Peptides having independently stabilized hydrophobic α-helical structures, peptide complexes containing the same, and uses thereof

Abstract

To provide a novel peptide that can produce a self-assembly with diverse structures through binding with hydrophilic molecules, while having an independently stabilized hydrophobic α-helical structure without external aid.SOLUTION: A hydrophobic peptide represented by the following general formula 1 or 2 is constituted. [General formula 1] -{[Aib]-[Xaa1]}m- [General formula 2] Xaa1-{[Aib]-[Xaa1]}m- (in the formula, Xaa1 is any one selected from the group consisting of Ala (A), Ile (I), Leu (L), Met (M) and Val (V); Xaa1 is a D- or L-type amino acid residue; and m is any one integer selected from 4 to 50).SELECTED DRAWING: Figure 44

Description

[Technical Field]

[0001] The present invention relates to a peptide having a hydrophobic α-helical structure that is independently stabilized without external aid, a peptide complex containing the same, and uses thereof. [Background technology]

[0002] Magnetically responsive materials are highly regarded for their potential biological and medical applications because they are radiation-free and use magnetic fields that are safe for humans. To date, a variety of stimuli-responsive self-assembling materials have been developed, including ferromagnetic / paramagnetic inorganic or inorganic / organic composites. However, the development of magnetically responsive self-assembling materials based solely on organic materials remains limited. While DNA and some proteins have been identified as magnetically responsive, their commercialization is hindered by the need for extremely strong magnetic fields.

[0003] Peptide self-assembly structures have attracted much attention due to their diverse biological application potential in fields such as medicine and nanotechnology. Because they are composed of short protein units, peptide self-assembly structures generally have high biological stability. These peptide monomers can form secondary structures such as α-helices or β-sheets, and can form various structures such as micelles, vesicles, nanofibers, and hydrogels through secondary bonds such as hydrophobic bonds, hydrogen bonds, electrostatic attraction, and π-π bonds.

[0004] As such, peptides have attracted much attention because they can be given not only diverse structures but also various functions depending on their properties, thereby realizing more precise and stable functional structures. However, as peptides are typical diamagnetic organic molecules and do not react even in strong magnetic fields of several tesla or more, they have been treated as non-magnetic substances.

[0005] Recently, it has been revealed that the α-helical structure, which is the secondary structure of peptide monomers, has some magnetic responsiveness. However, each peptide with α-helical structure has a problem that it cannot maintain the α-helical structure in the monomeric state or has the α-helical structure only when it is present in a protein.

[0006] One publication related to magnetically responsive peptides is Patent Document 1, which describes one-dimensional nanostructures created through the self-assembly of dipeptides whose arrangement can be controlled by a magnetic or electric field. However, the structure is limited to organic solvents and the peptide arrangement is restricted to Phe-Phe, preventing broad application. Therefore, additional modification processes are required to impart chemical and biological functionality.

[0007] That is, to use diamagnetic organic materials such as peptides as magnetically responsive organic materials that can control mechanical movement using magnetic force, a non-invasive stimulus source, it is necessary to form rod-structured peptide self-assemblies while maintaining the magnetic responsiveness of aligning along the direction of the magnetic field, but research on this topic has been very limited to date. Furthermore, for wide use through interaction with genetic materials, it is very important to stably maintain the secondary structure of peptides, but there has been little research on this, and there has been no research at all on controlling the formation and disassembly of peptide self-assemblies. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] Republic of Korea Patent Publication No. 10-2008-0102687 Summary of the Invention [Problem to be solved by the invention]

[0009] Therefore, the present invention has been made in consideration of the above problems, and an object of the present invention is to provide a novel peptide that has a hydrophobic α-helical structure that is independently stabilized without external aid, and can form self-assemblies with various structures through binding with hydrophilic molecules.

[0010] Another object of the present invention is to provide a peptide conjugate comprising the hydrophobic peptide.

[0011] Another object of the present invention is to provide a new system for safely storing and archiving nucleic acid information. [Means for solving the problem]

[0012] In order to achieve the above object, the present invention provides a hydrophobic peptide represented by the following general formula 1 or 2.

[0013] [General formula 1] -{[Aib]-[Xaa1]}m-

[0014] [General formula 2] Xaa1-{[Aib]-[Xaa1]}m-

[0015] In the above formula, Xaa1 is any one selected from the group consisting of Ala (A), Ile (I), Leu (L), Met (M) and Val (V), and Xaa1 is a D- or L-amino acid residue.

[0016] The m is an integer selected from 4 to 50.

[0017] In the formula 1, Xaa1 may be any one selected from the group consisting of Ala (A), Ile (I), Leu (L) and Met (M).

[0018] In the above formula 1, Xaa1 can be Ala (A) or Leu (L).

[0019] In the formula 1, m may be any one integer selected from 6 to 13.

[0020] In the formula 1, m may be any one integer selected from 6 to 10.

[0021] The hydrophobic peptide may be rod-shaped with an α-helical structure.

[0022] The hydrophobic peptide may have its sequence and orientation controlled in the direction of an external magnetic field.

[0023] To achieve the above object, the present invention provides a peptide conjugate comprising the hydrophobic peptide; and at least one hydrophilic polymer or hydrophilic peptide bound to both ends or one end of the hydrophobic peptide.

[0024] The hydrophilic polymer may be any one selected from the group consisting of polyethylene glycol (PEG), poly-N-(2-hydroxypropyl)methacrylamide, poly(2-(methacryloyloxy)ethylphosphorylcholines), poly(hydroxyalkyl L-asparagine), and poly(hydroxyalkyl L-glutamine).

[0025] The hydrophilic peptide is composed of a sequence of 3 to 20 amino acids, and the content of hydrophilic amino acids in the amino acid sequence is 70% to 100%. The hydrophilic amino acids may be any one or more selected from the group consisting of H (histidine), N (asparagine), Q (glutamine), S (serine), T (threonine), C (cysteine), G (glycine), K (lysine), R (arginine), E (glutamic acid), and D (aspartic acid).

[0026] The hydrophilic peptide may be any one selected from the group consisting of SEQ ID NOs: 98 to 105.

[0027] The peptide conjugate may be cyclic, with both ends of the peptide conjugate joined.

[0028] The N-terminus or C-terminus of the peptide conjugate may further contain a positively charged peptide consisting of 1 to 4 positively charged amino acid residues.

[0029] The positively charged peptide may be any one selected from SEQ ID NOs: 108 to 119.

[0030] The peptide conjugates may self-assemble in solution as spherical nanoparticles in a micellar or endoplasmic reticulum structure.

[0031] The peptide complexes can be self-assembled into anisotropic planar nanostructures or cross-shaped anisotropic nanostructures under the application of a magnetic field.

[0032] The magnetic field strength may be between 0.1T and 2T.

[0033] To achieve the above object, the present invention provides a composition for safely storing nucleic acid information, which comprises the peptide complex and a nucleic acid substance.

[0034] The peptide complex and the nucleic acid molecule may be self-assembled by any one interaction selected from the group consisting of non-covalent bonds, and may be manufactured into a nanostructure having a nanoribbon or artificial chromosome-like structure.

[0035] The peptide complex is bound to the nucleic acid material, thereby forming a nanoribbon structure through self-assembly, and the nanoribbon is folded and stacked to form a nanostructure of an artificial chromosome structure through self-assembly in stages.

[0036] The nucleic acid substance may be one or more selected from the group consisting of RNA, DNA, siRNA (short interfering RNA), aptamer, antisense ODN (antisense oligodeoxynucleotide), antisense RNA, ribozyme, and DNAzyme.

[0037] The nanostructures can be decomposed under magnetic field strength conditions of 0.1 T to 2 T, inducing the release of nucleic acid materials.

[0038] The magnetic field strength condition may be a rotating magnetic field condition of 0.1T to 0.5T. [Effects of the Invention]

[0039] The present invention relates to a novel hydrophobic peptide consisting of the shortest repeating amino acid sequence with a perfectly stabilized α-helical structure without external aid, which is non-cytotoxic and has magnetic reactivity, with its sequence and orientation controlled and regulated by a magnetic field. Furthermore, the hydrophobic peptide according to the present invention can not only form complexes with hydrophilic molecules in various structures, but also control its morphology using a magnetic field, and can provide nanostructures with stable structures, such as nanoribbon → artificial chromosome structures, through stepwise association with nucleic acid materials.

[0040] The peptide complex containing the hydrophobic peptide according to the present invention can completely protect nucleic acid materials from the external environment and provide a magnetically responsive material with a new structure whose assembly and disassembly can be controlled by a magnetic field.

[0041] The peptide conjugate of the present invention forms a nanostructure by association with a nucleic acid substance, which can be readily applied to analytical methods such as PCR to subsequently identify genetic information from the nucleic acid substance. Therefore, it can be useful not only in various therapeutic drug delivery systems, drug treatment techniques, and cosmetic compositions, but also in molecular machines, etc. [Brief explanation of the drawings]

