distinguish between different DNA sequences. The ability to metalize certain regions of nucleic acid strands, but not other regions, may be important in the development of DNA-based nanometer-sized electronic devices.
    [22] In addition, the literature describes DNA as a template for metallization to produce nanometer-sized metal wires. Pompe et al. (1999) Z. Metallkd. 90, 1085; Richter et al. (2000) Adv. Mater. 12, 507]. This metallization method involves treating the DNA with an aqueous solution of Pd (CH 3 COO) 2 for 2 hours and then adding a solution of dimethylamine borane (DMAB) as the reducing agent. Nanometer-sized palladium particles, 3 to 5 nm in diameter, grow on DNA within seconds after addition of the reducing agent. After about 1 minute, a semi-continuous coating is obtained and the size of the metallic aggregate is 20 nm.
    [23] Nucleic acid synthesis and modification techniques are the subject of countless publications. In particular, these methods are described in books ( Bioorganic Chemistry: Nucleic Acids (edited by SM Hecht, Oxford University Press, 1996; Bioconjugate Techniques (by GT Hermanson, Academic Press, 1996). "Annealing" and "Ligation" techniques have been described for combining double helix nucleic acids from smaller units (the chapter by M. Van Cleve in Bioorganic Chemistry: Nucleic Acids (Chapter 3, pages 75-104)). Chapter 8, pages 216-243, and chapters of Bioconjugate Techniques (Chapter 17, pages 639-671) by MJO'Donnell and LW McLaughlin in the literature. It describes chemical modifications of nucleic acids and oligonucleotides and covalent linkages of reporter groups (fluorophores, spin labels, etc.) These techniques are also attached to metal complexes, for example reducing agents and bond degradation. It used to act as a catalyst, but the metal is not as pragmatic as used.
    [24] One example of chemical modification is "bromine activation". Reaction with N-bromosuccinimide causes bromination at the C-8 position of the guanine residue and the C-5 position of the cytosine residue, for example (FIG. 7). The nucleophilic amine is then coupled to these positions by nucleophilic displacement to introduce various functional groups into the nucleic acid. Derivatized sites using this method do not involve hydrogen bonding during base pair formation, so hybridization capacity is not so disturbed.
    [25] The present invention as well as the two prior arts for the above mentioned DNA metallization use the principles commonly used in both the development of electrophotographic films and in electroless plating. These methods include the following two steps: (1) forming small nanometer-sized metal particles (or clusters) and (2) electrolessly depositing metals that may be the same or different than those used in step 1 Thereby enlarging the particles. Thus, the initially formed particles act as nucleation sites for subsequent metal deposition.
    [26] A "two step" electroless plating method is known, for example, from US Pat. Nos. 5,503,877 and 5,560,960. The substrate to be plated is first exposed to a solution containing the metal ion species, followed by a reducing agent solution that reduces the metal ion species to the metal catalyst. The catalyst metal is usually Pd, but can also be one or more of Pd, Cu, Ag, Au, Ni, Pt, Ru, Rh, Os and Ir, and is usually combined with an organic ligand containing one or more nitrogen atoms. The metal to be deposited may be magnetic and include, for example, B and P introduced by Co, Ni, Fe, and reducing agents (e.g., borohydride or hypophosphite; see US Pat. Nos. 3,986,901 and 4,177,253). It is an alloy which may contain.
    [27] Accordingly, the problem addressed by the present invention is, for example, through nanometer sized metal particles produced in situ, which can be used to form nanometer sized wires in electronic networks and circuits that enable high density arrangements. It is to provide an improved method of directly and selectively metallizing nucleic acids.
    [1] 1 shows the ultraviolet-visible absorbance spectra of Pt (II) -terpyridine-DNA conjugates and Pt-DNA complexes prepared according to Example 1. FIG.
    [2] FIG. 2 shows an AFM image of a Pt-DNA complex prepared according to Example 1, prior to treatment with a solution of GoldEnhance R according to Example 4. FIG.
    [3] FIG. 3 shows an AFM image of a Pt-DNA complex prepared according to Example 1 after treatment with a solution of GoldEnhance R according to Example 4. FIG.
    [4] 4 shows an AFM image of another area of the sample shown in FIG. 3.
    [5] FIG. 5 shows an AFM image of a Pt-DNA complex prepared according to Example 2 prior to treatment with a solution of GoldEnhance R according to Example 5. FIG.
    [6] FIG. 6 shows an AFM image of a Pt-DNA complex prepared according to Example 2 after treatment with a solution of GoldEnhance R according to Example 6. FIG.
    [7] Figure 7 shows the most preferred locations for "metallization" at the N-7 atoms of the purine nucleotides (G and A) of the nucleic acid.
    [8] 8 shows some variations of metal (M) -ligand (L 1 , L 2 and L 3 , X or Z) complexes (to simplify, charges are omitted).
    [9] 9 is a schematic representation of the process of metallizing a particular base in an oligonucleotide subunit at its original location (for simplicity, charges are omitted).
    [10] FIG. 10 schematically illustrates a process for metallizing a particular base in an oligonucleotide subunit at a position introduced by chemical modification (for the sake of brevity, charges are omitted).
