WO2008035787A1 - Procede de revetement de polymere charge au moyen de metal - Google Patents

Procede de revetement de polymere charge au moyen de metal Download PDF

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Publication number
WO2008035787A1
WO2008035787A1 PCT/JP2007/068467 JP2007068467W WO2008035787A1 WO 2008035787 A1 WO2008035787 A1 WO 2008035787A1 JP 2007068467 W JP2007068467 W JP 2007068467W WO 2008035787 A1 WO2008035787 A1 WO 2008035787A1
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Prior art keywords
polymer
dna
metal
state
color tone
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PCT/JP2007/068467
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English (en)
Japanese (ja)
Inventor
Anatoly Zinchenko
Ning Chen
Kenichi Yoshikawa
Koji Kubo
Damien Baigl
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Japan Science And Technology Agency
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Priority to JP2008514266A priority Critical patent/JPWO2008035787A1/ja
Publication of WO2008035787A1 publication Critical patent/WO2008035787A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means

Definitions

  • the present invention relates to a coating method of a polymer (in particular, a charged polymer such as DNA). More specifically, the present invention relates to a technique that enables a macroscopic observation of the higher-order structure (for example, conformation) and topology of a polymer (in particular, a charged polymer such as DNA) with the naked eye. By using the present invention, the coil globule phase transition of DNA can be observed with the naked eye.
  • Non-Patent Document 1 Non-Patent Document 2; Non-Patent Document 3; Non-Patent Document 4 and Non-Patent Document 4
  • Patent document 5 DNA strand templates are rarely used in such processes. Even recent research on DNA metallization is limited to short DNA fragments of less than a few kilobase pairs! /, DNA fragments (Non-Patent Document 6; Non-Patent Document 7; Non-Patent Document 8) .
  • Non-Patent Document 9 the ability to use short DNA oligomers to control the aggregation of metal nanostructures has been developed by Mirkin and co-workers (Non-Patent Document 9; Non-Patent Document 10). They also provide several examples using gold nanoparticles conjugated with oligonucleotides for analytical and biomedical purposes (Non-Patent Document 11; Non-Patent Document 12; Non-Patent Document 13). ). In fact, in view of the outstanding optical and electronic properties of nano-sized metal structures (Non-Patent Document 14), it is very useful in analytical protocol applications and in the production of new and superior materials. It can be said that it is a promising candidate substance.
  • Non-Patent Document 15 Non-Patent Document 16; Non-Patent Document 17
  • Non-Patent Document 18 Non-Patent Document 19; Non-Patent Document 20; Non-Patent Document 21; Non-patent document 22; Non-patent document 23; Non-patent document 24; Non-patent document 25; Non-patent document 19
  • DNA is folded!
  • the metal nanoparticles are coated on the DNA skifold by metallization of metals such as gold (Au), copper (Cu) and platinum (Pt). Formed (Non-patent document 26; Non-patent document 27; Non-patent document 28; Non-patent document 29; Non-patent document 30; Non-patent document 31; Non-patent document 32).
  • metals such as gold (Au), copper (Cu) and platinum (Pt).
  • Au gold
  • Cu copper
  • Pt platinum
  • Formed Non-patent document 26; Non-patent document 27; Non-patent document 28; Non-patent document 29; Non-patent document 30; Non-patent document 31; Non-patent document 32.
  • Such a metal coating is used to produce molecular nanowires. In general, it has been generally confirmed that this is a promising method in the future! (Non-Patent Document 19 and Non-Patent Document 33). It is highly desirable that metallization of the DNA strand template be applicable in addition to the preparation of
  • Non-patent Document 34 DNA strands larger than a few tens of base pairs (bp) undergo a very discontinuous transition from a disordered coiled state to an ordered compact state. , Shorter than the order of a few kilobase pairs (kbp)! /, Not seen in DNA molecules! /, (Non-Patent Document 35). Thus, the transitions in the folding of large DNA molecules are different from short DNA. In recent years, when using large DNA longer than lOOkbp, it has been demonstrated that the metal coating of a compact DNA strand proceeds through a process different from that of a linear metal coating (Non-patent Document 36).
  • Non-Patent Literature 1 Sun Y., Xia Y., Science 2002, 298, 2176-2179
  • Non-Patent Literature 2 Corbierre K. M., Cameron S. N., Lennox R. B., Langm uir 2004, 20, 2867— 2873
  • Non-Patent Document 4 Perumble N., Mingotaud A— F., Marty J— D., Rico— Lattes I., Mingotaud C., Chem. Mater. 2004, 16, 4856— 4858
  • Non-Patent Document 6 Mirkin CA, Letsinger RL, Mucic RC, Storhoff JJ, Nature 1996, 382, 607—609
  • Non-Patent Document 7 Storhoff JJ, Mirkin CA, Chem. Rev. 1999, 99, 1849 -1862
  • Non-Patent Document 8 Claridge S. A., Goh S. L., Frechet J. M.J., Williams S. C., Micheel C. M., Alivisatos A. P., Chem. Mater. 2005, 17, 1628— 1635
  • Non-Patent Document 9 Mirkin C. A., Letsinger R. L., Mucic R. C., Storhoff J. J., Nature 1996, 382, 607—609
  • Non-Patent Literature 10 Storhoff J. J., Mirkin C. A., Chem. Rev. 1999, 99, 1849 -1862.
  • Non-Patent Document 11 Shaikh KA, Ryu KS, Goluch ED, Nam J., Liu J., Thaxton CS, Thomas N., Barron AE, Lu Y., Mirkin CA, Liu C .. Proc. Natl. Acad. Sci USA 2005, 102 (28), 9745— 9750.
  • Non-Patent Document 12 Nam J., Thaxton CS, Mirkin CA, Science 2003, 3 01 (5641), 1884-1886
  • Non-Patent Document 13 Han MS, Lytton-Jean AKR, Oh B—K., Heo J., Mirkin CA, Angew. Chem. Intl. Ed. 2006, 45, 1807-1810
  • Non-Patent Document 14 Schmid G. ( Ed.) Nanoparticles: From Theory to Appli cations, VCH, Weinheim, 2004
  • Non-Patent Document 15 Kiriy A., Minko S., Gorodyska G., Stamm M., Nano Lett. 2002, 2, 881— 885
  • Non-Patent Document 16 Minko S., Kiriy A., Gorodyska G., Stamm M., J. Am. Chem. Soc. 2002, 124, 10192-10197
  • Non-Patent Document 17 Patolsky F., Weizmann Y., Lioubashevski O., Willner I., Angew. Chem. Int. Ed. 2002, 41, 2323— 2327
  • Non-Patent Document 18 Wang G., Murray RW, Nano Lett. 2004, 4, 95-101
  • Non-Patent Document 19 Nyamjav D., Kinsella JM, Ivanisevic A., Appl. Phys. Lett. 2005, 86, 093— 107
  • Non-Patent Document 20 Niemeyer CM, Simon U., Eur. J. Inorg. Chem. 2005, 18, 3641-3655
  • Non-Patent Document 21 Becerril H. A., Stoltenberg R. M., Monson C. F., Woolley A. T., Mater. Chem. 2004, 14, 611-616
  • Non-Patent Document 22 Ford W. E., Harnack O., Yasuda A., Wessels J. M., A dv. Mater. 2001, 13, 1793— 1797.
  • Non-Patent Document 23 Richter J., Mertig M., Pompe W., Monch I., Schacker t H. K., Appl. Phys. Lett. 2001, 78, 536—538
  • Non-Patent Document 24 Braun G., Inagaki K., Estabrook R. A., Wood D. K., Levy E., Cleland A. N., Strouse G. F., Reich N. O., Langmuir 200 5, 21, 10699-10701.
  • Non-Patent Document 25 Mertig M., Ciacchi L. C., Seidel R., Pompe W., Vita A. D., Nano Lett., 2002, 2, 841—844
  • Non-Patent Document 26 Nakao H., Shiigi H., Yamamoto Y., Tokonami S., Na gaoka T., Sugiyama S., Ohtani T., Nano Lett. 2003, 3, 1391-139 4.
  • Non-Patent Document 27 Karen K., Berman R., Braun E., Nano Lett. 2004, 4, 323-326.
  • Non-Patent Document 28 Monson C. F., Wooley A. T., Nano Lett. 2003, 3, 359 -363
  • Non-Patent Document 29 Xin H., Wooley A. T., J. Am. Chem. Soc. 2003, 125, 87 10-8711.
  • Non-Patent Document 30 Harnack O., Ford W. E., Yasuda A., Wessels J. M., Nano Lett. 2002, 2, 919— 923.
  • Non-Patent Document 31 Mertig M., Ciacchi L., Siedel R., Pompe W., Vita A. D., Nano Lett. 2002, 2, 841—844.