[0042] [Figure 1] 1 is a graph showing the results of MALDI-TOF analysis of the hydrophobic peptides of Examples 1 to 36. [Figure 2] 1 is a graph showing the results of MALDI-TOF analysis of the hydrophobic peptides of Examples 1 to 36. [Figure 3] 1 is a graph showing the results of MALDI-TOF analysis of the hydrophobic peptides of Examples 1 to 36. [Figure 4] 1 is a graph showing the results of MALDI-TOF analysis of the hydrophobic peptides of Examples 1 to 36. [Figure 5] 1 is a graph showing the results of MALDI-TOF analysis of the hydrophobic peptides of Examples 1 to 36. [Figure 6] 1 is a graph showing the results of MALDI-TOF analysis of the hydrophobic peptides of Examples 1 to 36. [Figure 7] 1 is a graph showing the results of MALDI-TOF analysis of the hydrophobic peptides of Examples 1 to 36. [Figure 8] 1 is a graph showing the results of MALDI-TOF analysis of the hydrophobic peptides of Examples 1 to 36. [Figure 9] 1 is a graph showing the results of MALDI-TOF analysis of the hydrophobic peptides of Examples 1 to 36. [Figure 10] 1 is a graph showing the results of MALDI-TOF analysis of the hydrophobic peptides of Examples 1 to 36. [Figure 11] 1 is a graph showing the results of MALDI-TOF analysis of the hydrophobic peptides of Examples 1 to 36. [Figure 12] 1 is a graph showing the results of MALDI-TOF analysis of the hydrophobic peptides of Examples 1 to 36. [Figure 13] 1 is a graph showing the results of MALDI-TOF analysis of the hydrophobic peptides of Examples 1 to 36. [Figure 14] 1 is a graph showing the results of MALDI-TOF analysis of the hydrophobic peptides of Examples 1 to 36. [Figure 15] 1 is a graph showing the results of MALDI-TOF analysis of the hydrophobic peptides of Examples 1 to 36. [Figure 16] 1 is a graph showing the results of MALDI-TOF analysis of the hydrophobic peptides of Examples 1 to 36. [Figure 17] 1 is a graph showing the results of MALDI-TOF analysis of the hydrophobic peptides of Examples 1 to 36. [Figure 18] 1 is a graph showing the results of MALDI-TOF analysis of the hydrophobic peptides of Examples 1 to 36. [Figure 19] 1 is a graph showing the results of MALDI-TOF analysis of the hydrophobic peptides of Examples 1 to 36. [Figure 20] 1 is a graph showing the results of MALDI-TOF analysis of the hydrophobic peptides of Examples 1 to 36. [Figure 21] 1 is a graph showing the results of MALDI-TOF analysis of the hydrophobic peptides of Examples 1 to 36. [Figure 22] 1 is a graph showing the results of MALDI-TOF analysis of the hydrophobic peptides of Examples 1 to 36. [Figure 23] 1 is a graph showing the results of MALDI-TOF analysis of the hydrophobic peptides of Examples 1 to 36. [Figure 24] 1 is a graph showing the results of MALDI-TOF analysis of the hydrophobic peptides of Examples 1 to 36. [Figure 25] 1 is a graph showing the results of MALDI-TOF analysis of the hydrophobic peptides of Examples 1 to 36. [Figure 26] 1 is a graph showing the results of MALDI-TOF analysis of the hydrophobic peptides of Examples 1 to 36. [Figure 27] 1 is a graph showing the results of MALDI-TOF analysis of the hydrophobic peptides of Examples 1 to 36. [Figure 28] 1 is a graph showing the results of MALDI-TOF analysis of the hydrophobic peptides of Examples 1 to 36. [Figure 29] 1 is a graph showing the results of MALDI-TOF analysis of the hydrophobic peptides of Examples 1 to 36. [Figure 30] 1 is a graph showing the results of MALDI-TOF analysis of the hydrophobic peptides of Examples 1 to 36. [Figure 31] 1 is a graph showing the results of MALDI-TOF analysis of the hydrophobic peptides of Examples 1 to 36. [Figure 32] 1 is a graph showing the results of MALDI-TOF analysis of the hydrophobic peptides of Examples 1 to 36. [Figure 33] 1 is a graph showing the results of MALDI-TOF analysis of the hydrophobic peptides of Examples 1 to 36. [Figure 34] 1 is a graph showing the results of MALDI-TOF analysis of the hydrophobic peptides of Examples 1 to 36. [Figure 35] 1 is a graph showing the results of MALDI-TOF analysis of the hydrophobic peptides of Examples 1 to 36. [Figure 36] 1 is a graph showing the results of MALDI-TOF analysis of the hydrophobic peptides of Examples 1 to 36. [Figure 37]FIG. 1 shows the structures of the peptide conjugates prepared in Examples 37 to 40 and the peptide conjugates prepared in Examples 125 and 128. [Figure 38] FIG. 1 shows the structures of peptide conjugates prepared in Examples 46 to 50. [Figure 39] FIG. 1 shows the structures of cyclic peptide conjugates prepared in Examples 98 to 102. [Figure 40] 1 is a graph showing the results of MALDI-TOG mass spectrometry for the peptide conjugates prepared in Examples 37, 39, 40, 46-50, 60, 98-102, 125, and 128. [Figure 41] 1 is a graph showing HPLC chromatography of the peptide conjugates prepared in Examples 37, 39, 40, 46-50, 60, 98-102, 125, and 128. [Figure 42](a) is a schematic diagram showing the diamagnetic anisotropy of peptide bonds in hydrophobic peptides according to the present invention, where B0 represents the external magnetic field (MF). (b) is a schematic diagram showing the α-helical structure and helical axis of hydrophobic peptides according to the present invention, indicating aligned peptide bonds, and the difference in diamagnetic susceptibility between the parallel and perpendicular directions due to this structure. (c) is a diagram showing the rod-coil structure of peptide complexes (αm-RGD) prepared in Examples 37 to 41. The peptide complexes according to the present invention form peptide complexes with a rod-coil structure by binding a hydrophobic peptide containing a repeating unit (α) consisting of two amino acid residues to a hydrophilic peptide (RGD). (d) is a CD spectrum for the peptide complexes (α4-RGD2, α7-RGD2, and α10-RGD2) shown in Examples 37, 39, and 40. (e) is a diagram showing the structure of the peptide conjugate (PEG16-α10-PEG16) prepared in Example 125. (f) is a CD spectrum of the peptide conjugate prepared in Example 125 as a function of concentration. (g) is a graph showing the helicity of the peptide conjugate prepared in Example 125 as a function of concentration. [Figure 43] These are AFM images of the peptide complexes (a, b, c, d, respectively) of Examples 37, 39, 40, and 125 (a4-(RGD)2, a7-(RGD)2, a10-(RGD)2, and PEG16-a10-PEG16), and are intended to compare the self-assembly behavior of peptide complexes with rod-coil structures and peptide complexes with coil-rod-coil structures. [Figure 44](a) is a 1H-15N IPAP-HSQC spectrum for the peptide complex (PEG30-α10-PEG30) of Example 128. (b) is a diagram showing the behavior of the peptide complex (PEG30-α10-PEG30) of Example 128 under magnetic field conditions. (c) is a diagram showing the difference in chemical structure between the peptide complexes of Examples 47 and 99. (d) is a CD and MCD (magnetic circular dichroism) spectrum for the peptide complex (L-α5-PEG10) of Example 47. (e) is a CD and MCD (magnetic circular dichroism) spectrum for the peptide complex (C-α5-PEG10) of Example 99. (f) is a diagram showing an experimental setup in which a peptide complex (specimen) dispersed in an aqueous solution is placed between neodymium magnets. (g) is an AFM image analyzing the self-assembly of the peptide complex (C-α5-PEG10) of Example 99 in the presence or absence of a magnetic field. The right side shows the case where a magnetic field is present, and the left side shows the case where a magnetic field is not present. (h) is an AFM image analyzing the self-assembly of the peptide complex (α10-(RGD)3) of Example 51 in the presence and absence of a magnetic field. The right side shows the case where a magnetic field is present, and the left side shows the case where a magnetic field is not present. (i) is a schematic diagram showing a model of the self-assembly process in the presence and absence of a magnetic field (with, without). [Figure 45] 700. 1H NMR spectrum of the peptide conjugate (PEG30-α10-PEG30) of Example 128 measured at 400 MHz (16.45 T). [Figure 46] 700. 1H-15N IPAP-HSQC spectrum of the peptide conjugate (PEG30-α10-PEG30) of Example 128 measured at 400 MHz (16.45 T). [Figure 47] 1H NMR spectrum of the peptide conjugate (PEG30-α10-PEG30) of Example 128 measured at 900.230 MHz (21.14 T). [Figure 48]1H-15N IPAP-HSQC spectrum of the peptide conjugate (PEG30-α10-PEG30) of Example 128 measured at 900.230 MHz (21.14 T). [Figure 49] (a) shows the structure of the peptide conjugate (R-a10-PEG16) of Example 60. In this peptide conjugate, there are two positive charges at the N-terminus of the hydrophobic peptide, one of which is derived from an arginine residue and the other from the N-terminal amine. (b) is an AFM image of linearized plasmid DNA (7.5 kb). (c) is an AFM image of the peptide conjugate (R-a10-PEG16) of Example 60. (d) is the result of an electrophoretic mobility shift assay (EMSA) for peptide-DNA nanostructures prepared by mixing linearized plasmid DNA (7.5 kb) with various concentrations of the peptide conjugate (R-a10-PEG16) of Example 60. (e) to (h) show the results of AFM and TEM measurements of the process of peptide-DNA nanostructure formation and the morphological changes of the nanostructure after mixing linear plasmid DNA (7.5 kb) with the peptide complex (R-a10-PEG16) of Example 60. (e) is an AFM image of the initial peptide-DNA nanostructure, (f) is an AFM image of the intermediate peptide-DNA nanostructure, (g) is a TEM image of the intermediate or later stage peptide-DNA nanostructure, and (h) is a TEM image of the later stage peptide-DNA nanostructure. (i) is a diagram showing the mechanism by which peptide-DNA nanostructures are formed into nanoribbon structures. [Figure 50] This is a cleavage map of the plasmid DNA used in the present invention, which was cleaved at the BamHI site. [Figure 51] 1 shows CD spectra for peptide-DNA nanostructures of the peptide complex of Example 60 and linear plasmid DNA. [Figure 52]Selected area electron diffraction (SAED) patterns for a peptide-DNA nanostructure in a nanoribbon state (a) and a peptide-DNA nanostructure in an artificial chromosome state (b). [Figure 53] 1 is a graph showing the results of WAXS (Synchrotron wide-angle X-ray scattering) analysis of the peptide complex of Example 60, linear plasmid DNA, and peptide-DNA nanostructure. [Figure 54] (a) CD spectrum of peptide-DNA nanostructures (artificial chromosome form). (b) Electrophoresis results analyzing the degradation of peptide-DNA nanostructures (artificial chromosome form) in the presence of DNase I. Here, the ladder is a DNA size marker, the DNA is linear plasmid DNA, the complex is the peptide-DNA nanostructure (artificial chromosome form), and the DNase is DNase I. The arrows indicate the peptide-DNA nanostructures (artificial chromosome form) confined in lanes 4 and 5. (c) Electrophoresis results analyzing the degradation of peptide-DNA nanostructures (artificial chromosome form) of Example 60 and linear plasmid DNA in the presence of DNase I over time (0 to 600 seconds). (d) PCR analysis results for peptide-DNA nanostructures (artificial chromosome form) of Example 60 and linear plasmid DNA. Lane 1 is a DNA size marker, Lane 2 is linearized plasmid DNA amplified by PCR, and Lane 3 is DNA amplified by PCR from peptide-DNA nanostructures (artificial chromosome form). [Figure 55](a) is a TEM image of the peptide-DNA nanostructure (nanoribbon form) of the peptide complex of Example 60 and linear plasmid DNA before exposure to a magnetic field. (b) is an experimental setup for generating a rotating magnetic field (RMF) using a permanent magnet. (c) to (i) are TEM images of the structural changes that occurred after the peptide-DNA nanostructure (nanoribbon form) of the peptide complex of Example 60 and linear plasmid DNA was exposed to a rotating magnetic field. [Figure 56] 1 shows AFM images of peptide-DNA nanostructures (nanoribbon form) of the peptide complex of Example 60 and linear plasmid DNA after exposure to a static magnetic field (0.1 T) for two weeks. [Figure 57a] 1 is an HPLC analysis graph of the peptide conjugate of Example 37 (1.286 mM). [Figure 57b] 1 is a MALDI-TOF mass spectrometry graph for the peptide conjugate of Example 37 (1.286 mM). [Figure 58a] 1 is an HPLC analysis graph for the peptide conjugate of Example 38. [Figure 58b] 1 is a MALDI-TOF mass spectrometry graph for the peptide conjugate of Example 38. [Figure 59a] 1 is an HPLC analysis graph for the peptide conjugate of Example 39. [Figure 59b] 1 is a MALDI-TOF mass spectrometry graph for the peptide conjugate of Example 39. [Figure 60a] 1 is an HPLC analysis graph for the peptide conjugate of Example 40. [Figure 60b] 1 is a MALDI-TOF mass spectrometry graph for the peptide conjugate of Example 40. [Figure 61] 1 is an HPLC analysis graph for the peptide conjugate of Example 53. [Figure 62a] 1 is an HPLC analysis graph for the peptide conjugates of Examples 60 to 63. [Figure 62b] 1 is a MALDI-TOF mass spectrometry graph for the peptide conjugates of Examples 60 to 63. In the figure, R1 is the peptide conjugate of Example 60, R2 is the peptide conjugate of Example 61, R3 is the peptide conjugate of Example 62, and R4 is the peptide conjugate of Example 63. [Figure 63a] 1 is an HPLC analysis graph for the peptide conjugate of Example 90. [Figure 63b] 1 is a MALDI-TOF mass spectrometry graph for the peptide conjugate of Example 90. [Figure 64a] 1 is an HPLC analysis graph for the peptide conjugate of Example 91. [Figure 64b] 1 is a MALDI-TOF mass spectrometry graph for the peptide conjugate of Example 91. [Figure 65a] 1 is an HPLC analysis graph for the peptide conjugate of Example 71. [Figure 65b] 1 is a MALDI-TOF mass spectrometry graph for the peptide conjugate of Example 71. [Figure 66] 1 is a MALDI-TOF mass spectrometry graph for the peptide conjugate of Example 72. [Figure 67a] 1 is an HPLC analysis graph for the peptide conjugate of Example 66. [Figure 67b] 1 is a MALDI-TOF mass spectrometry graph for the peptide conjugate of Example 66. [Figure 68a] 1 is an HPLC analysis graph for the peptide conjugate of Example 67. [Figure 68b] 1 is a MALDI-TOF mass spectrometry graph for the peptide conjugate of Example 67. [Figure 69a] 1 is an HPLC analysis graph for the peptide conjugate of Example 52. [Figure 69b] 1 is a MALDI-TOF mass spectrometry graph for the peptide conjugate of Example 52. [Figure 70] 1 is a MALDI-TOF mass spectrometry graph for the peptide conjugate of Example 53. [Figure 71a]1 is an HPLC analysis graph for the peptide conjugate of Example 46. [Figure 71b] 1 is a MALDI-TOF mass spectrometry graph for the peptide conjugate of Example 46. [Figure 72a] 1 is an HPLC analysis graph for the peptide conjugate of Example 47. [Figure 72b] 1 is a MALDI-TOF mass spectrometry graph for the peptide conjugate of Example 46. [Figure 73a] 1 is an HPLC analysis graph for the peptide conjugate of Example 48. [Figure 73b] 1 is a MALDI-TOF mass spectrometry graph for the peptide conjugate of Example 48. [Figure 74a] 1 is an HPLC analysis graph for the peptide conjugate of Example 49. [Fig. 74b] 1 is a MALDI-TOF mass spectrometry graph for the peptide conjugate of Example 49. [Figure 75a] 1 is an HPLC analysis graph for the peptide conjugate of Example 50. [Figure 75b] 1 is a MALDI-TOF mass spectrometry graph for the peptide conjugate of Example 50. [Figure 76a] 1 is an HPLC analysis graph for the peptide conjugate of Example 51. [Figure 76b] 1 is a MALDI-TOF mass spectrometry graph for the peptide conjugate of Example 51. [Figure 77] 1 shows CD spectrographs for the peptide conjugates of Examples 37(a), 39(b), and 40(c). [Figure 78] 1 is a CD spectrum graph for the peptide conjugate of Example 38. [Figure 79] 1 is a CD spectrum graph for the peptide conjugate of Example 40. [Figure 80a] 1A-1D are AFM images of the peptide conjugates of Examples 37(a), 38(b), 39(c) and 40(d). [Figure 80b]1A-1D are AFM images of the peptide conjugates of Examples 37(a), 38(b), 39(c) and 40(d). [Figure 80c] 1A-1D are AFM images of the peptide conjugates of Examples 37(a), 38(b), 39(c) and 40(d). [Figure 80d] 1A-1D are AFM images of the peptide conjugates of Examples 37(a), 38(b), 39(c) and 40(d). [Figure 81a] 1 shows an image of the peptide complex (R-α10-PEG16) of Example 60 taken with an AFM. [Figure 81b] This is an image of dsDNA taken with an AFM. [Figure 81c] The image was taken with an AFM 2 to 3 minutes after fabrication of the R-α10-PEG16 / dsDNA nanostructure. [Figure 81d] The image was taken with an AFM 2 to 3 minutes after fabrication of the R-α10-PEG16 / dsDNA nanostructure. [Figure 81e] This image was taken with an AFM 12 days after the R-α10-PEG16 / dsDNA nanostructure was fabricated. [Figure 81f] This image was taken with an AFM 22 days after the R-α10-PEG16 / dsDNA nanostructure was fabricated. [Figure 82a] The images were taken with a TEM 2 to 3 minutes after the R-α10-PEG16 / dsDNA nanostructure was fabricated. [Figure 82b] This is a TEM image taken 12 days after fabrication of the R-α10-PEG16 / dsDNA nanostructure. [Figure 82c] The image was taken by TEM 22 days after the R-α10-PEG16 / dsDNA nanostructure was fabricated. [Figure 82d] The image was taken by TEM 22 days after the R-α10-PEG16 / dsDNA nanostructure was fabricated. [Figure 83]1 shows CD spectra for the peptide complex (R-α10-PEG16) of Example 60, dsDNA, and R-α10-PEG16 / dsDNA nanostructure. [Figure 84] This is a selected area electron diffraction pattern (SAED) of the R-α10-PEG16 / dsDNA nanostructure with a long nanoribbon structure. [Figure 85] This is a selected area electron diffraction pattern (SAED) of the R-α10-PEG16 / dsDNA nanostructure with an artificial chromosome structure. [Figure 86] The results were obtained by UV-vis measurement after adding DNase I to dsDNA and R-α10-PEG16 / dsDNA nanostructures. [Figure 87] 10 is an AFM image of the peptide complex of Example 63. [Figure 88] AFM images were taken 40 minutes after fabrication of the R4-α10-PEG16 / dsDNA nanostructure. [Figure 89] This is an AFM image taken 111 days after fabrication of the R4-α10-PEG16 / dsDNA nanostructure. [Figure 90] CD spectrum of the R4-α10-PEG16 / dsDNA nanostructure. [Figure 91a] EMSA results for the nanostructure of the peptide complex (R-α10-PEG16) and dsDNA in Example 60. [Figure 91b] EMSA results for the peptide complex (R-α10-PEG16) and ssDNA nanostructure of Example 60. [Figure 91c] EMSA results for the nanostructure of the peptide complex (R-α10-PEG16) and pDNA in Example 61. [Figure 91d] EMSA results for the nanostructure of the peptide complex (R-α10-PEG16) and dsDNA in Example 61. [Figure 91e]EMSA results for the nanostructure of the peptide complex (R-α10-PEG16) and dsDNA in Example 62. [Figure 91f] EMSA results for the nanostructure of the peptide complex (R-α10-PEG16) and dsDNA in Example 63. [Figure 92a] HPLC (a), MALDI-TOF (b), and CD spectra (c) for the cyclic peptide conjugates of Examples 98 to 103, 106, and 109 to 111. [Figure 92b] HPLC (a), MALDI-TOF (b), and CD spectra (c) for the cyclic peptide conjugates of Examples 98 to 103, 106, and 109 to 111. [Figure 92c] HPLC (a), MALDI-TOF (b), and CD spectra (c) for the cyclic peptide conjugates of Examples 98 to 103, 106, and 109 to 111. [Figure 93a] HPLC (a), MALDI-TOF (b), and CD spectra (c) for the cyclic peptide conjugates of Examples 98 to 103, 106, and 109 to 111. [Figure 93b] HPLC (a), MALDI-TOF (b), and CD spectra (c) for the cyclic peptide conjugates of Examples 98 to 103, 106, and 109 to 111. [Figure 93c] HPLC (a), MALDI-TOF (b), and CD spectra (c) for the cyclic peptide conjugates of Examples 98 to 103, 106, and 109 to 111. [Figure 94a] HPLC (a), MALDI-TOF (b), and CD spectra (c) for the cyclic peptide conjugates of Examples 98 to 103, 106, and 109 to 111. [Figure 94b] HPLC (a), MALDI-TOF (b), and CD spectra (c) for the cyclic peptide conjugates of Examples 98 to 103, 106, and 109 to 111. [Figure 94c]HPLC (a), MALDI-TOF (b), and CD spectra (c) for the cyclic peptide conjugates of Examples 98 to 103, 106, and 109 to 111. [Figure 95a] HPLC (a), MALDI-TOF (b), and CD spectra (c) for the cyclic peptide conjugates of Examples 98 to 103, 106, and 109 to 111. [Figure 95b] HPLC (a), MALDI-TOF (b), and CD spectra (c) for the cyclic peptide conjugates of Examples 98 to 103, 106, and 109 to 111. [Figure 95c] HPLC (a), MALDI-TOF (b), and CD spectra (c) for the cyclic peptide conjugates of Examples 98 to 103, 106, and 109 to 111. [Figure 96a] HPLC (a), MALDI-TOF (b), and CD spectra (c) for the cyclic peptide conjugates of Examples 98 to 103, 106, and 109 to 111. [Figure 96b] HPLC (a), MALDI-TOF (b), and CD spectra (c) for the cyclic peptide conjugates of Examples 98 to 103, 106, and 109 to 111. [Figure 96c] HPLC (a), MALDI-TOF (b), and CD spectra (c) for the cyclic peptide conjugates of Examples 98 to 103, 106, and 109 to 111. [Figure 97a] HPLC (a), MALDI-TOF (b), and CD spectra (c) for the cyclic peptide conjugates of Examples 98 to 103, 106, and 109 to 111. [Figure 97b] HPLC (a), MALDI-TOF (b), and CD spectra (c) for the cyclic peptide conjugates of Examples 98 to 103, 106, and 109 to 111. [Figure 97c] HPLC (a), MALDI-TOF (b), and CD spectra (c) for the cyclic peptide conjugates of Examples 98 to 103, 106, and 109 to 111. [Figure 98a]HPLC (a), MALDI-TOF (b), and CD spectra (c) for the cyclic peptide conjugates of Examples 98 to 103, 106, and 109 to 111. [Figure 98b] HPLC (a), MALDI-TOF (b), and CD spectra (c) for the cyclic peptide conjugates of Examples 98 to 103, 106, and 109 to 111. [Figure 98c] HPLC (a), MALDI-TOF (b), and CD spectra (c) for the cyclic peptide conjugates of Examples 98 to 103, 106, and 109 to 111. [Figure 99a] HPLC (a), MALDI-TOF (b), and CD spectra (c) for the cyclic peptide conjugates of Examples 98 to 103, 106, and 109 to 111. [Figure 99b] HPLC (a), MALDI-TOF (b), and CD spectra (c) for the cyclic peptide conjugates of Examples 98 to 103, 106, and 109 to 111. [Figure 99c] HPLC (a), MALDI-TOF (b), and CD spectra (c) for the cyclic peptide conjugates of Examples 98 to 103, 106, and 109 to 111. [Figure 100a] HPLC (a), MALDI-TOF (b), and CD spectra (c) for the cyclic peptide conjugates of Examples 98 to 103, 106, and 109 to 111. [Figure 100b] HPLC (a), MALDI-TOF (b), and CD spectra (c) for the cyclic peptide conjugates of Examples 98 to 103, 106, and 109 to 111. [Figure 100c] HPLC (a), MALDI-TOF (b), and CD spectra (c) for the cyclic peptide conjugates of Examples 98 to 103, 106, and 109 to 111. [Figure 101a] HPLC (a), MALDI-TOF (b), and CD spectra (c) for the cyclic peptide conjugates of Examples 98 to 103, 106, and 109 to 111. [Figure 101b]HPLC (a), MALDI-TOF (b), and CD spectra (c) for the cyclic peptide conjugates of Examples 98 to 103, 106, and 109 to 111. [Figure 101c] HPLC (a), MALDI-TOF (b), and CD spectra (c) for the cyclic peptide conjugates of Examples 98 to 103, 106, and 109 to 111. [Figure 102] 1 is a graph showing an analysis of Caco-2 cell viability in response to the peptide complexes according to Examples 40, 60, and 102 and the nanostructures of artificial chromosome structures prepared in Experimental Examples 13 and 14. [Figure 103a] HPLC (a) and MALDI-TOF (b) for the peptide conjugates obtained in Examples 113 to 115, 119 to 121, 125, and 128. In these figures, when only one figure is present without a or b, it is the MALDI-TOF graph for the corresponding peptide conjugate. [Figure 103b] HPLC (a) and MALDI-TOF (b) for the peptide conjugates obtained in Examples 113 to 115, 119 to 121, 125, and 128. In these figures, when only one figure is present without a or b, it is the MALDI-TOF graph for the corresponding peptide conjugate. [Figure 104a] HPLC (a) and MALDI-TOF (b) for the peptide conjugates obtained in Examples 113 to 115, 119 to 121, 125, and 128. In these figures, when only one figure is present without a or b, it is the MALDI-TOF graph for the corresponding peptide conjugate. [Figure 104b] HPLC (a) and MALDI-TOF (b) for the peptide conjugates obtained in Examples 113 to 115, 119 to 121, 125, and 128. In these figures, when only one figure is present without a or b, it is the MALDI-TOF graph for the corresponding peptide conjugate. [Figure 105a]HPLC (a) and MALDI-TOF (b) for the peptide conjugates obtained in Examples 113 to 115, 119 to 121, 125, and 128. In these figures, when only one figure is present without a or b, it is the MALDI-TOF graph for the corresponding peptide conjugate. [Figure 105b] HPLC (a) and MALDI-TOF (b) for the peptide conjugates obtained in Examples 113 to 115, 119 to 121, 125, and 128. In these figures, when only one figure is present without a or b, it is the MALDI-TOF graph for the corresponding peptide conjugate. [Figure 106] HPLC (a) and MALDI-TOF (b) for the peptide conjugates obtained in Examples 113 to 115, 119 to 121, 125, and 128. In these figures, when only one figure is present without a or b, it is the MALDI-TOF graph for the corresponding peptide conjugate. [Figure 107] HPLC (a) and MALDI-TOF (b) for the peptide conjugates obtained in Examples 113 to 115, 119 to 121, 125, and 128. In these figures, when only one figure is present without a or b, it is the MALDI-TOF graph for the corresponding peptide conjugate. [Figure 108] HPLC (a) and MALDI-TOF (b) for the peptide conjugates obtained in Examples 113 to 115, 119 to 121, 125, and 128. In these figures, when only one figure is present without a or b, it is the MALDI-TOF graph for the corresponding peptide conjugate. [Figure 109a] HPLC (a) and MALDI-TOF (b) for the peptide conjugates obtained in Examples 113 to 115, 119 to 121, 125, and 128. In these figures, when only one figure is present without a or b, it is the MALDI-TOF graph for the corresponding peptide conjugate. [Figure 109b]HPLC (a) and MALDI-TOF (b) for the peptide conjugates obtained in Examples 113 to 115, 119 to 121, 125, and 128. In these figures, when only one figure is present without a or b, it is the MALDI-TOF graph for the corresponding peptide conjugate. [Figure 110] HPLC (a) and MALDI-TOF (b) for the peptide conjugates obtained in Examples 113 to 115, 119 to 121, 125, and 128. In these figures, when only one figure is present without a or b, it is the MALDI-TOF graph for the corresponding peptide conjugate. DETAILED DESCRIPTION OF THE INVENTION

[0043] The present invention will be described in detail below.