    [11] FIG. 11 shows an example of a substituted-inactive metal (M) complex attached to a nucleic acid interacting group of the formula INT-CON-LIG-M (L) n .
    [12] FIG. 12 shows covalent linkages of substitution-inactive metal complexes to specific bases in oligonucleotide subunits before or after hybridization in supplementary segments of longer nucleic acids (for the sake of brevity, charges are omitted).
    [13] FIG. 13 shows AFM images of unmodified non-platinumized DNA after treatment with a solution of GoldEnhance R. FIG.
    [14] FIG. 14 shows AFM images of unmodified unplated DNA after treatment with a solution of GoldEnhance R. FIG.
    [28] These challenges include reacting nucleic acid specific metal complexes with nucleic acids to produce metal complex-nucleic acid conjugates, removing unconjugated metal complexes and / or unconjugated by-products, and metal complex-nucleic acid conjugates. Is reacted with a reducing agent to produce a nanometer-sized metal particle-nucleic acid composite.
    [29] The present invention provides an improved method of directly and selectively metallizing nucleic acids such as DNA. After addition of the reducing agent, no clusters are observed to form on the DNA using AFM. This is in contrast to the method described by Richter et al., Wherein irregular clusters whose minimum diameter is approximately equal to the diameter of the DNA itself are formed on the DNA (which indicates that the growth of metal particles on the DNA is not controlled). to be. Treatment of the metalized DNA according to the invention with Gold Enhancer R further shows that the metallization is very dense as it is mainly limited to DNA. Nevertheless, metallized DNA can still be used for electroless metal deposition to make nanometer sized wires or other nanometer sized parts.
    [30] Although the metallization process described by Pompe et al. Was much improved over that of WO 99/04440, the initially grown nanometer-sized palladium particles are still considerably wider than the DNA itself (about 2 nm for double-stranded DNA). The present invention describes a means of producing nanometer-sized platinum particles on double helix DNA that is no wider than DNA, and these particles are catalytic to the electroless deposition of gold and can be expanded in a controlled manner. In addition, unlike the process of Pompe et al., Platinum particles / DNA complexes of sizes smaller than nanometers in nanometer sized particles, prepared according to the present invention, are stable for at least weeks or months. Thus, a single formulation of the composite can be used, for example, to manufacture nanometer sized wires at various times under various conditions. In addition, the present invention provides several types of nanometer-sized particle precursors and means for binding them to nucleic acids, thereby expanding the possibility of metallizing certain positions or segments within the nucleic acids.
    [31] According to the invention, the nucleic acid component can be reacted in a dissolved state, immobilized on a substrate or in a semi-solid state (eg a gel).
    [32] Nucleic acids for metallization include DNA, RNA, PNA, CNA, oligonucleotides, oligonucleotides of DNA, oligonucleotides of RNA, primers, A-DNA, B-DNA, Z-DNA, polynucleotides of DNA, polynucleotides of RNA , T-junctions of nucleic acids, triple nucleic acids, tetranucleic acids, domains of non-nucleic acid polymer-nucleic acid block copolymers, and mixtures thereof. Suitable non-nucleic acid polymers for block copolymers may be polypeptides, polysaccharides such as dextrose, or artificial polymers such as polyethylene glycol (PEG), which are commonly known to those skilled in the art. Nucleic acids can be double or single helix.
    [33] In a preferred method according to the invention, the metal complex-nucleic acid conjugate is formed by metallization and / or interactive ligand binding.
    [34] Nucleic acid specific metal complexes are attached, such as dichloro (2,2 ': 6', 2 "-terpyridine) platinum (II), cis-diaminodichloroplatinum (II), and insertion agents, groove binders and alkylating agents. More preferred is a method characterized in that it is selected from the group consisting of metal complexes with or combined nucleic acid interaction groups.
    [35] In a more preferred embodiment of the process according to the invention, the metal complex-nucleic acid conjugate is subjected to chromatography (eg gel filtration or ion exchange), precipitation (eg from unconjugated metal complex and / or unconjugated by-products), : Ethanol precipitation) or washing (eg washing with water or aqueous salt solution).
    [36] In a further aspect of the process according to the invention, the metal complex-nucleic acid conjugate is a boron hydride, borohydride salt, and a Lewis base: borane complex of formula L: BH 3 , wherein L is an amine, ether, phosphine or sulfide And hydrazine and derivatives thereof, hydroxylamine and derivatives thereof, hypophosphite, formate salt, dithionite salt and H 2 ).
    [37] A further preferred embodiment is characterized in that the reducing agent is used in the form of a gaseous reducing agent.
    [38] In general, the method according to the invention can be used to selectively metallize nucleic acids. Preferred nanometer sized metal particles are at least one metal selected from the group consisting of Fe, Co, Ni, Cu, Ru, Rh, Pd, Ag, Os, Ir, Pt, Au or mixtures of these metals (e.g. alloys). It is those containing.