  • Non-Patent Document 32 Ma Y., Zhang J., Zhang G., He H., J. Am. Chem. Soc. 2004, 126, 7097-7101
  • Non-Patent Document 33 Richter J., Seidel R., Kirsch R., Mertig M., Pompe W., Plaschke J., Schackert KH, Adv. Mater
  • Non-Patent Document 34 Wei G., Zhou H., Liu Z., Song Y., Wang L., Sun L
  • Non-Patent Document 35 Yoshikawa K., Takahashi S., Whyvskaya V. V., Kho khlov A. R., Phys. Rev. Lett. 1996, 76, 3029-3031
  • Non-Patent Document 36 Zinchenko A. A., Yoshikawa K., Baigl D., Adv. Mater
  • An object of the present invention is to provide a method by which a higher-order structure of a polymer can be easily observed with the naked eye.
  • the present invention allows a polymer to form a complex with a metal and can be used to determine the higher order structure of the complex.
  • High-order structures (such as conformation and topology) can be observed with the naked eye.
  • the coil globule phase transition of DNA can be observed with the naked eye.
  • the present invention has the following advantages, for example.
  • a giant DNA molecule that does not introduce a phase transition is manipulated in an aqueous solution, the DNA molecule is cleaved by the shear stress associated with the vortex generated in the aqueous solution. Therefore, in the conventional method, huge DNA molecules were embedded in agarose genome and all treatments were performed in agarose gel. It has become possible to determine with the naked eye whether such cuts can be prevented.
  • the present invention demonstrates that compact aggregates of DNA serve as torus-like templates for forming silver nanorings during the metal deposition process in silver. Therefore, in the present invention, is the difference in the metal deposition process in silver useful for easily monitoring the conformational change of the DNA strand and for determining the final shape of the DNA aggregate? Demonstrate whether to judge. In addition, the present invention also demonstrates that this approach provides a convenient way to monitor other related processes (eg, protein conformational changes during the folding process).
  • the present invention also provides an analytical method for examining DNA conformation and detecting transition points where DNA folding changes through the precipitation of silver and gold on the DNA strand skifold. Demonstrate that
  • the present invention provides the following.
  • a composite of a polymer and a metal (1) A composite of a polymer and a metal.
  • the complex includes DNA phase transition, charged polymer phase transition, protein folding and Item 10.
  • the complex according to any one of Items 1 to 9, which is for observing a state selected from the group consisting of DNA and / or protein aggregation.
  • the macromolecule is DNA having a size of lOkbp or more. ; The method according to any one of 15 to 15.
  • the above method is for observing a state selected from the group consisting of DNA phase transition, charged polymer phase transition, protein folding, and DNA and protein aggregation. 18. The method according to any one of 18.
  • the calculation includes determining whether it is symmetric, and the presence of two peaks in the wavelength curve indicates that the polymer is in a symmetric compaction state.
  • a term characterized by being determined! The method according to any one of! To 21.
  • the polymer is DNA, and the calculation includes determining whether it is a torus-like force, and the presence of two peaks in the wavelength curve is A term characterized in that the polymer is determined to be in a symmetric compaction state; ; The method according to any one of 22 to 22 above.
  • the calculation includes determining whether it is a torus shape, and the determination of the torus force is whether two peaks in the wavelength curve.
  • Existence is characterized in that the macromolecule is determined to be in a symmetric compaction state; ;! ⁇ 23! /, The method described in any way.
  • the step D) includes a step of determining the size of the compaction state of the polymer, and the determination is performed using a solution of a metal polymer composite of a known size as a control. Which is achieved by constructing and extrapolating the data of the polymer complex to be measured to the equilibration curve; ; The method according to any one of! To 24.
  • the calculation of the higher-order structure from the color tone includes a step of calculating the concentration at which the binder causes a phase transition of the compaction coil of the DNA. ;! The method described in any of 25.
  • step (C) the reducing agent is added to the mixed state of the metal ion and the polymer. ! ⁇ 26! /, The method described in any one of the above.
  • step C) further includes a step of irradiating with ultraviolet light in the presence of an alcohol; ; The method according to any of 27.
  • the irradiation with the ultraviolet light is performed for a time sufficient to induce the formation of the complex. ;
  • the higher-order structure includes a topology, and the topology is determined by performing a spectroscopic analysis of the color tone; ;
  • E) further comprising the step of determining a critical concentration of the binder that induces the polymer phase transition; ; The method according to any of 30.
  • the calculation includes determining whether or not it is symmetric, and the presence of two peaks in the wavelength curve indicates that the polymer is in a symmetric compaction state.
  • Item 43 The kit according to any one of Items 32 to 42, wherein the kit is determined to be present.
  • the polymer is DNA, and the calculation includes determining whether it is a torus-like force, and the presence of two peaks in the wavelength curve is Item 44.
  • the calculation includes determining whether it is a torus shape, and the determination of the torus force is whether the two peaks in the wavelength curve.
  • 45. The kit according to any one of Items 32 to 44, wherein presence is determined that the polymer is in a symmetric compaction state.
  • the means includes a means for determining the size of the compaction state of the polymer, and the determination is performed by creating and measuring an equivalence curve using a solution of a metal polymer composite of a known size as a control.
  • Item 46 The kit according to any one of Items 32 to 45, wherein the kit is achieved by extrapolating data of the polymer complex as a target to the equilibration curve.
  • the ultraviolet ray includes the ultraviolet ray having a wavelength of 254 nm, according to any one of items 32 to 49. Kit of the listed.
  • the polymer is charged and longer than the sustained length of the polymer
  • the metal is a noble metal or a metal having a charge equivalent to gold or silver
  • the composite includes a binder, and the polymer and the metal are bound by the binder.
  • ⁇ 4> The composite according to Item 1, wherein the binder includes at least one substance selected from the group consisting of a polyvalent cation and a surfactant.
  • composition for observing a higher-order structure of the polymer including the complex according to item 1. object.
  • composition according to item 7 wherein the complex is for observing a state selected from the group consisting of phase transition, folding and aggregation of the polymer.
  • composition according to item 7 wherein the higher-order structure is observed in order to determine a medical state, a biological state, a chemical state, or a physical state associated with the polymer.
  • the polymer is charged and longer than the sustained length of the polymer
  • the metal is a noble metal or a metal having a charge equivalent to gold or silver.
  • the higher-order structure calculation from the color tone indicates that when a color tone peculiar to the metal is observed, it is determined that the polymer is in a coil state, and when a color tone of a longer wavelength is observed, Item 10.
  • the higher-order structure calculation includes determining whether or not it is symmetric, and the presence of two peaks in the wavelength curve obtained from the color tone It is determined that the polymer is in a symmetric compaction state.
  • the macromolecule is DNA
  • the calculation includes determining whether the DNA force S torus-like force and two peaks in the wavelength curve.
  • Item 11 The method according to Item 10, wherein the presence of is determined that the polymer is in a symmetric compaction state.
  • D> includes the step of determining the size of the compaction state of the polymer, and the determination creates an equivalence curve using a solution of a metal polymer composite of a known size as a control.
  • Item 11 The method according to Item 10, which is achieved by extrapolating data of the polymer complex to be measured to the equilibration curve.
  • the calculation of the higher-order structure from the color tone includes a step of calculating a concentration at which the binder causes a phase transition of the compaction coil of the DNA.
  • the step D> further comprises a step of irradiating ultraviolet light in the presence of alcohol.
  • ⁇ 20> The method according to Item 10, further comprising: F> determining a critical concentration of the binder that induces the polymer phase transition.
  • the polymer is charged and longer than the sustained length of the polymer
  • the metal is a noble metal or a metal having a charge equivalent to gold or silver.
  • the composite includes a binder, and the polymer and the metal are bound by the binder.
  • kit ⁇ 22> The kit according to Item 21, wherein the reducing agent is sodium borohydride.
  • a drug for coloring, decoloring, or color modification comprising the complex according to item 1.
  • the polymer is charged and longer than the sustained length of the polymer
  • the metal is a noble metal or a metal having a charge equivalent to gold or silver.
  • the composite includes a binder, and the polymer and the metal are bound by the binder.
  • a method for easily and visually and colorimetrically detecting a nanostructure and a microstructure of a polygram electrolyte (DNA) of picogram order using a metal coating with a noble metal using a metal coating with a noble metal. Differences in the precipitation properties of precious metals on DNA templates due to various concentrations of binders such as spermine S, can be observed even clearly with the naked eye.
  • a single molecule can generally measure more than one unit of 100 monomers.
  • DNA phase transition, charged polymer phase transition, protein folding, DNA and protein aggregation, etc. can generally be observed.
  • the force of observing the structural change (ie, suspicion) of the compaction state from the actual polymer chain is used.
  • FIG. 1 shows the results of fluorescence observation of DNA molecules in an aqueous solution of Balta when spermine (4 +) was applied at four different concentrations.