[0044] The present invention provides magnetically responsive peptides and peptide nanostructures containing the same, which can be used in magnetic applications. To achieve the above-mentioned objectives, the present invention develops peptide monomers that tend to align parallel to an applied magnetic field and have a nonpolar and perfect α-helical structure (MNP-helix, monomeric, nonpolar, and perfect α-helix).

[0045] The hydrophobic peptide of the present invention has an α-helical structure, which is a common secondary structure. Similar to an aromatic ring, this structure has been confirmed to have significant diamagnetic anisotropy due to the partial double bond characteristics of the resonance-stabilized peptide bond (Figure 42(a)). Furthermore, it was found that the peptide bonds of the hydrophobic peptide of the present invention are oriented parallel to the helical axis, enhancing the overall diamagnetism of the α-helical structure compared to a single peptide bond (Figure 42(b)). The hydrophobic peptide of the present invention forms a rod structure with multiple α-helices aligned, which is a novel structure that responds sensitively even to low magnetic field strengths.

[0046] The hydrophobic peptides of the present invention act as supramolecular building blocks, and can provide peptide complexes with rod-coil structures by binding with hydrophilic molecules.

[0047] The hydrophobic peptides of the present invention act as hydrophobic blocks of rigid rod structures, which bind with hydrophilic blocks of flexible coil structures to form peptide complexes that assume the amphiphilic properties of rod-coil structures.

[0048] As mentioned above, peptide complexes with rod-coil structures have many advantages over conventional coil-coil molecules in terms of reactive self-assembly, mechanics, and well-ordered structure formation because the hydrophobic block composed of the hydrophobic peptides confers orientation and the entropic penalty associated with chain stretching in coil-coil structures is reduced in rod-coil structures.

[0049] Conventional peptides with α-helical rod structures have been unable to maintain an α-helical structure in the monomeric state. Furthermore, most α-helical peptide sequences present in proteins are only stabilized by noncovalent interactions with other residues present in the protein, and have the problem of being unable to maintain their structure independently and becoming separated. The α-helical structures of some peptides discovered or designed in proteins clearly have a helical shape in the monomeric state. However, these helical structures are all highly charged and hydrophilic, making them unsuitable for use as supramolecular building blocks.

[0050] To solve the above-mentioned problems, the present invention explores the synergistic effect of α-helix and rod-coil structures to develop organic molecules that respond to the magnetic field generated by a permanent magnet. To this end, we first designed and completed a new hydrophobic peptide with an MNP α-helix structure.

[0051] It was confirmed that the hydrophobic peptide of the present invention can control the self-assembly and disassembly processes even in a relatively weak magnetic field (0.07 T to 0.25 T) by using it as a rod block in a peptide complex with rod-coil amphiphilicity.

[0052] Furthermore, because the peptide complexes containing the hydrophobic peptides of the present invention are positively charged, they interact with various negatively charged substances, particularly forming nanostructures with structures similar to artificial chromosomes through self-assembly with genetic material (e.g., nucleic acids). These nanostructures are magnetically responsive organic molecules, a novel concept, that can safely store and transmit genetic material by densely packaging it, and whose release can be controlled by a magnetic field. The present invention was completed based on the discovery that these nanostructures, which are capable of controlling various magnetic responses, can be useful in a variety of applied research fields, such as medicine, cosmetics, stimuli-responsive molecular machines, and the control of the movement of organic materials.

[0053] One aspect of the present invention relates to a hydrophobic peptide represented by the following general formula 1:

[0054] [General formula 1] -{[Aib]-[Xaa1]}m-

[0055] [General formula 2] -[Xaa2]n-{[Aib]-[Xaa1]}m-

[0056] In the above formula, Xaa1 and Xaa2 are each independently any one selected from the group consisting of Ala (A), Ile (I), Leu (L), Met (M), and Val (V).

[0057] Xaa1 and Xaa2 are each independently an amino acid residue of D or L configuration.

[0058] The n is an integer of 1.

[0059] The m is an integer selected from 4 to 50.

[0060] As used herein, the term "peptide" or "polypeptide" refers to a linear molecule formed by the binding of amino acid residues to each other by peptide bonds, and is composed of 4 to 100 amino acid residues, preferably 4 to 80, more preferably 10 to 80, even more preferably 20 to 80, and most preferably 20 to 40.

[0061] The hydrophobic peptides represented by general formula 1 or 2 can be represented by sequence listings 121 and 122. They are not derived from conventional proteins, but were developed by trying numerous combinations of conventional amino acid residues so that they can stably maintain an α-helical structure, are hydrophobic, and have a rod structure. They are designed to contain a repeating unit of [Aib]-[Xaa1] or [Xaa1]-[Aib] (hereinafter also referred to as "α").

[0062] Through experiments, the inventors have confirmed that despite its short length, the sequence has a stable α-helical structure and a rod structure, and that it can be aligned in real time in response to a weak magnetic field, and that it forms a structure similar to an artificial chromosome through interaction with genetic material.

[0063] In the present invention, the hydrophobic peptide of formula 1 or 2 is a newly designed sequence that is not derived from any other source. Among the secondary structures of conventional peptides, the α-helices structure is known to become more stable as the length of the peptide chain increases. However, as the peptide length increases, not only does peptide synthesis become more difficult, but the synthesis of supramolecular building blocks with specific structures is also hindered. In fact, when peptides consisting of short repeating units were synthesized through the combination of various amino acid residues, it was found that most of them were unable to form an α-helix structure, or even if they did form an α-helix structure, they were unable to maintain it and remained in a decomposed state.

[0064] Through the above-mentioned preliminary experiments, the present invention has confirmed that a repeating unit (or repeating sequence) peptide prepared from a hydrophobic amino acid residue such as alanine (Ala or A) and Aib (2-aminoisobutyric acid, U) has a stable α-helical structure while having suitable hydrophobicity for self-assembly, and exhibits high orientation in response to a magnetic field.

[0065] According to one embodiment of the present invention, the hydrophobic peptide of the present invention has the advantage of having a perfectly stabilized α-helical structure while existing in solution in a monomeric form without aggregation.

[0066] According to one embodiment of the present invention, in the above formula, m may be any one integer selected from 4 to 50, more preferably, m may be any one integer selected from 6 to 13, even more preferably, m may be any one integer selected from 6 to 10, and most preferably, m may be 10.

[0067] According to one embodiment of the present invention, in the formula, Xaa1 may be any one selected from the group consisting of Ala (A), Ile (I), Leu (L) and Met (M), and preferably Ala (A) or Leu (L).

[0068] In the above formula, it is most preferable to use alanine (Ala or A) as Xaa1 in order to have a stable α-helical structure while having appropriate hydrophobicity for self-assembly and to have high orientation by a magnetic field.

[0069] According to one embodiment of the present invention, Xaa1 may be an L-amino acid residue or a D-amino acid residue, but is not particularly limited thereto.

[0070] According to one embodiment of the present invention, the hydrophobic peptide comprising the amino acid sequence represented by general formula 1 or 2 is a polypeptide composed of repeat units of the shortest sequence having a newly designed α-helical structure, and the hydrophobic peptide may be any one selected from the sequences represented by SEQ ID NOs: 1 to 36, preferably any one selected from SEQ ID NOs: 5 to 19, 24 to 36, and more preferably any one selected from the group consisting of SEQ ID NOs: 13 to 14, 32 to 33.

[0071] The hydrophobic peptide is characterized by its hydrophobicity, stable α-helical secondary structure, alignment in the direction of an external magnetic field, and excellent stimuli responsiveness, even in low magnetic fields of 1 T or less.

[0072] Due to the above-mentioned characteristics, the hydrophobic peptide according to the present invention is characterized by having a rigid rod structure that can stably maintain multiple α-helical structures.

[0073] The hydrophobic peptide according to the present invention has its alignment and orientation adjusted by a magnetic field, and as explained above, is aligned and oriented parallel to the direction of the external magnetic field.

[0074] Conventional peptides, because they are not contained within a specific structure, cannot maintain an α-helical structure, and are also very weak diamagnetic materials, so they have not been able to achieve magnetic field responsiveness. However, the present invention solves all of the above problems through a repeating unit (hereinafter also referred to as "α") consisting of the shortest amino acid sequence composed of [Aib]-[Xaa1] or [Xaa1]-[Aib]. This has achieved a significant effect in the relevant field by completing and providing a new magnetically responsive peptide that maintains a stable α-helical structure in the monomer state even without being constrained within a ring or a large protein, and is aligned and oriented parallel to the direction of an external magnetic field.

[0075] Furthermore, the hydrophobic peptide according to the present invention can be easily imparted with new functions without losing the above-mentioned characteristics through conjugation with a hydrophilic polymer or a hydrophilic peptide. It is useful for preparing an amphiphilic molecule and storing and delivering it as a micelle-shaped particle through self-assembly, and is therefore expected to be utilized in various fields such as medicine, stimuli-responsive molecular machines, and cosmetics.

[0076] Furthermore, the hydrophobic peptide according to the present invention has the advantage that even if it is mixed with a nucleic acid substance, it does not affect genetic information analysis methods such as PCR and does not act as noise, allowing for immediate analysis of nucleic acid substances without a separate purification process.

[0077] Another aspect of the present invention provides a peptide conjugate comprising the hydrophobic peptide; and at least one hydrophilic polymer or hydrophilic peptide bound to both ends or one end of the hydrophobic peptide.

[0078] As described above, the peptide conjugate of the present invention may have one or more hydrophilic polymers or hydrophilic peptides bound to one or both ends of the hydrophobic peptide.

[0079] The details regarding the hydrophobic peptides overlap with those described above, so please refer to the above contents.

[0080] The one end of the hydrophobic peptide means either the N-terminus or the C-terminus of the hydrophobic peptide, and both ends mean both the N-terminus and the C-terminus of the hydrophobic peptide.

[0081] The "N-terminus" refers to the first amino acid residue in the hydrophobic peptide sequence. The N-terminal residue contains a free α-amino group. The "C-terminus" refers to the last amino acid residue in the hydrophobic peptide sequence. The C-terminal residue contains a free carboxylate group.

[0082] In the present invention, a "polymer" refers to a macromolecule having repeating units linked by covalent bonds. Polymers can be hydrophilic, hydrophobic, or amphiphilic, but in the present invention, hydrophilic polymers are preferred. Polymers can include homopolymers, random copolymers, and block copolymers.

[0083] The "hydrophilic polymer" is not particularly limited as long as it is substantially miscible with water, but is preferably polyethylene glycol (PEG), poly-N-(2-hydroxypropyl)methacrylamide, poly(2-(methacryloyloxy)ethyl phosphorylcholines), poly(hydroxyalkyl L-asparagine), or poly(hydroxyalkyl L-glutamine), and is preferably polyethylene glycol (PEG).

[0084] The hydrophilic polymer is preferably an unbranched linear polymer, not crosslinked, and may have a molecular weight of about 1 kDa to about 100 kDa, preferably about 1 kDa to about 75 kDa, more preferably about 5 kDa to about 50 kDa, and even more preferably about 5 kDa to about 25 kDa.

[0085] The polyethylene glycol (PEG) contains a PEG monomer and can be represented by the chemical formula: CH3-O(CH2CHO)n- or -O(CH2CHO)n-, where n is a positive integer from about 10 to about 2,300.

[0086] The term "hydrophilic peptide" refers to a peptide that has hydrophilic properties, but is not limited thereto, and as understood by those skilled in the art, refers to a peptide that has a high ratio of polar amino acid or hydrophilic amino acid residues and has hydrophilic properties as a whole. The hydrophilic amino acids may include H (histidine), N (asparagine), Q (glutamine), S (serine), T (threonine), C (cysteine), G (glycine), K (lysine), R (arginine), E (glutamic acid), and D (aspartic acid).

[0087] The hydrophilic peptide may be characterized by being composed of an amino acid sequence of 3 to 100, preferably 3 to 60, more preferably 3 to 20, amino acids, and having a hydrophilic amino acid content of 70% to 100%.

[0088] The hydrophilic peptide is not particularly limited as long as it satisfies the above-mentioned conditions, and preferably may be an optional ligand which may or may not be present, such as RGD, transferrin, folate, a signal peptide or signal sequence, a localization signal or sequence, a nucleic localization signal or sequence (NLS), an antibody, a cell penetrating peptide (e.g., TAT or KALA), a receptor ligand (e.g., cytokines, hormones, growth factors, etc.), a small molecule (e.g., carbohydrates such as mannose, galactose, synthetic ligands), a small molecule agonist, and a receptor inhibitor or antagonist of a receptor. The hydrophilic peptide may be any one independently selected from the following amino acid sequences:

[0089] [Hydrophilic peptides]

[0090] [Table 1]

[0091] The hydrophobic peptide and the hydrophilic polymer or hydrophilic peptide are linked by a chemical bond, and they may be linked directly or via a separate linker.

[0092] The chemical bond or linkage via a linker may vary depending on the terminal sequence of the peptide and the type of the terminal of the polymer. Examples thereof include an amide bond, an ether bond, a thioether bond, an ester bond, a thioester bond, a carbonate bond, a carbamate bond, a phosphate bond, and an oxime bond, with an amide bond being preferred.

[0093] In this case, the linker can be any substance that can be linked to the end of the hydrophobic peptide, the end of the hydrophilic peptide, or the end of the hydrophilic polymer, and can form the above chemical bond.

[0094] Examples include dicyclohexylcarbodimide (DCC), 1,4-bis-maleimidobutane (BMB), 1,11-bis-maleimidotetraethyleneglycol (BM[PEO]4), 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC), succinimidyl-4-[N-maleimidomethylcyclohexane-1-carboxy-[6-amidocaproate]] (SMCC) and its sulfonated salt (sulfo-SMCC), succinimidyl Examples of suitable amines include succinidyl 6-[3-(2-pyridyldithio)-propionamido]hexanoate (SPDP) and its sulfonated salt (sulfo-SPDP), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) and its sulfonated salt (sulfo-MBS), and succinimidyl[4-(p-maleimidophenyl)butyrate] (SMPB) and its sulfonated salt (sulfo-SMPB). Dicyclohexylcarbodiimide (DCC) is preferred, and a linker peptide consisting of 1 to 10 amino acids may also be used.

[0095] The linker peptide may be a sequence of 1 to 10 residues consisting of H (histidine), G (glycine) and R (arginine).

[0096] The peptide sequences of the present invention may include silent mutations that may occur during the encoding process. Such silent mutations may be implicitly intended in each described sequence. Conservative substitutions that provide functionally similar amino acids in the peptide sequences of the present invention are widely known in the art, and sequence variations for such conservative substitutions are also included in the present invention.

[0097] The peptide conjugate of the present invention may further contain a positively charged peptide consisting of 1 to 4 positively charged amino acid residues at the N-terminus or C-terminus.

[0098] The positively charged peptide can be linked to the hydrophobic peptide portion of the peptide conjugate of the present invention. Preferably, the positively charged peptide is linked via a linker or a direct bond. This design provides a new approach that allows for easy formation of bonds with negatively charged molecules such as oligonucleotides for improved efficacy in delivery, treatment, storage, etc. of nucleic acid substances.

[0099] Even if the peptide complex of the present invention does not contain a positively charged peptide, it can easily be bound to a nucleic acid material later to form a nanostructure. However, if a positively charged peptide is further contained, the production of nanostructures with highly functional structures in the form of artificial chromosomes becomes more efficient.

[0100] The positively charged peptide may be any one selected from SEQ ID NOs: 108 to 119.

[0101] The peptide conjugate of the present invention may be an amphiphilic molecule having a rod-coil structure, i.e., since the peptide conjugate of the present invention has the properties of an amphiphilic molecule, it can form a self-assembly of a specific structure through self-assembly in an aqueous solution.

[0102] The peptide conjugate of the present invention is characterized by self-assembling in an aqueous solution into a micellar structure. The micellar structure refers to an aggregate nanostructure of uniform size and shape formed by amphiphilic molecules. Micelles are generally observed in a wide variety of shapes, ranging from spheres to ellipsoids, cylinders, rings, and lamellae. The peptide conjugate of the present invention may self-assemble into one or more shapes selected from the group consisting of spheres, ellipsoids, cylinders, rings, and lamellae.

[0103] The self-assembly of the peptide complex of the present invention can generally have an average diameter of 10 nm to 400 nm.

[0104] The peptide conjugate of the present invention can control the morphology of its self-assembly by adjusting its orientation using a magnetic field. That is, when an electric field is not applied, the peptide conjugate of the present invention is produced as spherical nanoparticles with the α-helical structure losing its orientation, but when an electric field is applied, the α-helical structure is oriented in the direction of the electric field, resulting in the formation of self-assemblies with an anisotropic planar nanostructure or a cross-shaped anisotropic nanostructure.