    [39] Preference is given to a process wherein the nanometer sized metal particles are catalytically active against electroless metallization. More preferred is a method in which nanometer-sized metal particles cannot be visualized by atomic force microscopy and / or the nanometer-sized metal particles have a diameter of less than 3 nm.
    [40] An object of the present invention is further provided by a method further comprising enlarging the nanometer-sized metal particles by treating the nanometer-sized metal particles in the nanometer-sized metal particle-nucleic acid composite with an electroless plating solution. Is achieved.
    [41] In another embodiment, the metal complex-nucleic acid complex is treated in a dissolved state, immobilized on a substrate, or in a semisolid state (eg a gel).
    [42] In a further preferred embodiment of the method according to the invention, the nanometer sized metal particles comprise Fe, Co, Ni, Cu, Ru, Rh, Pd, Ag, Pt, Au or mixtures of these metals (e.g. alloys), Electroless plating containing a metal mixture selected from the group consisting of magnetic and / or magnetized Fe, Co, Ni or mixtures of these metals (such as alloys) or mixtures of these metals and B or P (such as alloys) Treat with solution.
    [43] The problem faced by the present invention is further achieved by nanometer-sized metal particle-nucleic acid composites obtainable according to one of the methods of the present invention.
    [44] Preferably, the nanometer-sized metal particle-nucleic acid composite is characterized in that the nanometer-sized metal particles are less than 3 nm in diameter. More preferred are nanometer-sized metal particle-nucleic acid composites, characterized in that nanometer-sized particles cannot be visualized by atomic force microscopy.
    [45] In a further aspect of the present invention, an object of the present invention is to provide a nanometer-sized metal particle-nucleic acid composite according to the present invention (a) nanometer-sized particles by electroless deposition of a metal according to the invention. Growth, preferably controlled growth, step (b).
    [46] In a further aspect of the invention, the challenge is achieved by the nanometer sized metal particles or the linear arrangement of nanometer sized wires obtainable according to the method of the invention. Nanometer sized metal particles can be catalytic or magnetized. In a further aspect, the challenge is achieved by nanometer sized wires obtainable according to one of the methods of the invention. The nanometer sized wires of the present invention may form an electronic network comprising one or more nanometer sized wires according to the present invention or an electronic circuit comprising one or more electronic networks according to the present invention. In addition, the nanometer-sized wires of the present invention can be used as electronic components of a non-metallized form, with some insulating space between nanometer-sized individual particles disposed along nucleic acid strands. In other embodiments, the nanometer sized wire may be fully conductive or contain insulating sites at one or both ends, or the insulating sites may be within the wire itself, such that the nanometer sized wire may consist of a single conductive island. These structures of the present invention may form or form part of an electronic network or electronic circuit including one or more nanometer sized wires. In such electronic networks or electronic circuits, junctions are formed between two or more wires, each wire having a connecting segment adjacent to the junction comprising nanometer sized wires. In addition, a network comprising nanometers of wire may be part of a hybrid electronic structure.
    [47] In addition, the problem is achieved by the junction between two or more wires of the electronic circuit, with each wire having a terminal segment adjacent to the junction comprising nanometer sized wires according to the invention.
    [48] Aspects of the present invention essentially comprise the following four steps:
    [49] Step (1): binding of the metal complex to the nucleic acid to generate a metal complex-nucleic acid conjugate
    [50] The specificity of the metallization process for the nucleic acid and the specific domain within the nucleic acid is mainly determined by the nature of the binding in step (1). The most direct binding method is "metallization". This method represents a direct (“covalent”) linkage between a metal atom and the N-7 atom of a position on the nucleic acid, in particular purine nucleotides (G and A). This case is shown in FIG. 7. These reactions have been widely studied because of their association with the mechanism of anticancer agents, mainly Pt (II) or Pt (IV) complexes (“platinum”). Pt (IV) complexes are usually considered “prodrugs” because they are reduced in the body with the corresponding Pt (II) complexes before they become active.
    [51] Pt (II) complexes known to covalently bind to nucleic acids are usually of the formulas Pt (L 1 ) (L 2 ) (X) (Z) and of the formulas Pt (L 1 ) (L 2 ) (L 3 ) (X) Square planar 4-coordination species, wherein L 1 , L 2, and L 3 are relatively inactive ligands (“stable”) ligands for substitution, and X and Z represent relatively reactive (“unstable”) ligands for substitution. ]to be. In the formula, the ligands L 1 , L 2 and L 3 may be the same or different and the ligands X and Z may be the same or different. In addition, the ligands L 1 , L 2 and L 3 may be linked to each other or to the ligands X or Z via bridging groups. In addition, ligands X and Z may be in the "cis" or "trans" position with respect to the Pt (II) atom. The complex may also contain two or more Pt (II) atoms. Some of these variations are shown in FIG. 8.