  • the concentrations of spermine are 0 M (A), 0.4 ⁇ ( ⁇ ), and 2 ⁇ 5 ⁇ (ji), respectively.
  • the existence of coexistence means that this transition is a phenomenon of “all or no power” at the level of individual DNA molecules.
  • Figure 2 shows that a portion of a compact ⁇ 4 DNA molecule (1 H ⁇ , depending on the spermine concentration, determined by fluorescence microscopy analysis of the DNA's conformation for at least 100 DNA at each concentration. ).
  • Fig. 3 shows different concentrations (numbers 1 to 9 in the figure: 0 ⁇ ⁇ , 0 ⁇ 1 1, 0.2 2, 0 • 4 ⁇ , 0 ⁇ 5 ⁇ , 1 ⁇ , 1 ⁇ Gold with 100 M AgNO and 40 ⁇ NaBH in the presence of spermine (corresponding to 5 ⁇ , 2 ⁇ 0 ⁇ and 2 ⁇ 5 ⁇ )
  • FIG. 4 shows a schematic diagram of silver deposition in a folded DNA template (A) and a compact DNA template (B).
  • FIG. 5 is subjected to compaction by multivalent cations and multivalent electrolyte, it shows an image with a transmission electron microscope T4 DNA (10_ 6 M) in the different stages of their con Park Si-yeon.
  • a and B are compacted by spermine (20 ⁇ M) and NaBH
  • A. C is metallized with HAuCl (40 a M) and NaBH (40 ⁇ ⁇ )
  • D, E and F are compacted by spenoremin (5 ⁇ M) and metallized with HAuCl (40 ⁇ ⁇ ) with reduction treatment with NaBH (40 ⁇ ⁇ ).
  • G and ⁇ are the poly (L-lysine) (10- 6 ⁇ ) is induced compaction, metal object by NaBH (40, ⁇ M) HAuCl (20, ⁇ M) with reduction treatment by
  • the scale bar is 100 ⁇ m apart from showing 500 nm in G.
  • Figure 6 shows T4 metallized with NaBH (100 ⁇ M) and HAuCl (100 ⁇ M).
  • Fig. 7 shows different concentrations (numbers 1-9 in the figure: 0 ⁇ ⁇ , 0 ⁇ 1 ⁇ , 0.2 ⁇ , 0.4 ⁇ , 0.5 ⁇ , 1.0 ⁇ , respectively) , 1.5 ⁇ , 2.0 ⁇ and 2.5 ⁇ ) in the presence of 100M HAuCl and 100M NaBH
  • Figure 8 shows T4 metallized with NaBH (100 ⁇ M) and HAuCl (100 ⁇ M).
  • FIG. 9 shows the results of observation with a fluorescence microscope of the evolution of the conformational state of a single DNA molecule that interacts with poly (L-lysine).
  • Poly (L lysine) concentrations are 0 ⁇ ( ⁇ ), 0.4 4 ( ⁇ ), 0.8 ⁇ M (C), 1.4 M (D) and 3 ⁇ M (E), respectively.
  • A is the coil state
  • B and C are the contracted coil state
  • D is the globule state
  • E is the state where the expanded globules coexist.
  • FIG. 10 shows the average long axis length of T4 DNA molecule versus poly (L-lysine) concentration.
  • Statistical error was given as standard deviation.
  • FIG. 11 shows T4 DNA (1 M) in ImM Tris—HCl and various amounts of splenoremin (numbers;! To 8) (0 ⁇ , 0 ⁇ 4 ⁇ , 0 ⁇ 8M, 1M, 1 ⁇ 4M, 2M, 3M and 4M) with 100M AgNO and 40M
  • label refers to a means for imparting characteristics different from those of other substances in order to identify the substance.
  • labeled substance refers to a substance. A substance that gives it different characteristics for identification purposes.
  • polymer refers to any molecule having a large molecular weight, preferably charged, and preferably having a size of at least lOkbp, more preferably at least lOOkbp.
  • High molecular substances include those with linear molecules (linear polymers, such as high-density polyethylene), those with branched structures (such as low-density polyethylene), and three-dimensionally. There are reticulated ones (reticulated polymer, vulcanized rubber, phenol resin, etc.).
  • Specific polymers in the present invention include, for example, proteins, polypeptides, nucleic acids (for example, DNA, RNA, PNA, etc.), polysaccharides, glass, silica gel, polypropylene, polyurethane, polystyrene (PS), polyoxyethylene.
  • Examples include copolymers, poly (vinyl chloride), poly (vinylidene chloride), poly (vinyl acetate), poly (bull alcohol), polyimides, polyamides, polyethylene glycols, poly (acrylic acid), poly (methacrylic acid) and copolymers thereof, and complex molecules thereof. Can be on them Not a constant.
  • charged polymer or “charged polymer” refers to a polymer that can be ionized when dissolved in a liquid, or a polymer that is in such an ionized state. Point to. In the present invention, a polymer is regarded as a “charged polymer” when it is essentially inevitable to take an ionized state regardless of whether it is temporary or throughout the technical process. Or called “charged polymer”.
  • free solution electrophoresis procedures include:
  • Step 1 Fill the electrophoresis bowl with the solution that the polymer diffuses!
  • Step 3 Confirmation of whether the polymer migrates
  • the target polymer is charged by That is, whether the polymer is a charged polymer or not. In other words, if the polymer is not electrically neutral, it is a charged polymer. In the present invention, only the presence or absence of a charged state is important in carrying out the implementation, regardless of the amount of charge of the target polymer.
  • the target system is the Milliliter Nolesquenore (ml) scale and in the world of the Microlit Nore ( ⁇ 1) to the Nanolit Nore (nl) scale, ⁇ ⁇ ! ! Since the effect of thermal fluctuation is more effective in the scale, the effect given to the whole by the difference of several base pairs can be completely ignored in the physical property theory to be discussed in the present invention.
  • the charged polymer can also be identified by the following method.
  • Step 1 Correct the solution in which the charged polymer is diffusing to an appropriate concentration (generally several ⁇ ) (the solution means a material in which the already charged polymer is diffused, such as pure water) )
  • Process 2 Take 50 1 in a dedicated cell and measure with Zetasizer 1 (Malvern).
  • the composition for example, adenine, guanine, cytosine, thymine in the case of deoxyribonucleic acid
  • a macromolecule eg, deoxyribonucleic acid
  • the base composition in the case of DNA
  • the influence on the physical properties of the polymer as a whole is not affected in principle in the practice of the present invention.
  • the “string theory” is introduced, so that the general theory of physical properties is not dependent on the base composition. It is understood that the physical properties of a polymer having a size are generally equivalent.
  • RNA-DNA has a negative charge when macroscopically looking over the entire polymer. And, if somewhere somewhere in a different nature, if you exceed several hundred base pairs, they will be canceled. In the present invention, it has an important meaning that the polymer as a whole has a charge. No change to the basic principle of transition! /.
  • Examples of the charged polymer include, but are not limited to, DNA, RNA, and protein.
  • Examples of macromolecules of lOOkb or more include, but are not limited to, genomic DNA, phage DNA, chromosome, and the like.
  • Polymers such as lOOkb or more, such as DNA undergo a very discontinuous transition from a disordered coiled state to an ordered and compact state, and the effect of the phase transition affects the living body.
  • the power to do is to start.
  • the following are known effects of phase transitions on DNA. Large DNA is likely to be fragmented by shear stress generated when a globule solution is mixed with DNA in the solution. It is known that when a large DNA is embedded in a gel and undergoes a phase transition to a globule structure, the activity of the gel-degrading enzyme falls due to the effect of the globule solution, and the gel is not sufficiently degraded.
  • Persistence length is an index that determines the statistical properties of a polymer, and does not make much sense when the persistence length is longer than the total length of the molecule. Therefore, use a sufficiently long polymer to measure the persistence length.
  • a quantity called Khun length is also known as something similar to duration. The Khun length is defined differently from the duration, and its value is approximately twice the duration. In general, the persistence length and the Khun length are often used without being clearly distinguished. In this specification, the term “continuation length” is used. Shall be included.
  • Measure the sustained length as defined, for example, fix the position and angle of the polymer chain tip in an aqueous solution, and place a continuous marker at a distance from the tip on the polymer chain.
  • a method for experimentally determining the persistence length is, for example, by adsorbing a polymer chain once broken into a solution onto a smooth flat substrate, observing its structure with a high-resolution microscope such as AFM, and then using the molecular chain. There is a method of directly measuring the angular correlation above.
  • the most accurate way to determine the persistence length is to measure the angular correlation as described above, but in many practical cases and in the context of the present invention, a precise value of the persistence length is not necessarily required. In such a case, the following suboptimal means are often used.
  • Polymer chains that are significantly longer than the last length have rubber elasticity, and this elastic force depends on both the last length and the total length, so methods such as attaching beads to both ends of the polymer chain and pulling them with laser tweezers.