[0105] The magnetic field strength was 0.1 T to 2 T, and it was confirmed that the peptide complex of the present invention reacts sensitively to a very low magnetic field strength.

[0106] Yet another aspect of the present invention relates to a composition for safely storing nucleic acid information, comprising the peptide conjugate and a nucleic acid substance.

[0107] The peptide complex and the nucleic acid molecule may be self-assembled by any one interaction selected from the group consisting of non-covalent bonds, and may be manufactured into a nanostructure having a nanoribbon or artificial chromosome-like structure.

[0108] The composition of the present invention allows the nucleic acid material to be surrounded and coated by the peptide conjugate due to the interaction between the peptide conjugate and the nucleic acid material, thereby enabling the storage and transfer of genetic material in a highly stable manner. Furthermore, the above-mentioned process is advantageously easy and rapid, as it proceeds automatically through self-assembly in an aqueous solution. Furthermore, the peptide conjugate present in the composition of the present invention has no effect on analytical processes such as PCR, allowing genetic material information to be confirmed using DNA analysis methods useful in the biotechnology field without a separate purification process.

[0109] The peptide complex is bound to the nucleic acid substance, forming a nanoribbon structure through self-assembly, and the nanoribbon is folded and stacked, forming a nanostructure of an artificial chromosome structure through self-assembly in stages.

[0110] In the present invention, an artificial chromosome structure means a structure formed by condensation of thread-like chromatin composed of nucleic acids and proteins that carry the genetic information of an organism in the form of genes, such as human chromosomes. When considering the nanostructure in the present invention, it means that it is manufactured in the form of a nanoribbon by binding peptides and nucleic acid molecules, and that the condensation of the nanoribbon embodies a system similar to a chromosome that stores a large amount of genetic information in a minimum space.

[0111] The nucleic acid substance may be one or more selected from the group consisting of RNA, DNA, siRNA (short interfering RNA), aptamer, antisense ODN (antisense oligodeoxynucleotide), antisense RNA, ribozyme, and DNAzyme, and may be, for example, RNA, DNA, or cDNA. The nucleic acid sequence may be a coding region sequence or a non-coding region sequence (e.g., an antisense oligonucleotide or siRNA). The nucleotides serving as the nucleic acid cargo may be standard nucleotides (e.g., adenosine, cytosine, guanine, thymine, inosine, and uracil) or analogs (e.g., phosphorothioate nucleotides). For example, the nucleic acid cargo may be an antisense sequence or RNAi composed of phosphorothioate nucleotides.

[0112] The nanostructure can induce structural decomposition under magnetic field strength conditions of 0.1 T to 2 T, allowing the release of nucleic acid materials at a desired time and for a desired purpose. By utilizing this property, a delivery system that can control the long-term storage and release of nucleic acid materials can be realized.

[0113] The composition of the present invention requires 10 days or more for complete decomposition under static magnetic field conditions, so for short decomposition within one day, rotating magnetic field conditions are preferred. The rotating magnetic field strength is preferably 0.1 T to 0.5 T, but can be appropriately controlled depending on the time required for decomposition and the purpose.

[0114] According to one embodiment of the present invention, the composition of the present invention completely protects nucleic acid materials from the external environment and can be later recovered at any time using a genetic information analysis method such as PCR, making it very useful as a system for long-term storage, storage, and protection of information on nucleic acid materials.

[0115] The composition of the present invention has the advantage that the information of the nucleic acid material can be confirmed by immediately carrying out a genetic information analysis method such as PCR without a separate purification process.

[0116] The present invention will be described in more detail below with reference to preferred examples. However, it will be obvious to those skilled in the art that these examples are provided to more specifically explain the present invention and do not limit the scope of the present invention. [Example]

[0117] <Experimental Method>

[0118] CD spectroscopy (Circular dichroism spectroscopy)

[0119] To evaluate the structure of the linearized plasmid DNA and peptides, CD spectra were analyzed using a Chirascan CD spectrometer (Applied Photophysics, UK). The peptide concentrations ranged from 0.156 μM to 160 μM. The final concentration of linearized plasmid DNA was 30 ng / μl. All samples were dissolved in HO. To form the artificial chromosomes, the peptide and DNA mixtures were cultured for 12 days or more. Scans were performed using a cuvette with a 2 mm pathlength. Each scan was repeated three times, and the average was recorded. MRE (Mean Residue Ellipticity) was calculated based on the number of amino acid residues.

[0120] Magnetic circular dichroism (MCD) spectra were measured using a Jasco-815 159-L spectrophotometer (Jasco, Japan). The peptide concentration was 20 μM in distilled water. Scans were performed using a cuvette with a 2 mm path length. The magnetic field of the permanent magnet in the MCD accessory (PM-491) was 1.6 T. Each scan was repeated three times, and the average was recorded.

[0121] NMR analysis (Nuclear magnetic resonance spectroscopy)

[0122] 1 H NMR and 2 D 1 H- 15 N IPAP-HSQC (in-phase / anti-phase heteronuclear single quantum coherence) NMR spectra were obtained at 298 K using a Bruker AVANCE IV 900 MHz spectrometer and a Bruker AVANCE III HD 700 MHz spectrometer equipped with cryogenic probes. 30-α10- PEG 30 The NMR sample was dissolved in a 1:1 mixture of methanol-d3 / chloroform-d containing 0.05% TFA to a concentration of 4.26 mM. The IPAP acquisition parameters were 2048 t2 × 1024 t1 points, 8 scans, 0.2 recycle delay, and 1 14 ppm spectral width for H, 15 A spectral width of 26 ppm was used for N. The data were separated into two data sets, IP and AP, using TopSpin software (Bruker, Germany). Each spectrum was zero-filled to 8192 × 8192 points. Peak positions were analyzed using NMRFAM-SPARKY software.

[0123] Atomic force microscopy (AFM) analysis

[0124] AFM was performed using an NX10 system (Park Systems, Korea) in non-contact mode with a PPP-NCHR AFM probe (Nanosensors, Switzerland). The DNA concentrations ranged from 1 ng / μL to 30 ng / μL. The peptide concentrations ranged from 1 μM to 32 μM (in H2O). 2 μL of the sample solution was cast onto a fresh mica surface and then dried. The data were acquired using the SmartScan program (Park Systems, Korea) and analyzed using the XEI program (Park Systems, Korea).

[0125] TEM (Transmission electron microscopy) and electron diffraction

[0126] TEM was performed using a JEM-F200 multipurpose electron microscope (JEOL, Japan) at 200 kV. Two microliters of the sample solution was loaded onto a copper grid (carbon type-B grid or Formvar / silicon monoxide grid, 200-mesh copper grids with a 97-μm hole; Ted Pella, USA). After 1 hour, 2 μl of 1%–2% uranyl acetate was added to the dried sample. The negative staining solution was treated and filtered through filter paper before 1 minute had elapsed. To analyze the internal packing structures of the artificial chromosomes, electron diffraction (ED) patterns were observed in selected areas.

[0127] EMSA (Electrophoretic mobility shift assay)

[0128] The EMSA was performed to investigate the interaction mode and binding ratio between the peptide complex and linear plasmid DNA. The linear plasmid DNA concentration was fixed at 30 ng / μL, and the peptide complex concentration was increased from 1.5 μM to 6214 μM. The same mixing protocol was used as for the artificial chromosome formation. After incubation at room temperature, 10% glycerol was added to the sample for gel loading. Electrophoresis was performed on a 1% agarose gel at 90 V for 100 minutes. The bands were stained with SYBR Safe DNA Gel Stain (Invitrogen, USA) for visualization.

[0129] DNase I protection experiments

[0130] A mixture was prepared by mixing 0.1 μl (0.2 units) of DNase I and 1 μl of 10× DNase I reaction buffer with a sample (linear plasmid DNA or artificial chromosome) containing 60 ng of DNA (2 μl; 30 ng / μl). The volume of the mixture was 10 μl. After incubation at 37°C for 20 min, 0.3 μl of 0.05 M EDTA was added to inactivate the enzyme. Next, the sample was mixed with 1.7 μl of 60% glycerol and subjected to electrophoresis on a 1.1% agarose gel (120 min, 100 V). DNA was visualized by staining with SYBR Safe DNA Gel Stain (Invitrogen, USA). To evaluate the time-dependent kinetics of DNA degradation, UV absorbance at 260 nm was recorded over time after the addition of DNase I. Each sample (71, 2 μl) containing DNA (30 ng / μl) and 10×DNase I reaction buffer (8 μl) was mixed in a cuvette. DNase I (0.8 μl) was added to the cuvette, and the sample was mixed using a pipette for approximately 90–105 seconds. UV absorbance at 260 nm (A 260 The absorbance was measured using a V-650 UV-vis spectrophotometer (JASCO, Japan) using a quartz cuvette with a 10 mm penetration length. The absorbance was recorded at 10-second intervals.

[0131] Evaluation of information recovery from the artificial chromosomes

[0132] To evaluate the ability to restore DNA information stored in the artificial chromosome, polymerase chain reaction (PCR) was performed on the sample DNA to amplify DNA fragments. The specific 310 bp fragment was amplified using the sense primer 5'-ACG GAG ACT GGA GTC GAA GAG G-3 (SEQ ID NO: 106) and the antisense primer 5'-GTA GGG CAA CTA GTG CAT CTC CC-3 (SEQ ID NO: 107). A reaction mixture (50 μl) was prepared by mixing 50 ng of DNA, 10 pmol of each primer, and 25 μl of 2x Quick Taq HS DyeMix (Toyobo, Japan). PCR amplification (30 cycles) was performed using a T100 Thermal Cycler (Bio-Rad, USA). After amplification, the amplified product was electrophoresed on a 2% agarose gel at 80 V for 120 minutes (TBE system). DNA was stained using SYBR Safe DNA Gel Stain (Invitrogen, USA).

[0133] <Examples and Experimental Examples>

[0134] Examples 1 to 36. Hydrophobic α-helical peptides (α 4~13 ) synthesis

[0135] Hydrophobic α-helical peptides (α 4~13 ) was synthesized as follows. The peptide was separated using Rink Amide MBHA resin LL (100-200 mesh, 0.30-0.40 mmol g -1All Fmoc-amino acids, Fmoc-NH-PEG-propionic acid, and Fmoc-NH-PEG-propionic acid were purchased from AAPPTec (USA).

[0136] Coupling reagents such as HCTU (2-(6-chloro-1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium hexafluorophosphate), HOBt (1-hydroxybenzotriazole), and Fmoc-PEG2-Suc-OH (Fmoc-ebes-OH) were purchased from AnaSpec (USA). The synthesis scale was typically 0.1 mmol, and most syntheses were performed in 6 mL Resprep SPE (solid-phase extraction) tubes (Restek, USA). The synthesized peptides were purified by HPLC (high-performance liquid chromatography) using a C4 reversed-phase column (Waters, USA) at room temperature. Distilled water (0.1% TFA) and acetonitrile (0.1% TFA) were used as eluents. The molecular weight of the peptide was measured by matrix-assisted laser desorption / ionization time-of-flight (MALDI-TOF) mass spectrometry (Figures 1 to 36). The purity of the peptide was confirmed to be >95% by analytical HPLC.

[0137] Examples 37 to 45. Peptide complex (Wα m -RGD, Wα m -GS)

[0138] Peptide conjugates represented by SEQ ID NOs: 37 to 45 were prepared, and their structures are shown in Figure 37. The peptide conjugates were prepared by the same method as in Example 1, using Rink Amide MBHA resin LL (100-200 mesh, 0.30-0.40 mmol g -1 The peptides were synthesized by standard Fmoc solid-phase peptide synthesis (SPPS) using the Fmoc-based solid-phase peptide synthesis method (Fmoc solid-phase peptide syntheses, Novabiochem, Germany).

[0139] The molecular weight of the peptide conjugate was determined by matrix-assisted laser desorption / ionization time-of-flight (MALDI-TOF) mass spectrometry, and the purity of the peptide was confirmed to be >95% by analytical HPLC.

[0140] Examples 46 to 57. Peptide complex (Wα m -PEG, PEG-α m ) production

[0141] Peptide conjugates of Examples 46 to 57 were prepared by solid-phase peptide synthesis, in which (PEG2)5 or (PEG8)2 was bound to the peptides represented by SEQ ID NOs: 2, 8, 14, and 46 to 51 (Table 2). The structures of some of the peptide conjugates are shown in Figures 37 and 38. The solid-phase peptide synthesis was carried out using Rink Amide MBHA resin LL (100-200 mesh, 0.30-0.40 mmol g) in the same manner as in Example 1. -1 Standard Fmoc-based solid-phase peptide synthesis (SPPS) was used (by Novabiochem, Germany).

[0142] [Table 2]

[0143] Examples 58-97. Peptide complexes (Rα m-PEG, Eα m -PEG)

[0144] Using solid-phase peptide synthesis, peptide conjugates of Examples 58 to 97 were prepared in which (PEG8)2, (PEG8)3, (PEG8)4, (PEG8)5, or glucose was bound to peptides represented by SEQ ID NOs: 52 to 84 (Table 3). The solid-phase peptide synthesis was carried out using Rink Amide MBHA resin LL (100-200 mesh, 0.30-0.40 mmol g) in the same manner as in Example 1. -1 Standard Fmoc-based solid-phase peptide synthesis (SPPS) was used (by Novabiochem, Germany).

[0145] [Table 3] [Table 4]

[0146] Examples 98 to 111. Cyclic peptide conjugates (C-α m -PEG)

[0147] Using solid-phase peptide synthesis, peptide conjugates in Examples 98 to 103 were prepared, which have the same sequences as the peptide conjugates in Examples 46 to 51 but are cyclic (Table 4). The structures of some of the cyclic peptide conjugates are shown in Figure 39. The solid-phase peptide synthesis method was the same as in Example 1, except that 2-chlorotrityl chloride resin (100-200 mesh) (Novabiochem, Germany) was used instead of Rink Amide MBHA resin LL. Standard Fmoc-based solid-phase peptide synthesis (SPPS) was used.

[0148] [Table 5]

[0149] Examples 112-128. Peptide conjugates (PEG-α m -PEG)

[0150] Using solid-phase peptide synthesis, peptide conjugates of Examples 112 to 128 were prepared in which (PEG8), (PEG8), (PEG10), or (PEG10) was bound to the peptides represented by SEQ ID NOS: 2, 8, and 14, and SEQ ID NOS: 91 to 97 (Table 5). The solid-phase peptide synthesis was carried out using Rink Amide MBHA resin LL (100-200 mesh, 0.30-0.40 mmol g), as in Example 1. -1 Standard Fmoc-based solid-phase peptide synthesis (SPPS) was used (by Novabiochem, Germany).

[0151] [Table 6]

[0152] Experimental Example 1. MALDI-TOF mass spectrometry of purified peptides and peptide complexes

[0153] Figure 40 is a graph showing the results of MALDI-TOG mass spectrometry for the peptide complexes prepared in Examples 37, 39, 40, 46-50, 60, 98-102, 125, and 128, and Figure 41 is a graph showing HPLC chromatography for the peptide complexes prepared in Examples 37, 39, 40, 46-50, 60, 98-102, 125, and 128.

[0154] As shown in Figures 40 and 41, it was confirmed that the peptide conjugates prepared in Examples 37, 39, 40, 46-50, 60, 98-102, 125, and 128 were successfully synthesized.

[0155] Experimental Example 2. Design and analysis of peptides with MNP helical rod structure

[0156] The hydrophobic peptides of SEQ ID NOs: 1 to 36 have high hydrophobicity and are difficult to investigate their behavior in aqueous solution. Therefore, a peptide complex in which a hydrophilic substance is bound to one end of the hydrophobic peptide was used for analysis. The peptide complexes (α) prepared in Examples 37 to 40 were used. m -(RGD)2) was prepared and analyzed by CD spectroscopy.

[0157] Circular dichroism spectroscopy (CD spectroscopy) was performed as follows. m (RGD)2) was prepared at various concentrations (0.156–160 μM in HO) and analyzed using a Chirascan CD spectrometer (Applied Photophysics, UK). A 2 mm pathlength cuvette was used, and each scan was repeated three times, with the average recorded. The mean residue ellipticity (MRE) was calculated based on the number of each amino acid residue.

[0158] Figure 42(a) is a diagram showing the diamagnetic anisotropy of peptide bonds in a hydrophobic peptide according to the present invention, where B represents the external magnetic field (MF). Figure 42(b) is a diagram showing the α-helical structure and helical axis of a hydrophobic peptide according to the present invention, showing aligned peptide bonds, and the difference in diamagnetic susceptibility between the parallel and perpendicular directions due to this structure.

[0159] Figure 42(c) shows the peptide conjugates (α m42(d) shows the rod-coil structure of the peptide complexes (α4-RGD2, α7-RGD2, α7-RGD2, α4-RGD2, α7-RGD2) shown in Examples 37, 39, and 40. 10 -RGD2)

[0160] As shown in Figure 42, the peptide conjugates (α m It can be seen that the peptide complex (α-RGD) was formed by the successful binding of a rod portion having an α-helical structure and a portion having a coil structure. In particular, as the number of repeating sequences (α) consisting of U and A increased (Examples 37 to 40), the α-helical structure of the peptide complex gradually increased. Specifically, the peptide complex (α) prepared in Example 40 10 -RGD2) was confirmed to have the most perfect α-helical structure.

[0161] Experimental Example 3: Structural analysis of peptide complexes

[0162] An amphiphilic molecule is a molecule consisting of a hydrophilic portion and a hydrophobic portion, and each amphiphilic molecule can associate with each other in solution due to the hydrophobic effect to form a self-assembly. The peptide complexes (α4-RGD2, α7-RGD2, α 10 -RGD2) is also an amphiphilic molecule. Experimental Example 2 confirmed that the peptide conjugates prepared in Examples 37, 39, and 40 had an α-helical structure. However, this result merely indicates that the α-helical structure is stabilized within the peptide conjugate, and it was not possible to determine whether this was due to the α-helical structure stabilized by the hydrophobic peptide or the helical structure being stabilized through binding with the hydrophilic portion (RGD2).