    [52] The atoms in the stable ligands (L 1 , L 2 and L 3 ) directly coordinated to Pt (II) are usually N, P or S. Ligands that are not linked by bridging groups (s) are called "singular". When two ligands are linked, they are called "binary" and when three ligands are linked, they are called "trident". Monodentate N-ligands are typically amines, monodentate P-ligands are typically phosphines, and monodentate S-ligands are typically thiols, thioethers or thiocarbonyls. The amine ligand can be ammonia, primary amine, secondary amine or tertiary amine. These include aromatic amines such as pyridine and aniline. There are many examples of bidentate NN ligands in Pt (II) complexes known to covalently bind nucleic acids, including, for example, 1,2-diaminoethane, 1,2-diaminopropane, 1,3- Diaminopropane, 1,2-diaminocyclohexane and 2,2-bipyridine. Trident NNN ligands such as 2,2 ': 6', 2 "-terpyridine (terpy) and diethylenetriamine (diene) as well as examples of bidental NP and NS ligands are known.
    [53] Examples of labile ligands X and Z, which normally act as good leaving groups, include halides, water, (dialkyl) sulfoxides, nitrates, sulfates, carboxylates, dicarboxylates, carbonates, phosphates, pyrophosphates, phosphate esters, phospho Nates, nitrites, sulfites, sulfonates, β-diketonates, alkenes, selenates, squarates, ascorbates and hydroxides. These ligands can be bimodal, for example as in the case of selenate and dicarboxylate oxalate and 1,1-cyclobutanedicarboxylate. They may also be part of a molecule containing stable ligand (s) such as, for example, amino acids (carboxylates and primary amine groups) and picolinic acid (carboxylates and pyridine groups).
    [54] In addition to platinum, other metal complexes have been found to potentially be used as anticancer agents. These include complexes of Pd, Ru, Au, and Rh, which tend to be four-coordinated (eg rectangular planar geometry) or six-coordinated (eg octahedral geometry). As in the case of Pt (II) anticancer agents, they also have one or more leaving groups through which metallization of the nucleic acid takes place. Due to the strict criteria for anticancer drugs, only a few of these other metal complexes have been clinically successful. If the complex is too unstable, it is likely to interact with physiological nucleophilic substances (proteins, etc.) before reaching the site of action in the tumor, thereby inactivating or increasing the risk of toxicity. On the other hand, if the complex is excessively inactive, it may not be able to interact with the biomolecular targets required for anticancer effects. Complexes of Pd (II) are also usually unstable, whereas complexes of Rh (III) are usually too inert, and the problem with Au (III) complexes is the fact that they are readily reduced by physiological reducing agents. These properties cause problems when applying complex compounds as anticancer agents, while the problems are much less when applied to metallization of nucleic acids. Indeed, the increased reactivity of Pd (II) complexes is advantageous in this application compared to their Pt (II) homologues, and in the case of Au (III) complexes, foreign reducing agents can be avoided.
    [55] In addition to having one or more leaving groups, the metallization complex should be able to be reduced to a metal state that exhibits catalytic activity for the electroless plating process. In addition to Pt, this requirement usually appears to be best achieved by the complex of Pd and Au. However, complexes of Ru and Rh may also be used. The use of such metalizing agents provides greater selectivity for sequences or segments within nucleic acids and wider range of catalytic activity for electroless plating than conventional platinum agents.
    [56] In another embodiment of step (1) of the present invention, the specific base inside the oligonucleotide subunit is metalchloride. These subunits are combined by hybridizing to supplemental segments of longer nucleic acids. Metalchloride of target bases in oligonucleotide subunits can be performed before or after hybridization. Non-supplemented segments of longer nucleic acid components are not hybridized by metalchloride oligonucleotides, and such gaps may be filled with other supplementary oligonucleotides that are not, for example, metalchloride. Two variations of this embodiment are shown schematically in FIGS. 9 and 10. In one embodiment (FIG. 9) the metal chloride occurs at the position originally present in the nucleic acid, and in another embodiment (FIG. 10) the metal chloride occurs at the position introduced by chemical modification. Chemical modification of specific bases in oligonucleotide subunits can be performed before or after hybridization.
    [57] In the example illustrated schematically in FIG. 9, pentanucleotides having the sequence TTGTT are used as subunit targets for metal chlorides, and metal complexes having tridentate (N-N-N) ligands and leaving groups (X) are used as metalchlorides. Under mild conditions (eg room temperature and neutral pH), thymine (T) residues are essentially inert and only guanine (G) residues are metalchloride: Two pathways for combining metalchloride hybridization constructs are shown in the figure. have. In one method, the oligonucleotide is metalchlorideed (i) followed by hybridization (ii) to longer nucleic acid components. In another method, the oligonucleotide is first hybridized (iii) and then metalchloride (iv). In the second method, it may be necessary to use modified bases on the longer nucleic acid components during step (iv) to prevent the metallization of the longer nucleic acid components. In a preferred embodiment, the oligonucleotide subunit consists of 4 to 20 bases and the metalchloride is a complex of Pt, Pd, Au, Ru or Rh.