  • the duration can be determined. Based on the same principle, the average length of polymer chains is measured from scattering and direct observation, and the duration is determined from it.
  • a complex structure is a structure having a group of structures represented by several or more types of continuous parameters behind it.
  • it is a structure created on the basis of continuous contact and association within the molecular chain. More specifically, for example, a force that is a molecule having a structure spreading in the radial direction, a hollow (doughnut-like) structure, or a continuous structure thereof is not limited thereto.
  • the three-dimensional structure of a nanostructure that can take such a complicated structure can be visually determined. There is an effect.
  • the visualization of DNA nanostructures of the present invention is also useful for studies on the self-generation of DNA nanostructures that take on the characteristics of such long-chain DNA.
  • DNA of eukaryotes such as humans forms a complex with histone protein and is folded into the cell as chromatin.
  • histones an artificial biomimetic chromatin system using cationic synthetic nanoparticles with various sizes and charges has been constructed and researched.
  • This DNA-nanoparticle system has been shown to share many common properties with the actual chromatin in cells ("Compaction of Single-Chain DNA by Histone- Inspired Nanoparticles, AA Zinchenko, K. Yoshikawa , and D. Baigl, Phys. Rev.
  • DNA molecules with a length of several tens of kilobase pairs or more are tightly folded from the expanded coil state according to changes in the concentration of various condensing agents including polyvalent cations and neutral polymers. Transition to a globule state. This transition behavior has been shown to be discontinuous without going through an intermediate state.
  • DNA is also responsible for the genetic information of organisms, and the effect of such structural transitions on the expression of genes carved on DNA is interesting from a biological standpoint.
  • the present inventors simultaneously observed DNA and RNA transcribed in a cage shape at the single molecule level using a fluorescence microscope, and the structural transition of single-molecule long-chain DNA was observed. It was clarified that it causes on / off switching of transcriptional activity.
  • coiled DNA is stretched after the transcription reaction, RNA is synthesized on the DNA molecule.
  • the right is a globule state, and RNA is not seen.
  • observation of the phase transition of coinore globules is also important from a biological point of view.
  • the "higher order structure" or three-dimensional structure of a polymer means a three-dimensional space of the whole molecule or its aggregate in a polymer, particularly a biopolymer such as a protein or nucleic acid.
  • a structure eg, conformation
  • the sequence of amino acids and nucleotides in a polymer chain that is, the chemical structure based on a covalent bond is called a primary structure.
  • a local ⁇ -helix formed by hydrogen bonding or a ( The structure and secondary structure are fundamental, and one molecule created through hydrogen bonding, ionic bonding, hydrophobic interaction, etc.
  • the three-dimensional spatial structure of the whole molecule is tertiary structure, and molecules with tertiary structure like hemoglobin
  • the structure in which several particles are aggregated into one particle is called a quaternary structure, and the secondary and higher are combined into a higher order structure!
  • a structure governed by intermolecular forces is called a higher order structure, and it is understood that the higher order structure and higher order structure of the present invention include such an agglomerated (gububu) structure and a coil structure. .
  • the random coil is the simplest form representing the form of a linear polymer! Many unit chains of a certain length are connected, and can rotate freely at the connection point. In many cases, the shape of a linear polymer in an amorphous region of a solution or solid can be sufficiently approximated by the form of this random coil. Some types of synthetic polypeptides and proteins, however, may take the form of a helix due to interactions between molecular chains, and are represented by a helix model.
  • globule refers to a state that is almost spherical, and is a representative example of a state in which a polymer such as DNA is condensed.
  • the coil globule transition refers to a phase transition in the form of a polymer.
  • the polymer becomes a lump of string (coiled state, random coil model) swollen in the solvent.
  • the chain length is ⁇
  • the average size of one polymer is proportional to ⁇ ° ⁇ 6 due to the excluded volume effect.
  • the average size gradually decreases with decreasing temperature, and is proportional to ⁇ 1 / 2 at ⁇ temperature (called Gaussian chain).
  • Gaussian chain ⁇ temperature
  • Globule size is temperature It depends on N and does not depend on N.
  • the average size of the polymer is proportional to N 1/3 (similar to a solid).
  • the transition between the coil state and the globule state is almost a discontinuous primary phase transition if N is large.
  • DNA molecules in living organisms are in a complex globule state.
  • DNA exists as compact particles with a solenoidal arrangement of DNA strands (viruses), or through a compaction with histone proteins and through a five-step further compaction process, resulting in a more complex hierarchical structure in various fibers. It is known that it becomes like structure and has a final chromosomal structure (in the case of eukaryotes). Therefore, although it is important to distinguish between such a globule state and a coil state, until the disclosure of the present invention, observation of the coil globule transition has required use of an advanced instrument such as an electron microscope. .
  • the “composite” of a polymer and a metal refers to a polymer and a metal that are combined to form a single body.
  • the method of conjugation may be, for example, a hydrophobic interaction, an interaction such as a hydrogen bond, or the like, which may be a covalent bond.
  • a metal is “coated” with a polymer, it is understood that such a coating polymer also falls within the definition of “composite” because the metal and polymer are composited.
  • the polymer and the metal may form a complex.
  • a complex is a collective term for a group in which a ligand (atom / atomic group 'molecule or ion) is bound to an atom or ion of a metal element or metal-like element.
  • a ligand atom / atomic group 'molecule or ion
  • metal element or metal-like element In addition to complex ions and complex salts, it also includes non-electrolytes such as nickel-carbonyl (four carbon monoxide molecules coordinate to nickel atoms).
  • the polymer or particles used in the present invention may be particles having a diameter ranging from about 10 nanometers (nm) to about 100, OOOnm, and commercially available ones may be used. The most preferred diameter is between about lOnm and about 1, OOOnm, preferably between about 200nm and about 500nm.
  • the polymers or particles used in the present invention usually have a diameter in the range of about 0.01 to about 1000 micrometers (m).
  • the particles can take any size, but a preferred size is from about 0.1 to about 500 m, more preferably from about 1 m to about 200 m.
  • the particles are uniform (approximately the same size) or variable size so that the difference can be determined by size dependent properties such as light scattering or light refraction.
  • metal is used in the broadest sense defined in the art. Metals include noble metals and base metals.
  • non-noble metal refers to a metal that does not easily undergo chemical changes and is not easily oxidized when heated in air.
  • the power of ordinary gold, silver, and platinum groups (Ru, Rh, Pd, Os, Ir, Pt), which have a low ionization tendency, also falls within the category of noble metals in this specification.
  • a metal having a charge equivalent to that of a noble metal means a metal having a monovalent or divalent charge when expressed numerically.
  • a typical metal having a charge equivalent to that of a noble metal an ionized noble metal and a transition metal are used, but the metal is not limited thereto.
  • equivalent charge refers to the force S determined based on the basic properties shown in the periodic table of elements.
  • color is understood to mean various colors in the visible spectrum. Dyes exist in one of the media phases in a dispersed or solid state and are used to color objects (create or change color shades) and / or to make them opaque.
  • color tone refers to the level of intensity (shading) of color (color).
  • the absorbance is displayed at a specific wavelength. Since the color tone is observable with the naked eye, by using the present invention to correlate the color tone with the higher order structure (for example, topology) of the polymer, the higher order of the polymer such as the coil globule phase transition is used. Changes in the structure (eg topology) can be observed with the naked eye.
  • Structural Color is an optical phenomenon caused by a fine structure having a wavelength of light (typically, a visible region) or less, and diffraction of light "refraction". This is a phenomenon caused by interference-scattering.
  • the structural color is a force that is a coloring phenomenon caused by the cooperation of light with a fine structure of the wavelength of light or less and the color change according to the viewing direction.
  • industrial application research is progressing on fiber and automobile coating that cannot be decolored by ultraviolet rays! It is possible to modify the optical properties by manually creating a regular structure with a wavelength less than the wavelength of light.
  • the structural color can be obtained by utilizing various optical phenomena as described below.
  • Thin film interference Light reflected from the front and back of a thin film interferes and strengthens and weakens depending on the wavelength;
  • Multilayer interference Interference from multilayers is strongly reflected only by light of a certain wavelength for a specific incident 'reflection direction;
  • Diffraction Light is diffracted when the wavefront of light is blocked by matter. Diffraction angle depends on wavelength
  • Diffraction gratings Wavelength-dependent directional light diffraction by regularly arranged objects
  • Light scattering Light scattering by particles smaller than the wavelength is not directional, but the scattering intensity increases in proportion to the fourth power of the frequency;
  • Anisotropic material The material sandwiched between the polarizing plates is anisotropic (the refractive index varies depending on the direction), and the color changes depending on the rotation angle of the polarized light depending on the wavelength;
  • Refraction Light with different wavelengths is separated by refraction as the refractive index of the material varies (disperses) depending on the wavelength.