[0163] Therefore, the peptide conjugate prepared in Example 125 was prepared and its structure was analyzed (CD spectrum and AFM measurement). The peptide conjugate prepared in Example 125 has hydrophilic moieties attached to both ends of a hydrophobic peptide, eliminating the possibility that the α-helical structure would be stabilized by structural constraints due to self-assembly. The peptide conjugate prepared in Example 125 has a coil-rod-coil structure.

[0164] CD spectra were obtained in the same manner as in Experiment 2, and AFM was performed in non-contact mode using an NX10 system (Park Systems, Korea) with a PPP-NCHR AFM probe (Nanosensors, Switzerland). The peptide conjugate concentrations ranged from 1 μM to 32 μM (in H2O). 2 μL of the sample solution was cast onto a fresh mica surface and then dried. The data were acquired using the SmartScan program (Park Systems, Korea) and analyzed using the XEI program (Park Systems, Korea).

[0165] Figure 42(e) shows the peptide conjugate (PEG) prepared from Example 125. 16 -α 10 -PEG 16 42(f) shows the CD spectra of the peptide conjugate prepared in Example 125 as a function of concentration, and FIG. 42(g) shows the helicity of the peptide conjugate prepared in Example 125 as a function of concentration.

[0166] As shown in Figure 42(e), the peptide complex of Example 125 exists in a monomeric state with a coil-rod-coil structure, and the hydrophobic peptide was confirmed to have a rod structure while having an α-helical secondary structure.

[0167] As shown in Figures 42(f) and (g), the peptide conjugate of Example 125 (PEG 16 -α 10 -PEG 16) showed two clear peaks at 208 nm and 222 nm, which correspond to the α-helical structure.

[0168] Peptide conjugate of Example 125 (PEG 16 -α 10 -PEG 16 ) [θ] measured at 222 / [θ] 208 Since it was confirmed that the ratio was maintained at 1.1 to 1.2, the peptide conjugate (PEG) of Example 125 was 16 -α 10 -PEG 16 ) has a perfect α-helical structure, but exists independently in a monomeric state without forming self-assembly.

[0169] [θ] 222 / [θ] 208 If the ratio is greater than 1.0, it is considered to have a stable α-helical structure, i.e., a wound coil structure, in which the strands overlap each other. However, the peptide complexes of the present invention are confirmed to have a ratio of 1.0 or less, confirming that the α-helical structure exists independently.

[0170] Figure 43 shows the peptide conjugates (a, b, c, d, in order) of Examples 37, 39, 40, and 125 (a4-(RGD)2, a7-(RGD)2, a 10 -(RGD)2, PEG 16 -a 10 -PEG 16 ) and through this, we aimed to compare the self-assembly behavior of peptide complexes with rod-coil structures and peptide complexes with coil-rod-coil structures.

[0171] The peptide complexes of Examples 37, 39, and 40 have a rod-coil structure and self-assemble into spherical particles such as micelles in aqueous solution. On the other hand, the peptide complex of Example 125 has a coil-rod-coil structure and was confirmed not to self-assemble in aqueous solution. This indicates that the α-helical structures of the peptide complex of Example 125 do not interact with each other.

[0172] Based on the above results, it was confirmed that α10 (Example 14; SEQ ID NO: 14), which is composed of the shortest sequence repeat unit, has a monomeric, nonpolar, perfect α-helical structure (MNP-helix, monomeric & nonpolar & perfect a-helix).

[0173] Experimental Example 4: Analysis of peptide alignment structure under magnetic field conditions

[0174] Anisotropic diamagnetic molecules in an isotropic solution have the property of aligning when a magnetic field is applied. We also attempted to analyze the behavior of the peptide of the present invention under magnetic field conditions. To this end, we used NMR (nuclear magnetic resonance) under conditions of 16.45 T (700.400 MHz) and 21.14 T (900.230 MHz). 1 H- 15 Namide pairs ( 1 D NH We analyzed the residual dipolar couplings (RDCs) between the two groups.

[0175] Peptide conjugate of Example 128 (PEG 30 -α 10 -PEG 30 ) was used as the sample, and the NMR sample was prepared by dissolving it in a methanol-d3 / chloroform-d (1:1) mixture containing 0.05% TFA to a concentration of 4.26 mM.

[0176] in particular, 1 H NMR and 2 D 1 H- 15N IPAP-HSQC (in-phase / anti-phase heteronuclear single quantum coherence) NMR spectra were analyzed at 298 K using a Bruker AVANCE IV 900 MHz spectrometer and a Bruker AVANCE III HD 700 MHz spectrometer equipped with a cryogenic probe. NMR samples were prepared by dissolving them in a 1:1 mixture of methanol-d3 / chloroform-d containing 0.05% TFA to a concentration of 4.26 mM. The IPAP acquisition parameters were 2048 t2 × 1024 t1 points, 8 scans, and a 0.2 recycle delay. 1 14 ppm spectral width for H, 15 A spectral width of 26 ppm was used for N. The data were separated into two data sets, IP and AP, using TopSpin software (Bruker, Germany). Each spectrum was zero-filled to 8192 × 8192 points. Peak positions were analyzed using NMRFAM-SPARKY software.

[0177] Magnetic isotropy can be defined from Equation 1 below:

[0178] [Formula 1]

number

[0179] In the above formula,

number

[0180]

number

[0181]

number

number

number

[0182] The peptide conjugate (PEG) of Example 128 according to the present invention 30 -α 10 -PEG 30 ) was analyzed to determine whether the peptide of SEQ ID NO: 10 (α 10 )NH coupling for 21 amino acid residues ( 1 J NH + 1 D NH ) were measured and are shown in Figure 44(a), Figure 45, Figure 46, Figure 47, Figure 48, Table 1 and Table 2.

[0183] Figure 44(a) shows the peptide conjugate of Example 128 (PEG 30 -α 10 -PEG 30 ) 1 H- 15 Figure 44(b) shows the N IPAP-HSQC spectrum of the peptide conjugate (PEG) of Example 128 under magnetic field conditions. 30 -α 10 -PEG 30 ) is a diagram showing the behavior of

[0184] Figure 45 shows the peptide conjugate (PEG) of Example 128 measured at 700.400 MHz (16.45 T). 30 -α 10 -PEG 30 )of 146 shows the H NMR spectrum of the peptide conjugate (PEG) of Example 128 measured at 700.400 MHz (16.45 T). 30 -α 10 -PEG 30 )of 1 H- 15 Table 6 shows the N IPAP-HSQC spectrum of the peptide conjugate (PEG) of Example 128 measured at 700.400 MHz (16.45 T). 30 -α 10 -PEG 30 )of 1 J NH + 1 D NH Splitting.

[0185] [Table 7] [Table 8]

[0186] Figure 47 shows the peptide conjugate (PEG) of Example 128 measured at 900.230 MHz (21.14 T). 30 -α 10 -PEG 30 )of 1 48 shows the H NMR spectrum of the peptide conjugate (PEG) of Example 128 measured at 900.230 MHz (21.14 T). 30 -α 10 -PEG 30 )of 1 H- 15 Table 7 shows the N IPAP-HSQC spectrum of the peptide conjugate (PEG) of Example 128 measured at 900.230 MHz (21.14 T). 30 -α 10 -PEG 30 )of 1 J NH + 1 D NH This is a summary of the divisions.

[0187] [Table 9] [Table 10]

[0188] Table 8 shows the peptide conjugates of Example 128 (PEG 30 -α 10 -PEG 30 ) experimental RDCs ( 1 D NH,exp The experimental RDC is calculated from the following equation:

[0189] [Formula 2] ( 1 D NH,exp )=( 1 J NH + 1 D NH ) (21.14T) -( 1 J NH + 1 D NH ) (16.45T)

[0190] [Table 11]

[0191] As shown in Figures 44 to 48 and Table 8, the peptide conjugate of Example 128 (PEG 30 -α 10 -PEG 30 ) has RDC( 1 D NH,exp ) values ​​were all positive. 1 D) is

number

[0192] α present in the peptide complex of Example 128 10 The average RDC value of peptides is 2.007 Hz, which is quite high for organic molecules. 10 It is clear that all of the peptide bonds of the 21 residues present in the peptide (sequence 14) are involved in the formation of an α-helical structure.

[0193] To summarize the above results, it was found that the hydrophobic peptides of the present invention (sequences 1 to 36) have excellent magnetic responsiveness, and it was confirmed that the excellent responsiveness to a magnetic field can be maintained even when the hydrophobic peptides are prepared as amphipathic molecules and hydrophilic molecules by binding with hydrophilic polymers or hydrophilic peptides.

[0194] Experimental Example 5: Control of the self-assembly process of peptide complexes by magnetic field

[0195] To investigate whether the self-assembly process of the peptide conjugates according to the present invention can be controlled / regulated by a magnetic field, peptide conjugates having linear rod-coil structures (Examples 46 to 50) and cyclic rod-coil structures (Examples 98 to 102) were prepared. The chemical structures of the peptide conjugates of Examples 46 to 50 and Examples 98 to 102 are compared in Figure 44(c). In addition, changes in the α-helical structure were investigated by measuring CD and MCD spectra, and morphological changes were also investigated by AFM analysis with and without a magnetic field.

[0196] CD spectra were analyzed using a Chirascan CD spectrometer (Applied Photophysics, UK). The concentrations of the peptide conjugates ranged from 0.156 μM to 160 μM. All samples were dissolved in HO. Scans were performed using a cuvette with a 2 mm path length. Each scan was repeated three times, and the average was recorded. MRE (Mean residue ellipticity) was calculated based on the number of amino acid residues.

[0197] Magnetic circular dichroism (MCD) spectra were measured using a Jasco-815 159-L spectrophotometer (Jasco, Japan). The peptide conjugate concentration was measured after dissolving in distilled water to 20 μM. A 2 mm path length cuvette was used, and the magnetic field of the permanent magnet in the MCD accessory (PM-491) was set to 1.6 T. Each scan was repeated three times, and the average was recorded.

[0198] AFM was performed in non-contact mode using an NX10 system (Park Systems, Korea) with a PPP-NCHR AFM probe (Nanosensors, Switzerland). A peptide complex (specimen) dispersed in aqueous solution was placed between neodymium magnets, and measurements were performed before and after applying a magnetic field. The magnetic field strength was measured using a Model 450 Gaussmeter (Lake Shore Cryotronics, USA).

[0199] Figure 44(c) is a diagram showing the difference in chemical structure between the peptide conjugate of Example 47 and the peptide conjugate of Example 99. Figure 44(d) shows the difference in chemical structure between the peptide conjugate of Example 47 (L-α5-PEG 10 ) for Example 99. Figure 44(e) shows the CD and MCD (magnetic circular dichroism) spectra for the peptide conjugate (C-α5-PEG 10 ) and MCD (magnetic circular dichroism) spectra. Table 9 shows the CD spectra of the peptide complexes (L-α) prepared in Examples 46 to 50. 4~8 -PEG 10 ) and the peptide conjugates (C-α 4~8 -PEG 10 ) and the results of analyzing their morphological characteristics.

[0200] [Table 12]

[0201] α nd=not determined

[0202] As shown in Figure 44 and Table 9, the peptide conjugates (L-α m -PEG 10 ) and the peptide conjugates of Examples 98 to 102 (C-α 4~8 -PEG 10 ) shows a progressive increase in α-helical content with increasing α-peptide length.

[0203] α present in the peptide complex of the present invention m To confirm whether the helical stability of the peptides is affected by a magnetic field, analysis was performed using magnetic circular dichroism (MCD). The peptide complexes of Examples 47 and 99 were analyzed using a partially stable α-helical structure, as they have a partially stable α-helical structure. The peptide complexes of Examples 47 and 99 were prepared and subjected to a magnetic field of 1.6 T (Figures 44(d) and (e)). In both cases, the α-helical content, i.e., [θ] 222 / [θ] 208 It was confirmed that the α-helical structure of the peptide complex according to the present invention can be induced to change by a magnetic field.

[0204] Peptide conjugate of Example 47 (L-α5-PEG 10 ) (0.86 to 0.91) than the peptide conjugate of Example 99 (C-α5-PEG 10 ) (0.89 to 1.01) [θ] 222 / [θ] 208The increase in the ratio was even greater. From the above results, it can be seen that cyclic peptide complexes are more sensitive to magnetic fields than linear peptide complexes. In other words, even with the same sequence, structural differences can lead to greater sensitivity to magnetic fields.

[0205] Figure 44(f) shows an experimental setup in which a peptide complex (specimen) dispersed in an aqueous solution is placed between neodymium magnets, and Figure 44g shows the effect of the peptide complex (C-α5-PEG) of Example 99 with and without a magnetic field. 10 44(h) shows the AFM images of the peptide complex (α) of Example 51 in the presence or absence of a magnetic field. The right side shows the image in the presence or absence of a magnetic field, and the left side shows the image in the absence of a magnetic field. 10 Figure 44(i) is a schematic diagram of a model of the self-assembly process with and without a magnetic field.

[0206] Previous experiments have confirmed that the peptide conjugates of the present invention are highly sensitive to magnetic fields, allowing their α-helical structure to be regulated and controlled through magnetic fields. In this regard, we sought to determine whether magnetic fields also affect the self-assembly process of peptide conjugates. The peptide conjugates of Examples 51 and 99 were prepared and dissolved in aqueous solutions to induce self-assembly. To clearly observe the morphology, the aqueous solutions were treated with ultrasound to prevent aggregation. The aqueous solutions were placed in the test piece portion of the device shown in Figure 44(f) and incubated overnight before and after AFM analysis. AFM analysis was also performed before and after treatment with the device, and the results were compared before and after application of a magnetic field.

[0207] Examining Figure 44(g), the peptide conjugate of Example 99 (C-α5-PEG 10 ) is produced as spherical self-assemblies such as vesicles in the absence of a magnetic field, but when a magnetic field (0.25 T) is applied, it is found to form a cross-shaped anisotropic nanostructure that is not seen in general molecular assemblies.

[0208] Examining Figure 44(h), the peptide conjugate of Example 47 (L-α5-PEG 10 ) are produced as spherical self-assemblies such as micelles with very uniform sizes in the absence of a magnetic field, but when a magnetic field (0.07 T) is applied, they are confirmed to form anisotropic and unique planar nanostructures.

[0209] It was confirmed that the self-assembly behavior of each peptide complex according to the present invention was changed by a very low magnetic field of 0.07 T to 0.25 T. This is because the MNP helical structure present in the peptide complex is sensitive to magnetic fields, restricting the degree of freedom of movement of the rod-coil structure of the peptide complex, resulting in limited deformation of the self-assembly form. 1 H- 15 Considering the fact that molecules with N RDC values ​​are fully oriented under a magnetic field, the peptide complexes of the present invention should only be partially aligned in solution (α 10 (See experimental RDC values ​​of Table 3). However, the entropy penalty associated with self-assembly should be smaller for an aligned rod-coil structure than for a randomly tumbling rod-coil structure in solution (Figure 44(i)). Therefore, the aligned rod-coil structures participate in the self-assembly process more readily than others, altering the overall dynamics of supramolecular crystal growth. Therefore, since the peptide complex according to the present invention contains a hydrophobic peptide with an MNP-helical structure, it forms an aligned rod-coil structure when a magnetic field is applied, and it can be seen that the self-assembly structure can be regulated and controlled. It can be confirmed that the peptide complex according to the present invention is sensitive to a magnetic field, and not only its α-helical structure but also its self-assembly structure can be regulated and controlled.

[0210] Experimental Example 6: Analysis of structural changes in nanostructures containing peptide complexes and genetic material

[0211] We attempted to determine whether the peptide complex of the present invention could be formed as a complex through interaction with negatively charged genetic material by applying a magnetic field. To this end, we used the peptide complex (R-α) of Example 60, which uses a hydrophobic peptide with an additional positively charged arginine (Arg or R) residue. 10 -PEG 16 ) and linearized plasmid DNA were prepared (Figure 49(a)).

[0212] 1) Linear DNA preparation

[0213] Linearized plasmid DNA with sufficient length (estimated length: approximately 2.6 mm) was used to easily analyze the structure by AFM and TEM. The cleavage map is shown in Figure 50. Large-sized plasmid DNA (pTWIN-Rev; 7,528 bp) was prepared using the NucleoBond Xtra Maxi Plus kit (Macherey-Nagel, Germany). The plasmid DNA was digested into linear double-stranded DNA with BamH1 restriction enzyme (New England Biolabs, USA). The linearized plasmid DNA was purified from the restriction enzyme using a 1.1% agarose gel. Electrophoresis was performed in 1x TBE buffer at 120 V for 180 minutes. After electrophoresis, the DNA was stained with SYBR Safe DNA Gel Stain (Invitrogen, USA). The DNA band fragment corresponding to the 7.5 kb linear dsDNA was excised, and linear plasmid DNA was extracted using a MEGA quick-spin™ plus kit (iNtRON, Korea). Measurement using a Nanodrop 1000 spectrophotometer (Thermo Scientific, USA) confirmed that the DNA was of very high purity (A 260 / A 280 The ratio is 1.8, A 260 / A 230 The ratio was 2.0-2.2).

[0214] 2) Fabrication of peptide-DNA nanostructures

[0215] Peptide complex of Example 60 (R-α 10 -PEG 16 ) (34 μM in HO) was slowly injected into the same volume of linearized plasmid DNA (30 ng / μL in HO). Before mixing, the aqueous solution of the peptide conjugate was sonicated to prevent nonspecific aggregation of the peptide conjugate. The peptide-DNA mixture was mixed by gentle pipetting for 1 hour and then incubated at room temperature for an extended period. At each measurement time point, 2 μL of the mixture was sampled and analyzed for morphology by AFM and TEM.