    [58] In the example as shown in FIG. 10, pentanucleotides having the sequence TTC * TT are used as subunit targets for metalchloride, where C * is a metal ligand that is chemically modified to bind imidazole (Im) groups Cytosine residue. The imidazole group can be linked to the C-5 position of cytosine by, for example, nucleophilic displacement with bromine activation and 1- (3-aminopropyl) imidazole. Metal complexes having tridentate (NNN) ligands and leaving groups (X) are used as metal chlorides as in the example in FIG. 9. As in this example, two pathways are possible to combine metallized hybridization structures. In one method, the oligonucleotides are metalchloride (i) and then hybridized (ii). In another method, the oligonucleotide is first hybridized (iii) and then metalchloride (iv). In a second method, it may be necessary to use modified bases in the longer nucleic acid components during step (iv) to prevent metallization of the longer nucleic acid components. In a preferred embodiment, the oligonucleotide subunit consists of 4 to 20 bases and the metalchloride is a complex of Pt, Pd, Au, Ru or Rh.
    [59] In another embodiment of the invention, step (1) is carried out by a process wherein ligands coordinated to the metal in the complex are not substituted upon binding. This type of bond can be classified as an "external sphere" process. One example is a counterion exchange where a metal ion (eg Mg 2+ ) is replaced by a similarly charged metal complex (eg [Pt (NH 3 ) 4 ] 2+ ), but this simple exchange process Almost no distinction is made between nucleotide sequences or between nucleic acids and other negatively charged substances. Specificity for the nucleic acid and for specific domains within the nucleic acid is achieved by binding the nucleic acid interactive group to the metal complex. Such groups include intercalating agents, groove binders and alkylating agents known in the art. The nucleic acid interacting group may be the coalescing portion of the ligand coordinated to the metal ion (“as a metallization insert”) or covalently bound to the ligand. The main requirement of the metal complex used according to the invention is that the complex must be relatively stable against ligand exchange so that the complex can be delivered to the target nucleic acid binding site without damage. In addition, it should be able to be reduced to a metal state that exhibits catalytic activity for the electroless plating process. Both of these requirements are largely met by the complexes of metals of Groups 8 and IB of the Periodic Table.
    [60] Compounds useful in the embodiment of step (1) are compounds of the formula INT-CON-LIG-M (L) n , wherein INT is a nucleic acid interacting group, LIG is a stable ligand, and M (L) n is a coordination bond Unsaturated metal-ligand complex, which binds to LIG to meet the coordination requirements of metal M). The group CON may serve to connect the group INT and the group LIG, spatially separate the INT and LIG groups, and / or indicate their relative orientation.
    [61] Metallization insert complexes suitable for use according to this embodiment represent a special case of the formula INT-CON-LIG-M (L) n . Since the functions of INT and LIG are merged, CON cannot be defined as a separate group. Suitable metallization agents are complexes having the formula (ICL) M (L) n (e.g., ICL is a planar aromatic ligand, and M (L) n is a coordinating bond that binds to ICL to meet the coordination requirements of metal M). Unsaturated metal-ligand complex). Suitable metals M include Pt, Pd and Au. Planar aromatic bidentate ligands known to interact with nucleic acids by insertion of metal complexes include 8-hydroxyquinoline and α-diamines such as 2,2'-bipyridine, 1,10-phenanthroline, 2, 2-biquinoline, dipyrido [3,2-α: 2'3'-c] phenazine, and derivatives thereof). 2,2 ': 6', 2 "-terpyridine (Tupy) is an example of a tridentate ligand. The function of the ligand (s) in group M (L) n is mainly relatively substitution-inactive for metals. To provide a coordinating environment to enable a variety of stable single or multident N-, P- or S-ligands. Suitable bidentate ligands are 1,2-diaminoethane, 1,2-diaminopropane, 1, Diamines such as 3-diaminopropane and 1,2-diaminocyclohexane.
    [62] Specific examples of such compounds incorporating a complex of Pt (II), Pd (II) or Au (III) are shown in FIG. Such compounds are prepared by covalently binding the agent 1- (3-aminopropyl) imidazole to a nucleic acid interactivity group to provide an example of INT-CON-LIG, where the ligand is the N-3 atom of the bound imidazole group. Can be generated. The INT-CON-LIG compound is then reacted with a metal complex of the form M (diene) (X), wherein the diene is diethylenetriamine and X is a leaving group such as nitrate. The nucleic acid interacting groups in these examples consist of anthroquinones (insertants), cationic porphyrins (groove binders) and nitrogen mustards (alkylating agents).
    [63] In a further aspect of step (1) of the present invention, the substitution-inactive metal complex is covalently linked to a specific base in the oligonucleotide subunit. These subunits are combined by hybridization to supplemental segments of longer nucleic acids. Covalent modification of certain bases in oligonucleotides can be performed before or after hybridization. Unsupplemented segments of longer nucleic acid components are not hybridized by such modified oligonucleotides, and these gaps can be filled with other supplementary oligonucleotides to which no metal complexes are bound, for example. In the example shown in FIG. 12, a pentanucleotide having the sequence TTG * TT is used as a subunit target for metal chloride, where G * is chemically used to bind an amine group (-NH 2 ) as a covalent position. Modified guanidine residues are shown. The amine group can be linked to the C-8 position of guanidine by, for example, nucleophilic displacement with bromine activation and 1,4-diaminobutane. In this example, the substituted-inactive metal complex has a tridentate (NNN) ligand and a monodentate amine ligand. Monodentate amine ligands are used to bond free carboxylic acid groups (-COOH) to metal complexes. Condensation of a carboxylic acid group on a metal complex with an amine group on an oligonucleotide subunit to form an amide bond-(CONH-) links between these components. Such condensation can be carried out, for example, using carbodiimide as a coupling agent.