  • the red color is reflected light in the entire wavelength region of 600 nm or more
  • the yellow color is reflected in the entire wavelength region of 490 nm or more
  • the green color is from 460 to Reflected light in the entire wavelength region within 590 nm
  • blue light is reflected light in the entire wavelength region of 510 nm or less
  • purple light is absorbed in the entire wavelength region in the range of 460 to 590 nm, which is just the opposite of green.
  • Reflected light of all wavelengths other than, etc., and the reflected color (or reflected light color) of the specific color that the observer sees when irradiated with visible light is the corresponding specific wavelength region.
  • the reflected light color is the entire wavelength region of 600 nm or more
  • the yellow color is reflected in the entire wavelength region of 490 nm or more
  • the green color is from 460 to Reflected light in the entire wavelength region within 590 nm
  • blue light is reflected light in the entire wavelength region of
  • topology refers to various shapes related to the degree of entanglement between two polynucleotide strands, etc. found in a circular DNA molecule having a double helix structure. For example, if one strand of an unbroken double-stranded circular DNA is cut, one end is passed through the other uncut strand, and then joined to the original end again, strain is introduced into the entire DNA molecule. As a result, the strain energy accumulated in the DNA skeleton is minimized by supercoiling the double-stranded DNA to be twisted further and winding one strand around the other strand.
  • Diversity in higher-order structures occurs depending on the direction in which the chain passes (the direction in which the helix is rewound or vice versa) and the number of times. Depending on the direction in which the chain passes (direction to unwind the helix or vice versa) and the number of times, diversity in higher-order structures may occur.
  • the visible light wavelength region (380 to 780 nm)
  • the red color is reflected light in the entire wavelength region of 600 nm or more
  • the yellow color is reflected in the entire wavelength region of 490 nm or more
  • the green color is 460.
  • Reflected light in the entire wavelength region within ⁇ 590 nm blue light is reflected in the entire wavelength region of 510 nm or less
  • purple light is absorbed in the entire wavelength region in the range of 460 to 590 nm, which is just the opposite of green. Reflected light of all other wavelength regions, etc., and the reflected color (or reflected light color) of the specific color that we perceive when irradiated with visible light is the corresponding reflected light color of this specific wavelength region. is there.
  • Light absorption at wavelengths between 380 nm and 780 nm can be measured using any technique known in the art (eg, an absorptiometer).
  • an absorptiometer e.g., an absorptiometer
  • Such methods provide other methods for detection and analysis, including but not limited to visual inspection, digital (CCD) cameras, video cameras, photographic films, or laser scanning devices, fluorometers, Use of distribution equipment such as luminometers, photosensitive semiconductor elements (photodiodes), quantum counters, plate readers, epifluorescence microscopes, scanning microscopes, confocal microscopes, capillary electrophoresis detectors, or photo Multiplier tube or present
  • the "binder” refers to a molecule that causes aggregation of macromolecules such as DNA, and typically includes polycation molecules (for example, polylysine, supermin, etc.) also called DNA aggregating agents. Can do. Due to the presence of the binder, it is known that a polymer, typically DNA having a size of lOOkbp or more, causes a phase transition of globule coiling. When used in the present invention, this binder is generally used at a concentration of several mmol to several 11101, most preferably several mol when the charged polymer is made into a donut shape, for example. However, the concentration of the binder in carrying out the present invention is not limited to these values.
  • the present invention provides a composite of a polymer and a metal. It was found in the present invention that changes in the higher-order structure of this polymer can be seen with the naked eye by using such a complex.
  • the polymer include nucleic acids, proteins, polypeptides, polysaccharides and the like.
  • normally charged polymers are used in the present invention.
  • the charged polymer include DNA. Since DNA is known to have a complex globule structure, the present invention provides a technique for easily visualizing such a complex structure.
  • the polymer targeted by the present invention may be of at least lOkbp (kilo base pair) size.
  • the reason why the size of lOkbp or more is preferable is that it does not want to be bound by theory, but is a size that may cause a phase transition.
  • a phase transition is, for example, in DNA, its biological activity. This is because it is known to greatly affect (for example, transcriptional activity) (for example, on-off switch).
  • the polymer targeted by the present invention may be at least lOOkbp.
  • the size of at least 100 kbp is preferred because it does not want to be bound by theory but has a high probability of causing a phase transition. Examples of such a polymer include butteriophage T4 DNA.
  • the metal used in the present invention may be a noble metal.
  • the metal used in the present invention can be gold, silver, platinum, copper, palladium, and the like. Among these, silver and gold are preferable.
  • silver is preferred is that it has been found in the present invention that the colloidal state of the metal polymer composite is kept more stable, and the two peak spectra relating to the metallized torus state can be expressed well. is there.
  • Other metals are also available for visual detection and can be used to achieve good results in spectroscopic analysis.
  • the metal used in the present invention is gold.
  • gold is preferable is that it is not desired to be bound by theory, but in the present invention, it has been found that gold can be more clearly observed with the naked eye because it has a higher effect on color tone than silver. It is.
  • the composite of the present invention may further contain a binder. This is because the higher-order structure of the composite is changed due to the presence of the binder, and can be converted from a coil state to a globule state to be in a compact state.
  • the binder used in the present invention may be spermine, polylysine, polyamine, surfactant and the like. These binders are preferred because they are not desired to be bound by theory, but are known to easily cause aggregation when used with nucleic acids such as DNA.
  • the complex of the present invention is for observing the higher-order structure, topology, etc. of the complex itself.
  • Typical states in such higher-order structures include compact state (compaction state), agglomerated state, coiled state, and globule.
  • the state etc. can be mentioned. For example, when multiple large DNAs exist, even if a single molecule is in a compact state (compaction state), multiple large DNAs may aggregate to form one larger molecule. Good. If the technique of the present invention is used, these states can be observed with the naked eye.
  • the complex of the present invention may be for observing a phase transition of globule coiling.
  • biological functions such as transcription of DNA can be directly observed with the naked eye.
  • Such an effect is an exceptional effect that could not be achieved with conventional technology.
  • the present invention provides a method for observing a state of a higher order structure such as a topology of a polymer, comprising A) providing the polymer; B) metal ions in the polymer. Providing step; C) providing a reducing agent to the polymer to form a complex of the polymer and a metal included in the metal ion; D) increasing the polymer from the color tone of the complex. Including the step of calculating the next structure and topology. It was found in the present invention that changes in the higher order structure of this polymer can be seen with the naked eye. Examples of macromolecules include nucleic acids, proteins, polypeptides, polysaccharides and the like.
  • a polymer that is normally charged is used in the present invention.
  • the charged polymer include DNA. Since DNA is known to have a complex globule structure, the present invention provides a technique for easily visualizing such a complex structure.
  • the polymer to be detected by the present invention may have a size of at least lOkbp (kilobase pair).
  • a size of lOkbp or more is preferable is not to be bound by theory, but it is possible to cause a phase transition.
  • phase transitions are also known to have a significant impact (eg, on-off switch) on their biological activity (eg, transcriptional activity), for example, in DNA.
  • observation methods have been developed by Mirkin et al. For macromolecules with a size less than l Okbp (eg, DNA) by using short DNA oligomers to control the aggregation of metal nanostructures.
  • the polymer targeted by the present invention may be of at least 100 kb.
  • the size of at least lOOkbp is preferred because it does not want to be bound by theory but has a high probability of causing a phase transition.
  • DNA strands larger than a few tens of base pairs (bp) were the force S that undergoes a very discontinuous transition from a disordered coiled state to an ordered compact state, which was at this transition. The nature is shorter than the order of a few kilobase pairs (kbp)! / And is not found in DNA molecules! /, So observing these transitions is both physical and chemical.
  • the metal used in the present invention may be a noble metal.
  • the reason why the noble metal is preferable is that the detection method of the present invention improves detection sensitivity because it is not easily subjected to chemical changes and has a low ionization tendency that is not easily oxidized even when heated in air. Therefore, it is understood that copper or the like, which is considered to be hardly subject to chemical changes, is also included in the present specification even if it is unclear whether it normally falls within the category of noble metals.
  • the metal used in the present invention can be gold, silver, platinum, copper, palladium, and the like. Silver is preferred because it has been found in the present invention that the colloidal state of the metal polymer composite is kept more stable. With the method of the present invention, it is related to the metallized torus state. This is because one peak spectrum can be expressed well. Macroscopic examination of other metals It can be used in production, and good results can be obtained even in spectroscopic analysis.
  • the metal used in the present invention is gold.
  • gold is preferable is that it is not desired to be bound by theory, but in the present invention, it has been found that observation with the naked eye, which has a higher effect on the color tone of gold than silver, can be made clearer. Because.
  • the complex can be prepared by first applying a metal ion to the polymer and then applying a reducing agent.
  • metal ions can be selected appropriately depending on the target metal. For example, AgN 2 O (Ag—) is produced in the case of silver. ), If gold, aqueous solution of HAuCl (AuC14—ion is generated
  • any reducing agent can be used as long as it can reduce metal ions.