[0216] 3) EMSA (Electrophoretic mobility shift assay)

[0217] EMSA was performed using the peptide complex of Example 60 (R-α 10 -PEG 16 This study investigated the interaction mode and binding ratio between the peptide complex and linear plasmid DNA. The linear plasmid DNA concentration was fixed at 30 ng / μL, and the peptide complex concentration was increased from 0 μM to 6214 μM (charge ratio (+ / -) was 0, 0.03, 0.06, 0.13, 0.25, 0.5, 1, 2, 4, 8, 16, 32, 64, and 128, respectively). The mixing process was as described above. After incubation at room temperature, 10% glycerol was added to the sample for gel loading. Electrophoresis was performed on a 1% agarose gel at 90 V for 100 minutes. The bands were stained with SYBR Safe DNA Gel Stain (Invitrogen, USA) for visualization.

[0218] 4) Atomic force microscopy (AFM) analysis

[0219] AFM was performed using an NX10 system (Park Systems, Korea) in non-contact mode with a PPP-NCHR AFM probe (Nanosensors, Switzerland). 2 μl of the sample solution was cast onto a fresh mica surface and then dried. The data were acquired using the SmartScan program (Park Systems, Korea) and analyzed using the XEI program (Park Systems, Korea).

[0220] 5) TEM (Transmission electron microscopy) and electron diffraction

[0221] TEM was performed using a JEM-F200 multifunction electron microscope (JEOL, Japan) at 200 kV. Two microliters of sample solution was loaded onto a copper grid (carbon type-B grid or Formvar / silicon monoxide grid, 200-mesh copper grids with a 97-micron hole; Ted Pella, USA). After 1 hour, 2 microliters of 1%–2% uranyl acetate was added to the dried sample. Before 1 minute had elapsed, the sample was treated with negative staining solution and filtered through filter paper. To analyze the internal packing structure of the artificial chromosomes, electron diffraction (ED) patterns were observed in selected areas.

[0222] Figure 49(a) shows the peptide complex of Example 60 (Ra 10 -PEG 16 49(b) shows the structure of the peptide complex (Ra) of Example 60. In the peptide complex, there are two positive charges at the N-terminus of the hydrophobic peptide, one of which is derived from the arginine residue and the other from the N-terminal amine. Figure 49(b) shows an AFM image of linear plasmid DNA (7.5 kb), and Figure 49(c) shows the structure of the peptide complex (Ra) of Example 60. 10 -PEG 16 ) is an AFM image.

[0223] As shown in Figures 49(b) and (c), the linear plasmid DNA was confirmed to be a flexible molecule with a hair-like structure. 10 -PEG 16 ) was confirmed to form spherical self-assemblies similar to micelles through self-assembly. However, the peptide complex (R-α) of Example 60 10 -PEG 16 ) shows that electrostatic repulsion due to positive charges occurs in the micelle core, resulting in irregular spherical particles rather than perfect spheres.

[0224] Figure 49(d) shows the peptide complex (Ra) of Example 60 at various concentrations in linear plasmid DNA (7.5 kb). 10 -PEG 16 ) and the peptide-DNA nanostructure prepared by mixing them.

[0225] As shown in Figure 49(d), band broadening gradually occurred in EMSA depending on the charge ratio (+ / -). This indicates that peptide-DNA nanostructures were formed. Band broadening began to occur when the charge ratio (+ / -) was 1, indicating that the linear plasmid DNA (7.5 kb) and the peptide complex of Example 60 (Ra 10 -PEG 16 It has been found that peptide-DNA nanostructures are formed when the mixed charge ratio (+:-) of the peptides with the nucleotides is between 0.5:1 and 1:1.

[0226] Through the EMSA, the peptide complex of Example 60 (R-α 10 -PEG 16 It was confirmed that peptide-DNA nanostructures were formed when the mixed charge ratio (+:-) of the peptide complex (R-α) in Example 60 was between 0.5:1 and 1:1. 10 -PEG 16The peptide-DNA nanostructures were fabricated by mixing 1000-kJ / mL (peptide-DNA nanoparticles) and linear plasmid DNA at a charge ratio (+ / - ratio) of 0.7, i.e., a charge ratio (+:-) of 0.5:1. Samples were collected 1 hour, 24 hours, and 14 days after mixing and imaged by AFM and TEM.

[0227] Figures 49(e) to 49(h) show the results of the linear plasmid DNA (7.5 kb) and the peptide complex of Example 60 (Ra 10 -PEG 16 ) and then the process of forming a peptide-DNA nanostructure and the morphological changes of the nanostructure were measured by AFM and TEM. Figure 49(e) is an AFM image of the initial peptide-DNA nanostructure, Figure 49(f) is an AFM image of the intermediate peptide-DNA nanostructure, Figure 49(g) is a TEM image of the intermediate and later stages of peptide-DNA nanostructure, Figure 49(h) is a TEM image of the later stage of peptide-DNA nanostructure, and Figure 49(i) is a diagram illustrating the mechanism by which the peptide-DNA nanostructure is formed into a nanoribbon structure.

[0228] As shown in Figures 49(e) to 49(h), the peptide complex of Example 60 (Ra 10 -PEG 16 ) and linear plasmid DNA formed nanostructures in a stepwise manner, and each step was confirmed to occur very slowly.

[0229] First, the peptide complex of Example 60 (Ra 10 -PEG 16 We confirmed that the PD complex of ) and linear plasmid DNA successfully formed nanostructures even at a low charge ratio of 0.7.

[0230] Considering Figure 49(e), the peptide complex of Example 60 (Ra 10 -PEG 16 ) coated the linear plasmid DNA and formed a nanowire-shaped intermediate complex. 10 -PEG16 After mixing the linear plasmid DNA with the cellulose, it was confirmed that a rigid ribbon-shaped structure (nanoribbon) began to form one day later.

[0231] As described above, the peptide conjugates of the present invention self-assemble in cooperation with genetic material, and this self-assembly occurs stepwise to form ordered nanostructures. When the peptide-DNA nanostructures are in the form of nanoribbons, flat layers (terraces) and kinks are observed in the nanoribbons. This is due to the epitaxial growth mechanism of crystal formation. Due to the presence of steps and kinks between flat layers, the growth of the nanoribbons can be explained by the Terrace-Step-Kink (TSK) model for the formation of crystalline surfaces. The height of the steps corresponds to the diameter of DNA (2 nm) and multiples of it. Therefore, it can be confirmed that the peptide-DNA nanostructures are composed of multiple layers.

[0232] The nanoribbon-shaped peptide-DNA nanostructure according to the present invention possesses living properties. In polymer chemistry, the term "living" refers to the ability of an already formed polymer chain to react additionally with newly supplied monomers. Living behaviors during polymer synthesis, such as living polymerization and living supramolecular polymerization, have been widely known. Recently, organic molecules exhibiting living behavior have only been partially confirmed, such as living crystallization-driven self-assembly (CDSA) and fibrillation of amyloid-β peptides. The discovery of the present invention presents a new structure in this field and is therefore of significant impact.

[0233] That is, the peptide complex of Example 60 (Ra 10-PEG 16 The peptide-DNA nanostructure formed by mixing the peptide-DNA nanoparticles with linear plasmid DNA has a living nanoribbon structure.

[0234] It was confirmed that the peptide-DNA nanostructures with the living nanoribbon structure were finally (after 2 weeks) assembled into micrometer-scale nanostructures similar to the structure of human chromosomes. The above structure was named an "artificial chromosome" structure. It was confirmed that the peptide-DNA nanostructures in the form of "artificial chromosomes" were maintained for a long period of time without further structural changes.

[0235] Figure 50 shows the cleavage map of the plasmid DNA used in the present invention, which was cleaved at the BamHI region. Figure 51 shows the CD spectra of the peptide-DNA nanostructures of the peptide complex and linear plasmid DNA of Example 60. Figure 52 shows the selected area electron diffraction (SAED) patterns of the peptide-DNA nanostructures in nanoribbon form (a) and artificial chromosome form (b). Figure 53 shows the results of WAXS (Synchrotron wide-angle X-ray scattering) analysis of the peptide complex, linear plasmid DNA, and peptide-DNA nanostructures of Example 60.

[0236] As shown in Figures 51, 52, and 53, in the peptide-DNA nanostructure (PD complex), the peptide complex (Ra 10 -PEG 16 ) and B-DNA structure, which refers to linear plasmid DNA, DNA double helix structure, and α-helical structure were all confirmed.

[0237] Based on the above results, the peptide complex of Example 60 (Ra 10 -PEG 16It was confirmed that when the peptide complex (Ra) of Example 60 was mixed with linear plasmid DNA, it was produced as a nanostructure of artificial chromosome structure through the following steps: 10 -PEG 16 When the peptide complex is mixed with linear plasmid DNA, it is bound so that the peptide complex is surrounded by the linear plasmid DNA, forming a nanostructure with a living nanoribbon structure. The structure is gradually stabilized and finally produced as a nanostructure in the form of an artificial chromosome.

[0238] This is the peptide conjugate of Example 60 (Ra 10 -PEG 16 The formation of the nanostructure is due to the cooperative action of charge interactions between the arginine residues (positively charged amino acids) present in the MNP and DNA, and hydrophobic interactions between the MNP and helices. The final nanostructure, in the form of an artificial chromosome, consists of a crystalline core and a solvated corona, with a width of 200 nm to 500 nm and a length of 1 μm to 3 μm.

[0239] Experimental Example 7. Stability analysis of nanostructures in the form of artificial chromosomes

[0240] 1) Linear DNA preparation

[0241] Linearized plasmid DNA with sufficient length (estimated length: approximately 2.6 mm) was used to facilitate structural analysis by AFM and TEM. The cleavage map is shown in Figure 50. Large-sized plasmid DNA (pTWIN-Rev; 7,528 bp) was prepared using the NucleoBond Xtra Maxi Plus kit (Macherey-Nagel, Germany). The plasmid DNA was digested into linear double-stranded DNA with BamH1 restriction enzyme (New England Biolabs, USA). The linearized plasmid DNA was purified from the restriction enzyme using a 1.1% agarose gel. Electrophoresis was performed in 1x TBE buffer at 120 V for 180 minutes. After electrophoresis, the DNA was stained with SYBR Safe DNA Gel Stain (Invitrogen, USA). The DNA band fragment corresponding to the 7.5 kb linear dsDNA was excised, and linear plasmid DNA was extracted using a MEGA quick-spin™ plus kit (iNtRON, Korea). Measurement using a Nanodrop 1000 spectrophotometer (Thermo Scientific, USA) confirmed that the DNA was of very high purity (A 260 / A 280 The ratio is 1.8, A 260 / A 230 The ratio was 2.0-2.2).

[0242] 2) Fabrication of peptide-DNA nanostructures

[0243] Peptide complex of Example 60 (R-α 10 -PEG 16 The peptide-DNA nanostructures (in the form of artificial chromosomes) were prepared by gradually injecting the peptide complex solution (34 μM in HO) into the same volume of linearized plasmid DNA (30 ng / μL in HO). The aqueous peptide complex solution was sonicated to prevent nonspecific aggregation of the peptide complexes before mixing. The peptide-DNA mixture was mixed by gentle pipetting for 1 hour and then incubated at room temperature for 14 days to produce peptide-DNA nanostructures (in the form of artificial chromosomes).

[0244] 3) DNase I protection experiment

[0245] A mixture was prepared by mixing 60 ng (2 μl; 30 ng / μl) of linearized plasmid DNA or peptide-DNA nanostructures (artificial chromosome form) with 0.1 μl (0.2 units) of DNase I and 1 μl of 10× DNase I reaction buffer. The volume of the mixture was 10 μl. After incubation at 37°C for 20 min, 0.3 μl of 0.05 M EDTA was added to inactivate the enzyme. The sample was then mixed with 1.7 μl of 60% glycerol and subjected to electrophoresis on a 1.1% agarose gel (120 min, 100 V). DNA was visualized by staining with SYBR Safe DNA Gel Stain (Invitrogen, USA). To assess the time-dependent kinetics of DNA degradation, UV absorbance at 260 nm was recorded over time after the addition of DNase I. Each sample (71, 2 μl) containing DNA (30 ng / μl) and 10×DNase I reaction buffer (8 μl) was mixed in a cuvette. DNase I (0.8 μl) was added to the cuvette, and the sample was mixed using a pipette for approximately 90–105 seconds. UV absorbance at 260 nm (A 260 The absorbance was measured using a V-650 UV-vis spectrophotometer (JASCO, Japan) using a quartz cuvette with a 10 mm penetration length. The absorbance was recorded at 10-second intervals.

[0246] 4) Evaluation of information recovery from peptide-DNA nanostructures (artificial chromosomes)

[0247] We attempted to confirm whether DNA information stored in peptide-DNA nanostructures (in the form of artificial chromosomes) could be restored. Polymerase chain reaction (PCR) was performed to amplify a specific DNA fragment from linearized plasmid DNA. The specific 310-bp fragment was amplified using a sense primer, 5'-ACG GAG ACT GGA GTC GAA GAG G-3' (SEQ ID NO: 106), and an antisense primer, 5'-GTA GGG CAA CTA GTG CAT CTC CC-3' (SEQ ID NO: 107). A reaction mixture (50 μl) was prepared by mixing 50 ng of DNA, 10 pmol of each primer, and 25 μl of 2x Quick Taq HS DyeMix (Toyobo, Japan). PCR amplification (30 cycles) was performed using a T100 thermal cycler (Bio-Rad, USA). After amplification, the amplified product was electrophoresed on a 2% agarose gel at 80 V for 120 minutes (TBE system). DNA was stained using SYBR Safe DNA Gel Stain (Invitrogen, USA).

[0248] Figure 54(a) is a CD spectrum of a peptide-DNA nanostructure (artificial chromosome form) of the peptide complex of Example 60 and linear plasmid DNA.

[0249] As shown in Figure 54, in the peptide-DNA nanostructure (artificial chromosome form), the peptide complex of Example 60 (Ra 10 -PEG 16 ) tightly package DNA. Human chromosomes also contain 2 nm of DNA tightly packed within the chromosome and chromatin within the small cell nucleus (diameter: 6 μm) with the help of positively charged histone proteins, so peptide-DNA nanostructures (in the form of artificial chromosomes) are structurally very similar to human chromosomes.

[0250] The peptide-DNA nanostructure (in the form of an artificial chromosome) of the present invention has a size and shape similar to that of a human chromosome (length: approximately 2 μm to 13 μm). Due to the above-described structure, the peptide-DNA nanostructure (in the form of an artificial chromosome) of the present invention can store a large amount of biological data as genetic material in a minimal amount of space, is highly effective for stably storing biological data without damage, and allows for easy reconfirmation of genetic material information using common analytical methods such as PCR. Because the peptide-DNA nanostructure (in the form of an artificial chromosome) of the present invention can store DNA at high density, it can be used as a new carrier and storage medium for DNA data storage.

[0251] Figure 54(b) shows the results of electrophoresis analyzing the degradation of a peptide-DNA nanostructure (in the form of an artificial chromosome) of the peptide complex of Example 60 and linear plasmid DNA in the presence of DNase I. Here, the ladder is a DNA size marker, the DNA is linear plasmid DNA, the complex is a peptide-DNA nanostructure (in the form of an artificial chromosome), and the DNase is DNase I. The arrows indicate the peptide-DNA nanostructure (in the form of an artificial chromosome) confined in lanes 4 and 5.

[0252] Figure 54(c) shows the results of electrophoresis in which a peptide-DNA nanostructure (in the form of an artificial chromosome) consisting of the peptide complex of Example 60 and linear plasmid DNA was added in the presence of DNase I, and the presence or absence of decomposition was analyzed over time (0 seconds to 600 seconds).

[0253] Figure 54(d) shows the results of PCR analysis of the peptide-DNA nanostructure (artificial chromosome form) of the peptide complex and linear plasmid DNA of Example 60. Lane 1 is a DNA size marker, lane 2 is linear plasmid DNA amplified by PCR, and lane 3 is DNA amplified by PCR from the peptide-DNA nanostructure (artificial chromosome form).

[0254] For peptide-DNA nanostructures (in the form of artificial chromosomes) to be used for storing genetic and biological information, they must be able to stably store DNA, which is susceptible to enzymatic, chemical, and physical damage. To this end, we examined whether DNA could be maintained without degradation after exposing peptide-DNA nanostructures (in the form of artificial chromosomes) to DNase I for a long period of time.

[0255] FIG. 54(b) shows that the peptide-DNA nanostructure (artificial chromosome form) of the peptide complex of Example 60 according to the present invention and linear plasmid DNA effectively protected DNA from attack by DNase I.

[0256] As shown in Figure 54(c), it was confirmed that the peptide-DNA nanostructure (in the form of an artificial chromosome) of the present invention can be safely stored without DNA damage even when exposed to DNase I for more than 600 seconds. On the other hand, it was confirmed that when DNA alone is present, it is rapidly decomposed by the addition of DNase I.

[0257] As shown in Figure 54(d), a standard PCR protocol was performed on the peptide-DNA nanostructure (in the form of an artificial chromosome) according to the present invention, and a specific fragment of linear plasmid DNA was successfully amplified. 10 -PEG 16 ) was produced as a nanostructure in the form of an artificial chromosome when mixed with linear plasmid DNA, and it was confirmed that it not only stably stores genetic material such as DNA, but also maintains the genetic material such as DNA intact after storage and transfer. 10 -PEG 16 ) is extremely useful in that it does not interfere with the DNA amplification process by primers at all, and therefore PCR can be performed directly on the peptide-DNA nanostructure (artificial chromosome form) of the present invention without a separate purification process.

[0258] Experimental Example 8: Control of disassembly of nanostructures in the form of artificial chromosomes by magnetic fields

[0259] The peptide-DNA nanostructure (nanoribbon form) fabricated in Experimental Example 7 was prepared. The peptide-DNA nanostructure (artificial chromosome form) was placed on the test piece of the device shown in Figure 55(b). This device is designed to analyze the structural changes of the peptide-DNA nanostructure (nanoribbon form) due to a rotating magnetic field by attaching a permanent magnet to a motor to generate a rotating magnetic field (RMF). After exposure to a rotating magnetic field (RMF, 0.25 T, 1200 rpm) for 1 hour, the nanostructure was photographed using AFM and TEM. The AFM and TEM analysis conditions were the same as those in Experimental Examples 6 and 7.