    [64] Two pathways for combining hybridization structures are shown in FIG. 12. In one method, the oligonucleotides are coupled (i) with metal complexes and then hybridized (ii) with longer nucleic acid components. In another method, the oligonucleotides are first hybridized (iii) and then coupled (iv) to the metal complex. In a preferred embodiment the oligonucleotide subunit consists of 4 to 20 bases and the metalchloride is a complex of Pt, Pd, Ru, Au or Rh.
    [65] The preferred embodiment for step (2) depends on whether the metal complex-nucleic acid conjugate is in solution or immobilized on the substrate. When in solution, the conjugate can be separated from the unbound metal by some form of chromatography (eg gel filtration or ion exchange) or by precipitation (eg ethanol precipitation of the conjugate). If the conjugate is immobilized, the unbound metal complex may be removed by washing (eg, washing with water or an aqueous salt solution).
    [66] Relatively strong reducing agents may require step (3). Suitable compounds are boron hydrides, in particular borohydride (BH 4 ) salts, Lewis base: borane complexes of the formula L: BH 3 , wherein L is an amine, ether, phosphine or sulfide, hydrazine and derivatives thereof, hydroxylamine and Derivatives thereof, hypophosphite, dithionite salt, formate and H 2 ). Some of these formulations are suitable as gaseous reducing agents for nasal liquid phase treatment.
    [67] The process associated with step (4) is known in the prior art. Briefly, nanometer sized metal particles in the composite serve as catalyst sites for reducing metal ions in solution, which are deposited on nanometer sized particles to enlarge the particles. The deposited metal may be the same or different than the nanometer sized particles. This process can be used to increase the electrical conductivity of the composite or to impart magnetism to the particles.
    [68] The invention will now be described in more detail with reference to the accompanying drawings.
    [69] Two photographs of the Pt (turpy) -metal chloride DNA molecule are shown in FIGS. 3 and 4. 3 shows the presence of a continuous metal film covering an extended segment of DNA. The total thickness of these structures is in most cases 3-6 nm, but there are islands up to about 50 nm thick. 4 is the same sample showing the form of discrete strings of metal particles through an extended segment of DNA. Similar results are obtained when using cis-Pt (NH 3 ) 2 -metalchloride DNA as shown in FIG. 6.
    [70] Nanometer-sized Particle-DNA Complexes via Platinized Sodium Borohydride
    [71] Example 1
    [72] DNA (extracted from bovine thymus, Sigma-Aldrich Product No. D-1501) is dissolved in an aqueous solution containing 0.02 M HEPES / NaOH buffer (pH 7.5). The corresponding concentration of nucleotide base in the solution, calculated by ultraviolet-visible absorption spectrometer, is 80 μΜ. To 2.5 ml of this solution is added 2.5 μL of a 0.020 M solution of dichloro (2,2 ′: 6 ”, 2” -terpyridine) platinum (II) [Sigma-Aldrich Product No. 28, 809-8] in water. . The complex is known to bind DNA in two stages, the fast one involved the insertion of terpyridine (Tupy) ligands and the slow one associated with covalent formation (platinum). Peyratout et al. (1995) ) Inorg. Chem. 34, 4484]. The resulting solution is placed in the dark at room temperature for 24 hours. Subsequently, a cation exchange gel column (Sepadex-scope of Protection C-25, Sigma Aldrich Product No. 27 131-4) was used, using 0.02 M HEPES / NaOH buffer as solvent, to prevent conjugation to DNA. Remove the platinum complex. The ultraviolet-visible absorbance spectrum of the solution after this treatment, shown in FIG. 1, shows that the maximum identified is around 340 nm due to co-terminated platinum ligands and 260 nm mainly due to DNA. By comparing the intensity of absorbance at 340 nm with the value measured prior to ion exchange, it is estimated that 30% of the initial amount of the (Tupy) platinum complex is contained in the (Tupy) Platinum-DNA conjugate.
    [73] Sodium borohydride (2 mg, Sigma-Aldrich Product No. 21, 346-2) is dissolved in 0.02 M HEPES / NaOH buffer (100 μL) and 20 μL of this solution is added to 2.0 ml of (Tupy) Pt-DNA conjugate solution. Is added. The color of the solution changes directly from pale yellow to pale gray, but the solution remains optically clear. The change in ultraviolet-visible absorption spectrum, obtained after 30 minutes, is consistent with the formation of colloidal platinum (FIG. 1). The pH of the solution is 7.8.