  • any reducing agent can be used as long as it can reduce metal ions.
  • NaBH sodium borohydride
  • the ratio of the reducing agent to the solution containing metal ions may be any ratio as long as the metal coating (complexing) is possible.
  • the stoichiometric ratio is several times higher.
  • the binder used in the detection method of the present invention is added to change the higher order structure (eg, conformation) of the composite. This is because the higher-order structure of the composite is changed by the presence of the binder, and can be converted from the coil state to the globule state to be in a compact state.
  • higher order structure eg, conformation
  • the binder used in the present invention may be spermine, polylysine, polyamine, surfactant or the like. These binders are preferred because they are not desired to be bound by theory, but are known to easily cause aggregation when used with nucleic acids such as DNA.
  • the higher-order structure, topology, etc. of the complex itself are observed.
  • Typical examples of such higher-order structures include a compact state (compaction state), an agglomerated state, a coiled state, and a globule state.
  • compaction state when multiple large DNAs exist, a single molecule is compact. Multiple large DNAs that may be in a compact state (compaction state) may aggregate to form one larger molecule.
  • these states can be observed with the naked eye.
  • it has enabled observation at 10_ 12 mol (pmol) level. Since the same effect can be expected in other polymers, the use of the present invention, it can be said that the polymer generally is a 10- 12 mol (pmol) level of observation.
  • the present invention can observe the phase transition of a compaction-coil.
  • the color tone can be observed by measuring the absorbance at a specific wavelength.
  • the ratio of the absorption maximum at 400 nm and 500 nm (for the combination of DNA and silver), the ratio of the absorption maximum at 550 nm and 700 nm (for the combination of DNA and gold) can be measured.
  • the ultraviolet / visible light spectrum may be measured and compared.
  • the spectrum shape has a broad absorption region with a force of 600 nm to 900 nm that remains almost unchanged, and a weak but distinct peak exists at 530 nm.
  • Spectral shapes at wavelengths can be used.
  • the reducing agent is preferably added to a mixed state of the metal ion and the polymer.
  • complex formation proceeds efficiently by adding in this order.
  • the step C) further includes a step of irradiating ultraviolet light in the presence of alcohol.
  • Any alcohol may be used, and any of polyols may be used more preferably.
  • the ultraviolet light it is preferable to use strong ultraviolet light such as 254 nm, which may be of any wavelength.
  • “ultraviolet rays” refers to electromagnetic waves having a wavelength range of up to about 1 nm, with the upper limit being about 360 to 4 OOnm at the short wavelength end of visible light. The lower limit is not very clear, and several tens of squares or less overlap with soft X-rays. Therefore, it is understood in this specification that the range overlaps with X-rays.
  • Examples of light sources include quartz mercury lamps, carbon arc lamps, and sparks. Electricity, hydrogen or rare gas discharge, synchrotron radiation, and the like can be used, but are not limited thereto. If the wavelength of ultraviolet rays increases (for example, 365 nm), the reduction efficiency is reduced. Although not wishing to be bound by theory, it is because the formation of the complex proceeds efficiently by including this step.
  • the irradiation with ultraviolet light is performed for a time sufficient to induce formation of the complex. Such sufficient time varies depending on the combination of metal and polymer used, for example, ultraviolet light generates free radicals in the alcohol molecules, which reduce the metal ions. That is, the reducing agent is produced by irradiating ultraviolet rays to alcohol molecules. Subsequent metallization occurs in the same way as NaBH reduction.
  • the higher-order structure calculation from the color tone is selected from the group consisting of, for example, DNA phase transition, charged polymer phase transition, protein folding and DNA and protein cohesion. It may be for observing the condition being performed. In a preferred embodiment, it may be for observing the phase transition of the compaction coil.
  • the high-order structure calculation from the color tone is performed when the color tone peculiar to the metal (for example, yellow for silver and pink for gold) is observed. If it is determined to be in a state and a longer wavelength tone is observed (eg, pink for silver, blue / gray for gold), the polymer is determined to be in a compacted state. When the color tone of the intermediate wavelength (for example, orange for silver and violet for gold) is observed, it is determined that the coil state and the compaction state coexist.
  • the color tone peculiar to the metal for example, yellow for silver and pink for gold
  • the calculation includes determining whether or not it is symmetric, and the presence of two peaks in the wavelength curve indicates that the higher molecule is Determined to be in a symmetric compaction state.
  • the polymer in the detection method of the present invention, in the calculation of the higher order structure from the color tone, the polymer is DNA, and the calculation determines whether it is a torus-like force.
  • the presence of two peaks in the wavelength curve is determined to be that the polymer is in a symmetric connection state.
  • the calculation includes determining whether or not the torus-like force, and In the determination, the presence of two peaks in the wavelength curve determines that the polymer is in a symmetric compaction state. In this way, by calculating the higher-order structure from the color tone, the binder can calculate the concentration at which the phase transition of the DNA compaction coil occurs.
  • the method of the invention may include the step of determining the compaction state size of the polymer.
  • this determination process an equilibration curve is created using a solution of a metal polymer complex of a known size as a control, and the data of the polymer complex to be measured is added to the equilibration curve. Is made.
  • the present invention may include the step of calculating the higher-order structure from the color tone by calculating a concentration at which the binder causes a phase transition of the compaction coil of the DNA.
  • the present invention provides a kit for observing higher order structures such as polymer topology.
  • This kit consists of (optionally A) the target polymer); B) the metal ion; C) the reducing agent; D) the color of the complex formed from the polymer and the metal in the metal ion.
  • the kit of the present invention may further include means for determining a critical concentration of the binder that induces a polymer phase transition.
  • a polymer that is normally charged is used in the present invention.
  • the charged polymer include DNA. Since DNA is known to have a complex globule structure, the present invention provides a kit for easily visualizing such a complex structure.
  • kits generally refers to a unit in which parts to be provided (eg, reagents, enzymes, vertical nucleic acids, standards, etc.) are provided in two or more compartments. .
  • Compositions that should preferably be provided mixed and used preferably just before use The form of this kit is preferred when it is intended to provide Such kits preferably include the provided moieties (eg, reagents, enzymes, nucleotides, labeled nucleotides, nucleotides that stop the extension reaction (and their triphosphates), vertical nucleic acids, standards, etc.). It is advantageous to have instructions describing how to proceed.
  • the kit usually includes reagent components, buffers, salt concentrates, auxiliary means for use, instructions describing the method of use, etc. .
  • instructions describe to the user how to use the reagent of the present invention, how to react, and the like.
  • This instruction manual includes a word indicating a procedure such as an enzyme reaction of the present invention.
  • This instruction is prepared according to the format prescribed by the national supervisory authority where the present invention is implemented as necessary, and it is clearly stated that it has been approved by the supervisory authority. Instructions are so-called package inserts, which are usually provided on paper media, but are not limited to this, for example, films affixed to bottles, electronic media (eg homepages provided on the Internet) (Website), e-mail).
  • package inserts which are usually provided on paper media, but are not limited to this, for example, films affixed to bottles, electronic media (eg homepages provided on the Internet) (Website), e-mail).
  • the kit of the present invention it should be understood that the conditions that should be taken by the various constituent elements can take any form detailed in other forms of the present invention.
  • the complex of the present invention can be used as a material such as a fiber having a structural color to be exhibited, an additive for adjusting color development in cosmetics, or a material in a semiconductor manufacturing technology (for example, a dopant used in a doping process for a semiconductor).
  • the complex DNA structures such as toroids and rods are coated with metal using the present invention.
  • an alternating electric field is applied to a suspension of such a metal-coated DNA structure, the DNA structure is aligned so as to align in a certain direction, and in this state, packing (adhesive to the solution and high transparency! Add) to produce a sheet in an aligned state, and laminate the sheet to produce a multilayer film.
  • This multilayer structure creates a structural color due to multilayer interference.
  • Sheets made in this way can be used in the textile and clothing field by either weaving the sheet (mm size) or adhering it to the surface of the shaped cloth when weaving the cloth from yarn. Applicable. In this case, a sheet with a multilayer structure has already been created! /, So it will exhibit a structural color in any direction.
  • the change in the higher-order structure of a polymer on the order of nanometers to micrometers can be visually observed by metallization in a liquid according to the present invention.
  • the color tone is controlled by using the present invention.
  • the fact that the micro / nanoscale structure is visible means that the structure color is seen. Control the size of the particles in the system (shape when metallizing) or change the particle concentration 'change the color by changing the metal.
  • DNA and protein are DAPI (4,6—diamidino—2-phenylindole),
  • Ribo green Condensing agent multiple cation such as spermine or surfactant
  • DAPI Excitation wavelength 358 nm, fluorescence wavelength 461 nm.
  • Ribogreen Excitation wavelength: 485 nm, fluorescence wavelength: 530 nm.