[0260] Figure 55(a) is a TEM image of the peptide-DNA nanostructure (nanoribbon form) of the peptide complex of Example 60 and linear plasmid DNA before exposure to a magnetic field, and Figure 55(b) shows the experimental setup for generating a rotating magnetic field (RMF) using a permanent magnet. Figures 55(c) to 55(i) are TEM images of the structural changes observed after exposure of the peptide-DNA nanostructure (nanoribbon form) of the peptide complex of Example 60 and linear plasmid DNA to a rotating magnetic field. Figure 56 is an AFM image of the peptide-DNA nanostructure (nanoribbon form) of the peptide complex of Example 60 and linear plasmid DNA after exposure to a static magnetic field (0.1 T) for two weeks.

[0261] As shown in Figure 56, the peptide-DNA nanostructure (nanoribbon form) according to the present invention was confirmed to be degraded when exposed to a magnetic field. However, it was confirmed that it took more than two weeks under static magnetic field (0.1 T) conditions.

[0262] As shown in Figure 55, when the peptide-DNA nanostructure (nanoribbon form) according to the present invention is exposed to a rotating magnetic field, it is decomposed within one hour. Examination of Figures 55(a) to 55(i) reveals that the peptide-DNA nanostructure (nanoribbon form) according to the present invention is decomposed to release DNA, peptide aggregates, and partially decomposed nanoribbon forms. It was confirmed that some of the released DNA exists as single strands or multiple DNA strands. It can be seen that some DNA strands are aligned parallel to each other (Figures 55(h) and (i)).

[0263] In other words, it can be seen that the assembly or disassembly process of the peptide-DNA nanostructure according to the present invention can be efficiently controlled and regulated by a magnetic field.

[0264] Experimental Example 9. Analysis of α-helical structure of hydrophobic peptides from Examples 1 to 36

[0265] The CD spectra of the hydrophobic peptides of Examples 1 to 36 were measured, and 222nm / θ 208nm The ratio was analyzed. The results are shown in Table 10. 222nm / θ 208nm When the ratio is 1.0 to 1.2, it can be evaluated as having a stabilized helical structure.

[0266] [Table 13]

[0267] As shown in Table 10, the hydrophobic peptides of Examples 6 to 19 and Examples 25 to 36 were confirmed to have a complete and stable α-helical structure. Therefore, it was confirmed that the α-repeat units constituting the hydrophobic peptide are preferably repeated 6 to 13 times, and most preferably 6 to 10 times, and that this is the most stable α-helical structure.

[0268] Experimental Example 10. Purification and molecular weight analysis of peptide complexes

[0269] The peptide conjugates of Examples 37-40, 46-53, 60-63, 66-67, 71-72, and 90-91 were purified by HPLC and analyzed by MALDI-TOF under the following conditions: C4 semiprep TFA ACN 15-35% 10-60 min, mobile phase: ACN (0.1% TFA), gradient: 15-35% / 10-60 min (+0.4% / min), flow rate: 2,000 mL / min, injection volume: 4.5 mL, injection solvent: ACN 5%, temperature: 25°C, UV detecting: 230 nm.

[0270] Figure 57a is an HPLC analysis graph for the peptide conjugate of Example 37 (1.286 mM), and Figure 57b is a MALDI-TOF mass spectrometry graph for the peptide conjugate of Example 37 (1.286 mM), which confirmed that the molecular weight of the peptide conjugate of Example 37 was 1670.78 g / mol and the purity was >95%.

[0271] Figure 58a is an HPLC analysis graph for the peptide conjugate of Example 38, and Figure 58b is a MALDI-TOF mass spectrometry graph for the peptide conjugate of Example 38, which confirms that the peptide conjugate of Example 38 was successfully synthesized and its purity was >95%.

[0272] Figure 59a is an HPLC analysis graph for the peptide conjugate of Example 39, and Figure 59b is a MALDI-TOF mass spectrometry graph for the peptide conjugate of Example 39. From these figures, it was confirmed that the peptide conjugate of Example 39 was successfully synthesized and had a purity of >95%. The molecular weights of the peptide conjugate of Example 39 were confirmed to be molecular weight calc. = 2139.33 g / mol and molecular weight mes. = 2137.71 g / mol, respectively.

[0273] Figure 60a is an HPLC analysis graph for the peptide conjugate of Example 40, and Figure 60b is a MALDI-TOF mass spectrometry graph for the peptide conjugate of Example 40. From these figures, it was confirmed that the peptide conjugate of Example 40 was successfully synthesized and had a purity of >95%. The molecular weight of the peptide conjugate of Example 40 was confirmed to be 2607.88 g / mol.

[0274] FIG. 61 is an HPLC analysis graph of the peptide conjugate of Example 53, which shows that the peptide conjugate of Example 53 was successfully synthesized.

[0275] Figure 62a is an HPLC analysis graph for the peptide conjugates of Examples 60 to 63, and Figure 62b is a MALDI-TOF mass spectrometry graph for the peptide conjugates of Examples 60 to 63. In the figure, R1 is the peptide conjugate of Example 60, R2 is the peptide conjugate of Example 61, R3 is the peptide conjugate of Example 62, and R4 is the peptide conjugate of Example 63.

[0276] As shown in Figure 62, the peptide conjugates of Examples 60 to 63 were successfully synthesized with a purity of >95%. The molecular weights of the peptide conjugates of Examples 60 to 63 were confirmed to be 2653.15 g / mol, 2809.34 g / mol, 2965.53 g / mol, and 3121.72 g / mol, respectively.

[0277] Figure 63a is an HPLC analysis graph for the peptide conjugate of Example 90, and Figure 63b is a MALDI-TOF mass spectrometry graph for the peptide conjugate of Example 90, which confirms that the peptide conjugate of Example 90 was successfully synthesized and its purity was >95%.

[0278] Figure 64a is an HPLC analysis graph for the peptide conjugate of Example 91, and Figure 64b is a MALDI-TOF mass spectrometry graph for the peptide conjugate of Example 91, which confirms that the peptide conjugate of Example 91 was successfully synthesized and its purity was >95%.

[0279] Figure 65a is an HPLC analysis graph for the peptide conjugate of Example 71, and Figure 65b is a MALDI-TOF mass spectrometry graph for the peptide conjugate of Example 71. This confirms that the peptide conjugate of Example 71 was successfully synthesized and had a purity of >95%. The molecular weight of the peptide conjugate of Example 71 was confirmed to be 2833.17 g / mol.

[0280] FIG. 66 is a MALDI-TOF mass spectrometry graph for the peptide conjugate of Example 72, which confirms that the peptide conjugate of Example 72 was successfully synthesized and had a molecular weight of 2833.17 g / mol.

[0281] Figure 67a is an HPLC analysis graph for the peptide conjugate of Example 66, and Figure 67b is a MALDI-TOF mass spectrometry graph for the peptide conjugate of Example 66, which confirms that the peptide conjugate of Example 66 was successfully synthesized and its purity was >95%.

[0282] Figure 68a is an HPLC analysis graph for the peptide conjugate of Example 67, and Figure 68b is a MALDI-TOF mass spectrometry graph for the peptide conjugate of Example 67, which confirms that the peptide conjugate of Example 67 was successfully synthesized and its purity was >95%.

[0283] Figure 69a is an HPLC analysis graph for the peptide conjugate of Example 52, and Figure 69b is a MALDI-TOF mass spectrometry graph for the peptide conjugate of Example 52, which confirms that the peptide conjugate of Example 52 was successfully synthesized and its purity was >95%.

[0284] FIG. 70 is a MALDI-TOF mass spectrometry graph for the peptide conjugate of Example 53, which confirms that the peptide conjugate of Example 53 was successfully synthesized.

[0285] Figure 71a shows the HPLC analysis graph for the peptide conjugate of Example 46, and Figure 71b shows the MALDI-TOF mass spectrometry graph for the peptide conjugate of Example 46. These graphs confirm that the peptide conjugate of Example 46 was successfully synthesized and had a purity of >95%. The molecular weights of the peptide conjugate of Example 46 were determined to be 2021.34 g / mol (calculated) and 2041.08 g / mol (observed), respectively.

[0286] Figure 72a shows the HPLC analysis graph for the peptide conjugate of Example 47, and Figure 72b shows the MALDI-TOF mass spectrometry graph for the peptide conjugate of Example 46. These graphs confirm that the peptide conjugate of Example 47 was successfully synthesized and had a purity of >95%. The molecular weights of the peptide conjugate of Example 47 were determined to be 2177.53 g / mol (calculated) and 2197.30 g / mol (observed), respectively.

[0287] Figure 73a shows the HPLC analysis graph for the peptide conjugate of Example 48, and Figure 73b shows the MALDI-TOF mass spectrometry graph for the peptide conjugate of Example 48. These graphs confirm that the peptide conjugate of Example 48 was successfully synthesized and had a purity of >95%. The molecular weights of the peptide conjugate of Example 48 were determined to be 2333.71 g / mol (calculated) and 2353.09 g / mol (observed), respectively.

[0288] Figure 74a shows the HPLC analysis graph for the peptide conjugate of Example 49, and Figure 74b shows the MALDI-TOF mass spectrometry graph for the peptide conjugate of Example 49. These graphs confirm that the peptide conjugate of Example 49 was successfully synthesized and had a purity of >95%. The molecular weights of the peptide conjugate of Example 49 were determined to be 2489.90 g / mol (calculated) and 2508.38 g / mol (observed), respectively.

[0289] Figure 75a is an HPLC analysis graph for the peptide conjugate of Example 50, and Figure 75b is a MALDI-TOF mass spectrometry graph for the peptide conjugate of Example 50. These graphs confirm that the peptide conjugate of Example 50 was successfully synthesized and had a purity of >95%. The molecular weights of the peptide conjugate of Example 50 were confirmed to be 2646.08 g / mol (calculated) and 2664.37 g / mol (observed), respectively.

[0290] Figure 76a is an HPLC analysis graph for the peptide conjugate of Example 51, and Figure 76b is a MALDI-TOF mass spectrometry graph for the peptide conjugate of Example 51. These graphs confirm that the peptide conjugate of Example 51 was successfully synthesized and had a purity of >95%. The molecular weights of the peptide conjugate of Example 51 were confirmed to be 2802.27 g / mol (calculated) and 2820.75 g / mol (observed), respectively.

[0291] Experimental Example 11. Analysis of the α-helical structure of the peptide complexes of Examples 37 to 97

[0292] CD spectra were determined for the peptide conjugates of Examples 37 to 97, from which θ 222nm / θ 208nm The ratio was analyzed, and the results are shown in Figures 77 to 79 and Table 11. 222nm / θ 208nm When the ratio is 1.0 to 1.2, it can be evaluated as having a stabilized helical structure.

[0293] [Table 14] [Table 15]

[0294] In Table 11, the peptide conjugates of Examples 67, 72, and 75 did not show minima at 222 nm and 208 nm in the RGD spectra. These results confirm that most of the peptide conjugates according to the present invention have a stable α-helical structure. The peptide conjugates of Examples 37, 47, 52, 55, and 58 have an α-helical structure in which the hydrophobic peptide contains 4 α-repeat units and less than 0.5 α-repeat units. Therefore, it is preferable to use the peptide conjugates of Examples 39-45, 48-51, 53-55, 56-57, and 59-97, in which the hydrophobic peptide contains 6 to 13 α-repeat units.

[0295] Experimental Example 12. AFM analysis of peptide complexes of Examples 37 to 40

[0296] The peptide complexes of Examples 37 to 40 were photographed with an AFM microscope. 25 μM of the peptide complex was dissolved in distilled water, sonicated for 5 minutes, and incubated overnight before being dropped onto mica for measurement.

[0297] Figure 80 shows AFM images of the peptide conjugates of Examples 37(a), 38(b), 39(c), and 40(d), and the average diameters of these self-assemblies were measured and shown in the table below. AFM images were also taken of peptide conjugates other than those of Examples 37 to 40, and the average diameters and particle morphologies of the self-assemblies were confirmed, and then recorded together in a table.

[0298] [Table 16] [Table 17]

[0299] 80(a) to (d), the peptide complex of Example 37 was confirmed to exist in the form of irregular aggregates and vesicles, while the peptide complex of Example 38 was confirmed to exist in the form of spherical particles. In particular, the peptide complex of Example 39 was confirmed to exist in the form of micellar particles with lengths of 6 to 10 nm, average diameters of 20 nm, and average heights of 2 to 4 nm. The peptide complex of Example 40 was confirmed to exist in the form of micellar particles with lengths of 7.2 to 12.4 nm and average diameters of 20 nm.

[0300] As shown in Table 12, the hydrophobic peptide having an α-helix according to the present invention can be successfully and safely self-assembled into spherical particles in liquid, not only with the hydrophilic peptide RGD but also with hydrophilic polymers such as PEG.

[0301] Furthermore, the peptide conjugate according to the present invention is self-assembled in an aqueous solution and exists in the form of spherical nanoparticles, the average diameter of which is 20 nm to 400 nm.

[0302] However, if the number of repeating units of the hydrophobic peptide having an α-helical chain is six or less, irregular aggregation occurs, so it is preferable that the number of repeating units of the hydrophobic peptide having an α-helical chain is six or more.

[0303] Experimental Example 13: Peptide-DNA nanostructure of the peptide complex of Example 60 and dsDNA

[0304] 1) Linear dsDNA

[0305] A NucleoBond Xtra Maxi Plus kit (Macherey-Nagel, Germany) was used to prepare large-sized plasmid DNA (pTWIN-Rev; 7,528 bp). The plasmid DNA was digested into linear double-stranded DNA using BamH1 restriction enzyme (New England Biolabs, USA). The DNA was purified from the restriction enzyme using a 1.1% agarose gel. Electrophoresis was performed in 1x TBE buffer at 120 V for 180 minutes. After electrophoresis, the DNA was stained with SYBR Safe DNA Gel Stain (Invitrogen, USA). The DNA band corresponding to the 7.5 kb linear dsDNA was excised and linear plasmid DNA was extracted using a MEGA quick-spin™ plus kit (iNtRON, Korea). Measurement using a Nanodrop 1000 spectrophotometer (Thermo Scientific, USA) confirmed that the DNA was highly pure (A 260 / A 280 The ratio is 1.8, A 260 / A 230 The ratio was 2.0-2.2).

[0306] 2) Peptide-DNA nanostructures

[0307] The peptide complex of Example 60 was dissolved in distilled water and treated with ultrasound. The same volume of dsDNA (1.8 ng / μl) was slowly injected into the peptide complex, and R-α 10 -PEG 16 The / dsDNA nanostructures were prepared and analyzed by primary AFM and TEM after 2-3 minutes of reaction at room temperature, secondary AFM and TEM after 12 days of reaction, and tertiary AFM and TEM after 22 days of reaction.

[0308] 3) DNase I protection experiment

[0309] dsDNA 60ng (2μl; 30ng / μl) or R-α 10 -PEG 16A mixture was prepared by mixing 0.1 μl (0.2 units) of DNase I and 1 μl of 10× DNase I reaction buffer with the dsDNA nanostructure (artificial chromosome form). The mixture volume was 10 μl. After incubation at 37°C for 20 minutes, 0.3 μl of 0.05 M EDTA was added to inactivate the enzyme. Next, the sample was mixed with 1.7 μl of 60% glycerol and subjected to electrophoresis on a 1.1% agarose gel (120 minutes, 100 V). DNA was visualized by staining with SYBR Safe DNA Gel Stain (Invitrogen, USA). To evaluate the time-dependent kinetics of DNA degradation, UV absorbance at 260 nm was recorded over time after the addition of DNase I. Each sample (71 μl, 2 μl) containing DNA (30 ng / μl) and 8 μl of 10× DNase I reaction buffer was mixed in a cuvette. DNase I (0.8 μl) was added to the cuvette, and the sample was mixed with a pipette for approximately 90 to 105 seconds. UV absorbance at 260 nm (A 260 The absorbance was measured using a V-650 UV-vis spectrophotometer (JASCO, Japan) using a quartz cuvette with a 10 mm penetration length. The absorbance was recorded at 10-second intervals.

[0310] Figure 81a shows the peptide complex of Example 60 (R-α 10 -PEG 16 ) was photographed by AFM, and Figure 81b is an image of dsDNA photographed by AFM. Figures 81c and 81d show the R-α 10 -PEG 16 Figure 81e shows the AFM image of R-α / dsDNA nanostructure taken 2 to 3 minutes after fabrication. 10 -PEG 16 Figure 81f shows an AFM image of the R-α / dsDNA nanostructure taken 12 days after fabrication. 10 -PEG 16 This image was taken with an AFM 22 days after fabrication of the / dsDNA nanostructure.

[0311] As shown in Figures 81a and 81b, the peptide complex of Example 60 (R-α 10 -PEG 16 ) has a length of 1 nm to 1.25 nm, and dsDNA has a long linear structure with a width of 23 nm and a length of 2.2 μm to 2.3 μm.

[0312] As shown in Figures 81c and 81d, R-α 10 -PEG 16 In the case of dsDNA nanostructures, it was confirmed that they self-assembled into nanoribbon shapes with width, height, and length of 33 nm to 48 nm, 6 nm to 10 nm, and 0.8 μm to 3 μm, respectively.

[0313] As shown in Figure 81e, R-α 10 -PEG 16 After 12 days, the R-α / dsDNA nanostructures transformed into short and thick nanoribbon structures with widths and heights ranging from 234 nm to 1172 nm and 26 nm to 132 nm. 10 -PEG 16 / dsDNA nanostructure is a peptide complex (R-α 10 -PEG 16 They confirmed that the chromosomes are formed in an "artificial chromosome"-like structure, surrounded and protected by a protective membrane.