    [74] Example 2
    [75] Basically using the same procedure as in Example 1, except that 0.013M of cis-diaminedichloroplatinum (II) ["cisplatin", Sigma-Aldrich Product No. P-4394] in 67% water-33% dimethylsulfoxide 3.8 μL of solution is used instead and only 2.5 hours are left before the (diamine) Pt-DNA conjugate is separated by cation exchange. Cisplatin is known to covalently bind to DNA, forming an adduct in the bifunctional strand between N-7 atoms of the surrounding G-G pair or G-A pair (Kelland (2000) Drugs 59 Suppl. 4,1).
    [76] Gold Enhancement RAtomic force microscope measurements before and after treatment
    [77] Example 3
    [78] Abrasive surface of a piece of silicon wiper (for semiconductor, p-type, boron doped, native surface oxide) is treated with O 2 -plasma (Gala Instruments PlasmaPrep-5) for 4 minutes (0.4 mbar, approximately 33 Watts, low power) ). The treated wiper is then mounted on a spin-coating machine (Mikasa Spin-Coater 1H-D3). A few drops of the solution of the Pt-DNA complex obtained in Example 1 are applied to the substrate. After 2 minutes, the sample is spun at 1000 rpm for 10 seconds, then at 5000 rpm for 90 seconds. Two drops of water are added dropwise onto the sample during the second rotation step to remove salt. The sample is investigated by tapping mode AFM (Digital Device, Multimode Nuclear Microscopy) using a silicon nitride cantilever (Olympus Optical, MicroCantilever OMCL-AC160TS-W, about 250 kHz resonance frequency, about 25 N / m spring constant). An image (eg, shown in FIG. 2) represents an extended segment of DNA without indicating the presence of platinum particles at all.
    [79] Example 4
    [80] A solution of Goldenhance R (Nanoprobes, catalog number 2113) is applied to the substrate surface from Example 3 for 10 minutes, then the surface is washed with water and dried with air vapor. Two AFM images of this sample are shown in FIGS. 3 and 4. 3 shows the presence of a continuous metal film covering an extended segment of DNA. The total thickness of these structures is generally 3-6 nm, but there are also islands that reach about 50 nm in thickness. 4 is an image of another area for the same sample showing discrete strings of metal particles along an extended segment of DNA. Although the total thickness of these structures is 2-6 nm, some islands may be up to about 50 nm thick. This is evident from images in which some segments of DNA are not metallized. Both images indicate that there are no metal deposits on the surface of the silicon substrate, ie the metallization is mainly limited to DNA.
    [81] Example 5
    [82] Another silicon wiper was prepared as in Example 3 using a Pt-DNA complex solution from Example 2. AFM images (shown in FIG. 5) also show only extended segments of DNA with no signs of platinum particles present.
    [83] Example 6
    [84] The sample of Example 5 is treated with a GoldEnhanced R solution as described in Example 4. The AFM image obtained after this treatment is shown in FIG. 6. Similar to FIG. 4, this image shows that the metal particles form a discontinuous string form along an extended segment of DNA having a total thickness of 2 to 6 nm with nonmetallized segments having a thickness of 0.7 to 0.9 nm. The silicon wiper surface is essentially free of metal deposits.
    [85] Example 7
    [86] Unmodified ct-DNA is immobilized and dried on a silicon substrate as described in Example 3. This is then treated with GoldEnhanced R solution for 15 minutes. AFM images such as those shown in FIGS. 13 and 14 show relatively large particles on the surface, but no particles are observed on the DNA itself. These results show that the particles biased in the DNA shown in FIGS. 3, 4 and 6 require platinum.
    [87] The present invention provides an improved method for direct and selective metallization of nucleic acids through nanometer sized metal particles produced in situ, and nanometer sized metal particle-nucleic acid complexes produced by such improved methods. Can be effectively used to form nanometer-sized wires in electronic networks and circuits that enable high density arrangements.
    权利要求:
    Claims (23)
    [1" claim-type="Currently amended] Reacting the nucleic acid specific metal complex with the nucleic acid to produce a metal complex-nucleic acid conjugate,
    Removing unconjugated metal complexes and / or unconjugated by-products, and
    Reacting the metal complex-nucleic acid conjugate with a reducing agent to produce a nanometer-sized metal particle-nucleic acid composite.
    [2" claim-type="Currently amended] The method of claim 1, wherein the nucleic acid component reacts in a dissolved state, immobilized on a substrate, or in a semisolid state (eg, a gel).
    [3" claim-type="Currently amended] 3. The polynucleotide of claim 1, wherein the nucleic acid is DNA, RNA, PNA, CNA, oligonucleotide, oligonucleotide of DNA, oligonucleotide of RNA, primer, A-DNA, B-DNA, Z-DNA, poly of DNA. Nucleotides, polynucleotides of RNA, T-junctions of nucleic acids, triple nucleic acids, quadruple nucleic acids, domains of non-nucleic acid polymer-nucleic acid block copolymers, and mixtures thereof.
    [4" claim-type="Currently amended] The method of any one of claims 1 to 3, wherein the nucleic acid is double helical or single helical.
    [5" claim-type="Currently amended] 5. The method of claim 1, wherein the metal complex-nucleic acid conjugate is formed by metallization and / or interactive ligand binding. 6.