  • the charged polymer to be used at this time should be a uniform object (standard).
  • Step 3 This graph makes it possible to predict the structure and the color emitted by Mie scattering.
  • examples of usage as a cosmetic or fiber material include the following.
  • the composite Since the composite has already been molded into a multi-layer structure! /, It can be incorporated into the material without problems using the same mixing method as V, so-called lame (thin metal piece).
  • lame thin metal piece
  • mixing powder talc 'sericite' titanium oxide, etc. and nylon powder
  • foundation, rouge, eye shadow, etc. it is completed by stirring and mixing like other powders and compression molding.
  • a method of using the present invention as a semiconductor material will be described below.
  • One suitable example in which the present invention can be used is not limited to the force S, which can be used as a dopant used in the doping process in manufacturing a light emitting diode.
  • Light-emitting diodes are semiconductors that effectively convert electrical energy into spontaneous and non-coherent visible and near-infrared electromagnetic radiation by electroluminescence at a PN junction biased in the forward direction. It is a diode. Also called LED (Light Emitting Diode).
  • the PN junction is a portion of a semiconductor where a p-type region and an n-type region are in contact.
  • pure semiconductors have low conductivity as they are, so they are processed to increase the carrier density by doping semiconductor impurities (dopants) (doping) and to have appropriate conductivity and properties.
  • n-type and p-type are distinguished depending on whether the majority carrier is an electron or a hole.
  • the n-type semiconductor is an impurity semiconductor in which the concentration of conduction electrons is higher than the concentration of holes
  • the p-type semiconductor is an impurity semiconductor in which the hole density is higher than the conduction electron density.
  • the color of light emitted from the light-emitting diode varies depending on the material used, and it can be manufactured from the infrared region to the visible light region and the ultraviolet region.
  • Materials include aluminum gallium arsenide (AlGaAs), gallium arsenide phosphorus (GaAsP), indium gallium nitride (InGaN) / gallium nitride (GaN) / aluminum gallium nitride (A1G aN), gallium phosphide (GaP), zinc selenide (ZnSe), aluminum indium gallium phosphide (AlGalnP), etc., and the color is selected by selecting the material.
  • silicon carbide (SiC), sapphire (Al 2 O 3), cai are used as substrates.
  • the basic structure of a light emitting diode is a PN junction, but a double heterojunction structure or a quantum well junction structure is used for the purpose of increasing luminous efficiency.
  • a method of stacking thin films on a substrate by chemical vapor deposition is used.
  • doping means a process of adding impurities to a substance! /. Doping is a process used to control the physical properties of a semiconductor. This added impurity is called “dopant”.
  • the “donor” is an impurity that forms an n-type semiconductor
  • the “acceptor” is an impurity that forms a p-type semiconductor.
  • a charged polymer can be coated with gold, platinum, silver, etc., and added as an impurity, and can be used as a new semiconductor material. Since the charged polymer metallized by the present invention has stable physical properties! /, It can be used to change the electrical characteristics of the semiconductor by using it in conventional doping processes in semiconductor manufacturing technology. Can do.
  • the composite according to the present invention is suitable as a dopant in the following points, for example.
  • the conductor using the normal metal is desired to be uniform and has no uneven conductivity.
  • conventional semiconductor materials used for doping include those harmful to the human body and the environment.
  • the use of the charged polymer coated with a noble metal according to the present invention makes it harmful. It is understood that it is possible to replace metals with less hazardous materials.
  • the present invention it is possible to freely control the structure and metallize, and thus it is possible to realize a color tone that has been impossible until now with a single element.
  • Batateriophage T4 DNA (approx. 166,000 base pairs, Nitsubon Gene (Japan)), AgNO (purity 99 ⁇ 999%; Aldrich (Japan)), HAuCl ⁇ 3 ⁇ Cl (Nacalai
  • TEM transmission electron microscope
  • the metal coating was carried out as follows. First, an appropriate amount of fluorescent dye, condensing agent and, if necessary, buffer solution was added to distilled water and mixed well. DNA was added to the resulting solution and gently mixed so as not to break long DNA. After preparing the DNA, observation with a fluorescence microscope (FM) was performed for 15-30 minutes. The metal coating procedure was as follows. First, a metal salt solution was added, the solution was gently mixed, and then NaBH was added and mixed to reduce the metal in the solution. The solution is
  • Tetravalent cationspermine is one of the most common condensing agents that induces the DNA folding transition.
  • FM real-time fluorescence microscope
  • Figure 1 shows fluorescence microscopy images of 10_M T4 DNA solutions (in nucleotides) at various concentrations of spermine. At low concentrations of spermine (up to 0.2 M), all of the DNA molecules were observed as uncoiled (unfolded) coiled states. This spreading! / Coil state is characterized by a large thermal fluctuation of the DNA molecule segment ( Figure 1A). Increasing the spermine concentration to 0.4 M causes the DNA molecule to co-exist with a coiled state and a folded (compact) state, and this compact DNA has observable intramolecular fluctuations. In addition, it showed significant translational Brownian motion and emitted fluorescence that was stronger than its coiled state (Fig. 1B).
  • the inventors used the silver strand pretreated with spermine as described above with gold.
  • the genus was coated. Differences in the precipitation characteristics of noble metals on DNA templates with different concentrations of spermine could be observed clearly and clearly even with the naked eye.
  • the procedure is to add silver nitrate (AgNO, final concentration 100 M) and water as the reducing agent to the experimental sample.
  • the molar ratio with sodium borohydride was selected to ensure fast kinetic characteristics for silver reduction and DNA metallization.
  • the reducing agent (8 molecules of AgNO 3 vs. 1 molecule of NaBH), which is several times the stoichiometric excess, was optimal. Up
  • the solution developed in a few minutes and remained in color for at least one day.
  • the color of the liquid changed from yellow to pink.
  • a photograph of the cell containing the sample is shown in FIG.
  • the final solution color was yellow when spermine was low as described above and pink when spermine was high.
  • the observed color change is very cooperative and abrupt, and the solution is present in a narrow concentration range (0 ⁇ 2 to 0.3 ⁇ ⁇ ).
  • the silver solution is yellow, it corresponds to the formation of silver nanoparticles of several to several tens of nanometers (nm). If the solution undergoes a red shift in its color, it indicates that relatively large metal nanoparticles are formed in the solution!
  • this characteristic shape determines the shape of its spectrum, where the absorbance observed near 400 nm is the silver “ The diameter of the “ring-shaped part” (dl: 30-40 nm in Fig. 3) corresponds to absorption around 400 nm, and the absorption around 500 nm corresponds to the outside diameter of the nanoring (d2; 80 to 90 nm) (See Fig. 3).
  • Two peak patterns in the UV'visible spectrum are reported for noble metal nanorods (Mohamed M. B., Volkov V., Link S., El-Sayed MA, Chem. Phys. Lett.
  • Gold nanorod morphology is characterized by two geometric dimensions (length and diameter), resulting in an ultraviolet-visible spectrum pattern in the nanorod depending on the size of the nanorod. (38. Mohamed MB, Volkov V., Link S., El-Sayed MA, Chem. Phys. Lett. 2000, 317, 517-523). Single nanoparticles were produced as a byproduct in 1S reduction and were found to contribute somewhat to the intensity at 400 nm. The ratio of the above two absorptions (absorbance ratio) varies depending on the experimental conditions.
  • the optical change can be obtained by measuring by UV spectroscopy. It was expressed as the absorbance ratio at m and 500 nm. This absorbance ratio at 4 OOnm and 500nm, as measured by UV spectroscopy, is used to clearly show dramatic changes in the spectra of silver nanostructures (Storhoff JJ, Mirkin CA, Chem. Rev. 1999, 99). , 1849— 1862). From the comparison of Fig. 2 and Fig. 6, it is clear that the change in the DNA conformation observed by the single-molecule fluorescence microscope itself is adequate.
  • the observed phenomenon is that by monitoring the state of silver deposition on DNA, information on the state of DNA conformation is provided, and transitions in the conformation of biopolymers occur. Demonstrate that it is useful in determining the concentration of DNA binders. It should be noted that the power of silver ions as cationic compounds. The compaction of DNA with silver nitrate itself does not occur even in mil-molar order according to previous theoretical studies on monocations (Wilson, RW, Bloomfield, VA Biochemistry 1979, 18, 2192-2196). Therefore, the influence of silver ions on the transition of DNA confectionary formations can be ignored.
  • metallization with gold was performed.
  • HAuCl solution is used as a source of gold (Au)
  • gold with silver is used.
  • NaBH NaBH was used as the reducing agent, just like the genus coating.
  • NaBH NaBH was used at a somewhat higher ratio compared to silver. Use the following experiment with silver
  • FIG. 7 shows the color change in DNA solutions containing various concentrations of spermine after metallization using gold.