[0314] Figure 82a shows R-α 10 -PEG 16 Figure 82b shows the TEM image taken 2 to 3 minutes after fabrication of the / dsDNA nanostructure. 10 -PEG 16 TEM images taken 12 days after fabrication of the / dsDNA nanostructures are shown in Figures 82c and 82d. 10 -PEG 16 This is a TEM image taken 22 days after fabrication of the / dsDNA nanostructure.

[0315] As shown in Figures 82a to 82d, R-α 10 -PEG 16The / dsDNA nanostructures were initially long linear structures, but over time they self-assembled into long nanoribbon forms, gradually increasing in width and decreasing in length, resulting in the production of nanostructures with an "artificial chromosome"-like morphology (width and length 600 nm and 2 μm to 3 μm, respectively).

[0316] Based on the above results, the peptide complex of Example 60 (R-α 10 -PEG 16 ), dsDNA and R-α 10 -PEG 16 We attempted to compare and analyze the α-helical structures of dsDNA nanostructures.

[0317] Figure 83 shows the peptide complex of Example 60 (R-α 10 -PEG 16 ), dsDNA and R-α 10 -PEG 16 1. CD spectrum of the / dsDNA nanostructure, which shows that the peptide complex (R-α 10 -PEG 16 ) and R-α 10 -PEG 16 It was confirmed that the / dsDNA nanostructure maintained a stable α-helical structure. 10 -PEG 16 It can be seen that the α-helical structure is contained within the complex even when the dsDNA nanostructure is formed. In the figure, R1 only represents the peptide complex (R-α) of Example 60. 10 -PEG 16 ) alone, and the mixture (Mix) is R-α 10 -PEG 16 / dsDNA nanostructures.

[0318] Figure 84 shows the R-α 10 -PEG 16 85 shows the SAED (Selected Area Electron Diffraction Pattern) of the / dsDNA nanostructure.10 -PEG 16 This is the SAED (Selected Area Electron Diffraction Pattern) of the / dsDNA nanostructure. 10 -PEG 16 When the / dsDNA nanostructure was formed, R-α 10 -PEG 16 It can be seen that the α-helical structure is completely contained within the / dsDNA nanostructure without any deformation.

[0319] Through the DNase I protection experiment mentioned in this example, R-α 10 -PEG 16 We also sought to analyze whether the / dsDNA nanostructures could stably protect DNA from DNases.

[0320] Figure 86 shows the relationship between dsDNA and R-α 10 -PEG 16 The results were obtained by adding DNase I to the / dsDNA nanostructure and measuring it with UV-vis. When only dsDNA was present (naked DNA), the dsDNA was completely decomposed, but when R-α 10 -PEG 16 When the dsDNA is present as a nanostructure, it can be confirmed that the dsDNA is maintained without being decomposed.

[0321] Experimental Example 14: Peptide complex of Example 63 (R4-α 10 -PEG 16 ) Analysis

[0322] 1) Linear dsDNA

[0323] First, large-sized plasmid DNA (pTWIN-Rev; 7,528 bp) was prepared using the NucleoBond Xtra Maxi Plus kit (Macherey-Nagel, Germany). The plasmid DNA was digested into linear double-stranded DNA with BamH1 restriction enzyme (New England Biolabs, USA). It was purified using a 1.1% agarose gel. Electrophoresis was performed in 1x TBE buffer at 120 V for 180 minutes. After electrophoresis, the DNA was stained with SYBR Safe DNA Gel Stain (Invitrogen, USA). The DNA band corresponding to the 7.5 kb linear dsDNA was excised and linear plasmid DNA was extracted using the MEGA quick-spin™ plus kit (iNtRON, Korea). Measurement using a Nanodrop 1000 spectrophotometer (Thermo Scientific, USA) confirmed that the DNA was highly pure (A 260 / A 280 The ratio is 1.8, A 260 / A 230 The ratio was 2.0-2.2).

[0324] 2) Peptide-DNA nanostructures

[0325] The peptide conjugate of Example 63 was dissolved in distilled water and treated with ultrasound. The same volume of dsDNA (145.2 ng / μl in H2O) was slowly injected into the peptide conjugate at a concentration of 22.6 μM, and R4-α 10 -PEG 16 The / dsDNA nanostructures were prepared and analyzed by AFM and TEM after 40 minutes of reaction at room temperature, and by AFM and TEM after 111 days of reaction.

[0326] Figure 87 shows an AFM image of the peptide complex of Example 63, and Figure 88 shows an AFM image of R4-α 10 -PEG 16 Figure 89 shows an AFM image taken 40 minutes after fabrication of the R4-α / dsDNA nanostructure. 10 -PEG 16This is an AFM image taken 111 days after fabrication of the / dsDNA nanostructure.

[0327] Inspection of Figure 87 reveals that the peptide conjugate of Example 63 self-assembles in the form of spherical particles in aqueous solution.

[0328] 88 and 89, it can be seen that the peptide complex of Example 63 is prepared as a nanoribbon-shaped nanostructure through self-assembly when mixed with dsDNA, and then completed as an artificial chromosome-shaped nanostructure. It can also be seen that even when the peptide complex contains more positively charged amino acids than the peptide complex of Example 60 described above, it is prepared as a nanostructure according to the present invention.

[0329] Figure 90 shows R4-α 10 -PEG 16 The CD spectrum of the dsDNA nanostructure shows that the peptide complex of Example 63, which has a strong positive charge, also forms nanostructures through cooperative interactions with dsDNA. In the figure, R4 is the peptide complex of Example 63, and Mix is ​​R4-α. 10 -PEG 16 R4-α after 1 hour of incubation with dsDNA nanostructures 10 -PEG 16 The R4-α / dsDNA nanostructures were incubated for 1 h. 10 -PEG 16 It was confirmed that the / dsDNA nanostructure retained both the α-helical structure of the peptide and the DNA structure even after being fabricated in the form of an artificial chromosome.

[0330] Experimental Example 15. Analysis of nanostructures fabricated with the peptide conjugate of Example 60 and various genetic materials

[0331] 1) Preparation of plasmid DNA, linear dsDNA, and ssDNA

[0332] First, large-sized plasmid DNA (pTWIN-Rev; 7,528 bp) was prepared using the NucleoBond Xtra Maxi Plus kit (Macherey-Nagel, Germany). The plasmid DNA was digested into linear double-stranded DNA with BamH1 restriction enzyme (New England Biolabs, USA). It was purified using a 1.1% agarose gel. Electrophoresis was performed in 1x TBE buffer at 120 V for 180 minutes. After electrophoresis, the DNA was stained with SYBR Safe DNA Gel Stain (Invitrogen, USA). The DNA band corresponding to the 7.5 kb linear dsDNA was excised and linear plasmid DNA was extracted using the MEGA quick-spin™ plus kit (iNtRON, Korea). Measurement using a Nanodrop 1000 spectrophotometer (Thermo Scientific, USA) confirmed that the DNA was highly pure (A 260 / A 280 The ratio is 1.8, A 260 / A 230 The ratio was 2.0-2.2).

[0333] As the ssDNA, a short oligomer of 20 nt length (SEQ ID NO: 120) custom-made by BIONEER was used.

[0334] 2) Peptide-DNA nanostructures

[0335] The peptide complex of Example 60 was dissolved in distilled water and sonicated. dsDNA (30 ng / μL in H2O) or ssDNA (30 ng / μL in H2O) was gradually added to the peptide complex of Example 60 at various concentrations, and R-α complexes with nucleic acid to peptide charge ratios (+ / -) of 0, 0.03, 0.06, 0.12, 0.25, 0.5, 1, 2, 4, 8, 16, 32, 64, and 128 were prepared. 10 -PEG 16 / dsDNA and R-α 10 -PEG 16 The / ssDNA nanostructures were prepared and measured by EMSA after 1 hour of incubation.

[0336] 3) EMSA

[0337] For EMSA (electrophoretic mobility shift assay), 10% glycerol was added to the sample. Electrophoresis was performed on a 1% agarose gel at 100 V for 120 minutes. The bands were visualized by staining with SYBR Safe DNA Gel Stain (Invitrogen, USA).

[0338] Figure 91a shows the peptide complex of Example 60 (R-α 10 -PEG 16 ) and dsDNA nanostructures. Figure 91b shows the EMSA results for the peptide complex (R-α 10 -PEG 16 ) and ssDNA nanostructures. In each figure, the ladder is a nanostructure fabricated by mixing nucleic acid materials (dsDNA, ssDNA) and peptide complexes at charge ratios (+ / -), where the charge ratios (+ / -) are 0, 0.03, 0.06, 0.12, 0.25, 0.5, 1, 2, 4, 8, 16, 32, 64, and 128.

[0339] 91a and 91b, it was confirmed that the peptide conjugate according to the present invention forms nanostructures through interaction with nucleic acid substances, regardless of the type and length of the nucleic acid substances.

[0340] Experimental Example 16: Analysis of nanostructures of peptide complexes and linear plasmid DNA from Examples 61 to 63

[0341] Peptide-DNA nanostructures were prepared by mixing the peptide complexes of Examples 61 to 63 with dsDNA. Peptide-DNA nanostructures were prepared by mixing the peptide complexes of Example 61 with plasmid DNA (circular dsDNA, pTWIN-rev=7.5 kbp). The preparation process was the same as in Experimental Example 15. The nanostructures were prepared by mixing the nucleic acid material and peptide so that the charge ratio (+ / -) was 0, 0.03, 0.06, 0.12, 0.25, 0.5, 1, 2, 4, 8, 16, 32, 64, 128, and 256, and R-α 10 -PEG 16 / pDNA, R-α 10 -PEG 16 The / dsDNA nanostructures were prepared and incubated for 1.5 hours before measurement by EMSA.

[0342] For EMSA (electrophoretic mobility shift assay), 10% glycerol was added to the samples. Electrophoresis was performed on a 1% agarose gel at 100 V for 120 minutes. The bands were visualized by staining with SYBR Safe DNA Gel Stain (Invitrogen, USA).

[0343] Figure 91c shows the peptide complex of Example 61 (R-α 10 -PEG 16 ) and pDNA nanostructures. Figure 91d shows the EMSA results for the peptide complex (R-α 10 -PEG 16 ) and dsDNA nanostructures. Figure 91e shows the EMSA results for the peptide complex (R-α 10 -PEG 16 ) and dsDNA nanostructures. Figure 91f shows the EMSA results for the peptide complex (R-α 10 -PEG 16) and dsDNA nanostructures. In each figure, the ladder is a nanostructure fabricated by mixing nucleic acid materials (dsDNA, ssDNA) and peptide complexes at charge ratios (+ / -), where the charge ratios (+ / -) are 0, 0.03, 0.06, 0.12, 0.25, 0.5, 1, 2, 4, 8, 16, 32, 64, 128, and 256.

[0344] As shown in Figures 91c to 91f, it was confirmed that the peptide polymers of Examples 61 to 63 according to the present invention also successfully formed nanostructures by binding with nucleic acid materials.

[0345] Experimental Example 17. Structural analysis of cyclic peptide complexes

[0346] The cyclic peptide conjugates of Examples 98 to 111 were purified by HPLC and analyzed by MALDI-TOF and CD spectroscopy under the following conditions: C4 semiprep TFA ACN 15-35% 10-60 min, mobile phase: ACN (TFA 0.1%), gradient: 15-35% / 10-60 min (+0.4% / min), flow rate: 2,000 mL / min, injection volume: 4.5 mL, injection solvent: ACN 5%, temperature: 25°C, UV detecting: 230 nm.

[0347] 92a to 101c show HPLC (a), MALDI-TOF (b), and CD spectra (c) for the cyclic peptide conjugates of Examples 98-103, 106, and 109-111.

[0348] Table 13 shows the results of analyzing the morphological characteristics of the cyclic peptide complexes of Examples 98 to 111 as measured by CD spectroscopy.

[0349] [Table 18]

[0350] α nd=not determined

[0351] As shown in Figures 92a to 101c and Table 13, the cyclic peptide complexes of Examples 98 to 103, 106, and 109 to 111 were successfully formed. It was also found that the α-helical content of the cyclic peptide complexes gradually increased as the α-peptide length increased. Cyclic peptide complexes (Examples 98, 104, and 105) with fewer than six repeating units (α) of [Aib]-[Xaa1] or [Xaa1]-[Aib] were found to have an α-helical tendency of less than 0.5 and were therefore unstable. It was also found that the cyclic peptide complexes of Examples 99 to 103 and 106 to 111, preferably having 6 to 10 repeating units, possessed the most stable α-helical structure.

[0352] Experimental Example 18. Biostability analysis of peptide conjugates

[0353] Through the above experiments, we investigated the self-assembly behavior of the peptide conjugates according to the present invention and nanostructured artificial chromosome structures prepared therefrom, investigated the manner in which they interact with genetic material, and confirmed their potential use as genetic material storage media and systems. When used in fields such as cellular medicine and food for the stable storage of genetic material, cytotoxicity is also very important. Therefore, we attempted to analyze the cytotoxicity of the peptide conjugates according to the present invention and nanostructured artificial chromosome structures prepared therefrom.

[0354] To confirm the presence or absence of cytotoxicity, toxicity was examined by WST-1 assay using HeLa cells. First, HeLa cells were subcultured in DMEM supplemented with 10% FBS (Fetal Bovine Serum), and 5 × 10 cells were added to each well. 4HeLa cells were plated per well and cultured for 16 hours, then treated with various concentrations of samples (5 μM, 10 μM, 20 μM) and cultured for 24 hours at 37°C. Four hours after adding WST-1 solution to the cultured cells, the absorbance at 450 nm was measured using a microplate reader (Bio-Tek Instrument Co., WA, USA).

[0355] The peptide complexes of Examples 40, 60, and 102 were prepared as samples. Similarly to Experimental Examples 13 and 14, peptide-DNA nanostructures (in the form of artificial chromosomes, cultured for 12 days) were prepared by mixing the peptide complexes of Examples 60 and 63 with linear plasmid DNA. Untreated normal cells were used as a control.

[0356] Figure 102 is a graph showing an analysis of cell viability for peptide complexes of Examples 40, 60, and 102 and nanostructures of artificial chromosome structures prepared from Experimental Examples 13 and 14. It was confirmed that not only the peptide complexes of various structures prepared by the present invention, but also the nanostructures do not induce toxicity in cells and are stable to cells.

[0357] Experimental Example 19. Structural analysis of peptide complexes with coil-rod-coil structure

[0358] Peptide conjugates in Examples 112 to 128 were prepared. These have a coil-rod-coil structure. The peptide conjugates were purified by HPLC and analyzed by MALDI-TOF and CD spectroscopy. The HPLC conditions were as follows: C4 semiprep TFA ACN 15-35% for 10-60 min, mobile phase: ACN (TFA 0.1%), gradient: 15-35% / 10-60 min (+0.4% / min), flow rate: 2,000 mL / min, injection volume: 4.5 mL, injection solvent: ACN 5%, temperature: 25°C, UV detecting: 230 nm.

[0359] Figures 103a to 110 show HPLC (a) and MALDI-TOF (b) graphs for the peptide conjugates of Examples 113 to 115, 119 to 121, 125, and 128. When only one figure is shown without a or b, it is the MALDI-TOF graph for the corresponding peptide conjugate. Table 10 shows the results of analyzing the morphological characteristics of the peptide conjugates of Examples 116 to 128 as measured by CD spectroscopy.

[0360] [Table 19]

[0361] As shown in Figures 102 to 110 and Table 14, the peptide complexes of Examples 112 to 128 with a coil-rod-coil structure were successfully synthesized. It was also found that the α-helical content of the peptide complexes with a coil-rod-coil structure gradually increased as the α-peptide length increased. Cyclic peptide complexes (Examples 98, 104, and 105) with fewer than six repeating units (α) of [Aib]-[Xaa1] or [Xaa1]-[Aib] were found to have an α-helical tendency of less than 0.5 and were unstable. It was confirmed that the peptide complexes of Examples 112 to 118, 120 to 122, and 124 to 128, preferably with 6 to 10 repeating units, possess the most stable α-helical structure.

Claims

1. a hydrophobic peptide; and at least one hydrophilic polymer attached to one end of the hydrophobic peptide; The hydrophobic peptide is a peptide of the following general formula 1: [General formula 1] -{[Aib]-[Ala]}m- Ala is a D- or L-amino acid residue, and m is an integer selected from 6 to 8. The hydrophilic polymer is a polyethylene glycol (PEG) polymer (PEG 2 ) 5; the other end of the hydrophobic peptide is bound to a positively charged peptide consisting of one positively charged amino acid residue W; The peptide complex is characterized in that it self-assembles into an anisotropic planar nanostructure or a cross-shaped anisotropic nanostructure under the condition that a magnetic field is applied.

2. The peptide complex according to claim 1 , wherein the peptide complex is cyclic with both ends of the peptide complex joined together.

3. The peptide conjugate according to claim 1, which is self-assembled in solution as spherical nanoparticles with a micellar or endoplasmic reticulum structure.

4. The peptide complex according to claim 1, wherein the magnetic field has a strength of 0.1T to 2T.

5. A composition for safely storing and archiving nucleic acid information, comprising: a peptide complex according to claim 1; and a nucleic acid material; the peptide complex and the nucleic acid substance are self-assembled by any one interaction selected from the group consisting of non-covalent bonds to produce a nanostructure having a nanoribbon or artificial chromosome-like structure; The peptide complex is bound to the nucleic acid substance, thereby forming a nanoribbon structure by self-assembly, and the nanoribbon is folded and stacked to form a nanostructure of an artificial chromosome structure by self-assembly in stages, The composition, wherein the nanostructure is decomposed under a magnetic field strength of 0.1 T to 2 T, thereby inducing the release of a nucleic acid substance.

6. The nucleic acid substance may be RNA, DNA, siRNA (short interfering RNA), or the like. The composition according to claim 5, characterized in that the compound is at least one selected from the group consisting of an antisense oligodeoxynucleotide (ODN), an antisense RNA, a ribozyme, and a DNAzyme.

7. The composition according to claim 5, wherein the magnetic field strength condition is a rotating magnetic field condition of 0.1T to 0.5T.

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