    [6" claim-type="Currently amended] The method according to any one of claims 1 to 5, wherein the specific base of the nucleic acid is metalchloride.
    [7" claim-type="Currently amended] The nucleic acid specific metal complex according to any one of claims 1 to 6, wherein the nucleic acid specific metal complex is dichloro (2,2 ': 6', 2 "-terpyridine) platinum (II), cis-diaminodichloroplatinum (II). And a metal complex with a bound or joined nucleic acid interacting group, such as an insertion agent, groove binder and alkylating agent.
    [8" claim-type="Currently amended] 8. The metal complex-nucleic acid conjugate according to claim 1, wherein the metal complex-nucleic acid conjugate is separated from the non-conjugated metal complex and / or unconjugated by-products, eg, by gel filtration or ion exchange, precipitation ( Eg ethanol precipitation) or washing (eg washing with water or an aqueous salt solution).
    [9" claim-type="Currently amended] 8. The metal complex-nucleic acid conjugate according to claim 1, wherein the metal complex-nucleic acid conjugate is selected from boron hydride, borohydride salts, and Lewis base: borane complexes of the formula L: BH 3 , wherein L is an amine, an ether, Phosphine or sulfides, hydrazines and derivatives, hydroxylamines and derivatives, hypophosphites, formate salts, dithionite salts and H 2 ), Way.
    [10" claim-type="Currently amended] 10. The method of claim 9, wherein the reducing agent is used in the form of a gaseous reducing agent.
    [11" claim-type="Currently amended] The method of claim 1, wherein the nanometer-sized metal particles comprise Fe, Co, Ni, Cu, Ru, Rh, Pd, Ag, Os, Ir, Pt, Au and mixtures of these metals ( For example, an alloy).
    [12" claim-type="Currently amended] The method of claim 1, wherein the nanometer sized metal particles are catalytically active against electroless metallization.
    [13" claim-type="Currently amended] The method of claim 1, wherein the nanometer-sized metal particles cannot be visualized by atomic force microscopy and / or the diameter of the nanometer-sized metal particles is less than 3 nm.
    [14" claim-type="Currently amended] 14. The method of any one of claims 1 to 13, further comprising enlarging the nanometer sized metal particles by treating the nanometer sized metal particles in the nanometer sized metal particle-nucleic acid composite with an electroless plating solution. Including as.
    [15" claim-type="Currently amended] The method of claim 14, wherein the metal complex-nucleic acid complex reacts in a dissolved state, immobilized on a substrate, or in a semisolid state (eg, a gel).
    [16" claim-type="Currently amended] The method of claim 14 or 15, wherein the nanometer sized metal particles are Fe, Co, Ni, Cu, Ru, Rh, Pd, Os, Ir, Ag, Pt, Au and mixtures of these metals (e.g. alloys). And electroless plating solution comprising at least one metal selected from the group consisting of:
    [17" claim-type="Currently amended] The method according to claim 14 or 15, wherein the nanometer sized metal particles are magnetic and / or magnetized Fe, Co, Ni, mixtures of these metals (e.g. alloys) and mixtures of these metals with B or P (e.g. An electroless plating solution comprising at least one metal selected from the group consisting of: alloys).
    [18" claim-type="Currently amended] Nanometer-sized metal particle-nucleic acid composite, obtainable according to the method according to any one of claims 1 to 13.
    [19" claim-type="Currently amended] The method of claim 18, wherein the nanometer sized metal particles are less than 3 nm in diameter and / or cannot be visualized by atomic force microscopy.
    [20" claim-type="Currently amended] The nanometer-sized particles are grown, preferably by providing a nanometer-sized metal particle-nucleic acid composite according to claim 18 or 19 and by electroless deposition of the metals according to claims 16 or 17. Controlled growth. A method of making nanometer sized wires.
    [21" claim-type="Currently amended] A linear arrangement of nanometer sized metal particles or nanometer sized wires obtainable according to the method of claim 20.
    [22" claim-type="Currently amended] Small network or electronic circuit comprising at least one nanometer sized wire according to claim 21.
    [23" claim-type="Currently amended] Use of a method according to any one of claims 1 to 17 for selectively metallizing nucleic acids.
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    同族专利:
    公开号 | 公开日
    CN1356336A|2002-07-03|
    CN100436469C|2008-11-26|
    EP1209695A1|2002-05-29|
    US8227582B2|2012-07-24|
    US20020065242A1|2002-05-30|
    JP2002371094A|2002-12-26|
    EP1209695B1|2004-10-06|
    DE60014678D1|2004-11-11|
    DE60014678T2|2005-10-13|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    法律状态:
    2000-11-24|Priority to EP00125823.5
    2000-11-24|Priority to EP20000125823
    2001-11-24|Application filed by 소니 인터내셔널(유로파) 게엠베하
    2002-05-30|Publication of KR20020040650A
    优先权:
    申请号 | 申请日 | 专利标题
    EP00125823.5|2000-11-24|
    EP20000125823|EP1209695B1|2000-11-24|2000-11-24|Selective metallisation of nucleic acids via metal nanoparticles produced in-situ|
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