  • the purple line of gold nanoparticles The absorbance of visible light is located on the long wavelength side compared to the absorbance of silver nanoparticles, and the initial solution containing unfolded DNA, metallized with gold, is pink. The maximum absorbance is around 520nm. Were present. This result suggests the formation of gold nanoparticles measured at about 10 nm (41. Storhoff JJ, Elghanian R., Mucic RC, Mirkin CA, Letsinger RL, J. Am. Chem. Soc. 1998, 120, 1959— 1964.).
  • the color change of the solution is observed even when the concentration of spermine is low, and its absorbance shifts to the longer wavelength side, and accordingly, the color changes from purple.
  • This change in color reflects the difference in the size of gold nanoparticles formed after DNA metallization or nanoparticle aggregation after DNA metallization at various spermine concentrations.
  • a compact DNA metallization with gold provides a blue-gray solution. The force with which the color change characteristics of the solution differ between silver and gold. The transition point of the DNA conformation can easily be tracked in either case. In other words, after the compatible DNA conformation appears, the color of the solution does not change.
  • the corresponding UV'visible spectrum is shown in FIG.
  • the metal precipitates in the form of a smooth layer or in the form of particles depends on the stability of the corresponding nanoparticles of large size.
  • large nanoparticle forces are observed under the same experimental conditions and are produced without the seed of DNA in solution. It proceeds in the form of a smooth metal shell.
  • small metal nanoparticles are produced under the same conditions in a DNA-free solution, the metal precipitates on the toroid surface in the form of the nanoparticles, as found in gold. To do.
  • the present invention can be used to systematically analyze the relationship between experimental conditions and final nanostructure morphology.
  • Metallization with gold is similar to metallization with silver because of the property of rapid transitions and almost no spectral change even after the transition of the DNA structure.
  • the conformational transition of the DNA can be easily monitored by the following spectral changes.
  • FIG. 8 shows the change in the ratio of absorbance at 500 nm and 750 nm as correlated to spermine concentration. This change in spectral shape with respect to the intensity ratio is similar to the curve change for silver in Figure 6.
  • DNA compaction and continuous metal coating on the toroid is clearly shown by the shape of the ultraviolet (UV) spectrum, where silver is the compact DNA It is more useful to detect the form.
  • DNA aggregating agents affect the mechanism of DNA compaction, the final aggregate shape, and the resulting metal nanostructures after metallization of DNA.
  • DNA aggregates do not necessarily have a torus-like form. In fact, stronger condensing agents such as polycations have a lower tendency to form DNA toroids, and DNA aggregates often have a globule (spherical) form (Perales J. C. , Ferkol ⁇ ., Beegen ⁇ ., Ratnoff ⁇ . D., Hanson, RW, Proc. ⁇ atl. Acad. Sci. USA 1994, 91, 4086—4090).
  • the final product of DNA compaction and metallization with polycations has an average particle size of about 150 nm to 200 nm. It is a metal particle having a diameter, and no ring structure is observed.
  • the inventors have shown that the conformational power of sufficiently long DNA (eg, T4 DNA) determines the nature of precious metal deposition on DNA polymer templates. That is, it shows that this technique can be used for analytical detection of biopolymers.
  • the fine morphology of the resulting metal nanostructure can be controlled by various parameters (eg, chemical structure of DNA condensing agent, metal type, etc.).
  • an important feature of investigating DNA conformation by metal coating is a clear change in absorbance of metal nanostructures and in conformation of DNA polymer chains. It is to respond to.
  • this metallization method Force It has become clear that it can be used to study the phase transitions of long chain biopolymers. Specifically, using this metal coating method, (i) the critical concentration of flocculant that induces a conformational transition in the biopolymer; (ii) the properties of this transition; (iii) the final aggregate And (iv) the compact biopolymer aggregate morphology can be evaluated.
  • the noble metals silver and gold are suitable candidates for this purpose.
  • Precious metals are known to complex with various ligands, but the composition in solution can vary over a wide range while maintaining the relationship between changes in DNA conformation and changes in UV spectra. .
  • concentration of up to about 10- 3 M concentration of up to about 10- 3 M
  • noble metals to obtain significant effect against rigidity of the DNA, and, The electrostatic characteristics are also affected.
  • changes in the ultraviolet spectrum cannot correspond to the outcome for its actual transition concentration.
  • the present invention relies on the modal force S to precipitate noble metals on long DNA strands when reduced, the conformation of the DNA strands, and is very sensitive. Monitor biopolymer conformational changes in very dilute solutions by colorimetric, spectroscopic and macroscopic observations due to differences in precious metal nanostructures and outstanding optical properties Various means for providing are provided.
  • the metallization process of the present invention is useful for detecting important conformational changes, such as the conformational state of any sufficiently long macromolecule, using the method of colorimetric high throughput.
  • the fast and simple detection method of the present invention for polymer conformation is DNA or tampering. It is a promising tool for the analysis of large libraries of compounds involved in the regulation of protein conformation.
  • the present invention relating to metal deposition is also used to investigate the structural asymmetry of compact DNA, RNA or proteins with submicron resolution that is sensitive to the shape and size of compact biopolymer chains. Useful.
  • mitochondria are collected by the cell fraction obtained by density gradient centrifugation after the force to physically disrupt the cell tissue, enzymatic necrosis, and density gradient centrifugation (standard method). After that, it is generally necessary to confirm the morphology with an electron microscope. However, using this technique, it is possible to confirm the change in morphology by metallization in a solution that does not allow the use of an electron microscope. It can be applied as a quick judgment kit.
  • the color tone is supposed to change according to the type of dopan.
  • the force S which changes the color tone by changing the metal to be metallized, can be mixed by controlling nanorods and nanorings having different resistances at the center and the outer edge. .
  • non-uniform energization in the electrode is created due to the difference in shape, and the color tone changes due to a gap profile different from that of existing materials. (Rod and ring shapes have different colors)
  • Patents, patent applications, and documents cited in this specification should be incorporated by reference as if the contents themselves were specifically described in the present specification. Understood.
  • the present invention By using the present invention, the following advantages are obtained: (1) Metallization can be performed while floating (or suspended) in the liquid. (2) From the electron microscope image, it was confirmed that the surface was smoother than the previous coating. Specifically, it is so smooth that only undulations of several tens of pixels (typically 10 to 20 pixels) (corresponding to several nm) can be seen when an electron microscope image is analyzed. Since the conventional metallization has a degree of smoothness of about several hundred pixels, the present invention has been improved until the unevenness on the surface becomes about 1/10.
  • kits for diagnosing a disease in which a three-dimensional structural change of a macromolecule in a living body is related to its predisposition are assumed, and in the biochemistry field, it is used for research. It can be used as a kit.
  • the present invention can be used in the textile industry and the like as industrial materials such as fibers having the structural color exhibited by the composite of the present invention.
  • the present invention can be used as an additive for color adjustment in cosmetics.
  • the present invention can be used as a material in semiconductor manufacturing technology (for example, a dopant used in a doping process for a semiconductor).

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Abstract

L'invention concerne un procédé permettant d'examiner aisément à l'œil nu la structure d'ordre supérieur d'un polymère. Ce procédé consiste : A) à obtenir le polymère ; B) à fournir au polymère des ions métalliques ; C) à fournir au polymère un agent réducteur pour former un composite de polymère avec le métal contenu dans les ions métalliques ; et D) à calculer la structure d'ordre supérieur du polymère à partir du ton de couleur du composite. L'utilisation d'ions métalliques et d'un agent réducteur dans l'examen à l'œil nu d'une structure d'ordre supérieur de polymère est une nouveauté.
PCT/JP2007/068467 2006-09-22 2007-09-21 Procede de revetement de polymere charge au moyen de metal WO2008035787A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017189930A1 (fr) 2016-04-27 2017-11-02 Quantum Biosystems Inc. Systèmes et procédés de mesure et de séquençage de biomolécules
CN109031482A (zh) * 2018-09-01 2018-12-18 哈尔滨工程大学 一种制备微透镜结构的方法

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
NING CHEN ET AL.: "Nanostructures via DNA scaffold metalization", IEEE INTERNATIONAL SYMPOSIUM, 2005, pages 71 - 74, XP010890606 *
NING CHEN ET AL.: "Probing Biopolymer Conformation by Metallization with Noble Metals", NANOTECHNOLOGY, vol. 17, no. 20, 28 September 2006 (2006-09-28), pages 5224 - 5232, XP020104240 *
ZINCHENKOO A.A. ET AL.: "DNA-templated silver-nanorings", ADV. MATER., vol. 17, 2005, pages 2820 - 2823, XP003021858 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017189930A1 (fr) 2016-04-27 2017-11-02 Quantum Biosystems Inc. Systèmes et procédés de mesure et de séquençage de biomolécules
CN109031482A (zh) * 2018-09-01 2018-12-18 哈尔滨工程大学 一种制备微透镜结构的方法

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