KR20150081685A - Analysis Method for DNA Interaction with p53 Protein Using Single Plasmonic Nanosensor - Google Patents

Abstract

The present invention relates to a method for effectively analyzing an interaction between DNA and p53 protein by using a single plasmonic nanosensor. More particularly, the invention relates to a method for analyzing a difference of an interaction between a wild-type p53 protein and a mutant-type p53 protein both bonded to GADD45 promoter and a difference of a bonding force therebetween by using a sensor having a single gold nanoparticle fixed thereto. The analysis method by using a single plasmonic nanosensor of the present invention is capable of directly analyzing a difference of an interaction and a bonding force of the p53 protein bonded to a particular promoter without a separate labeling process, and capable of identifying importance of each part in accordance with amino acid mutation by comparing a difference of a bonding force between the wild-type p53 protein and the mutant-type p53 protein. Accordingly, the invention may be applied and used in the analysis field for diagnosing a patient with a high potential to develop a tumor at an early stage.

Description

Technical Field [0001] The present invention relates to a method for analyzing the interaction between DNA and p53 protein using a single nanoplasmonic sensor,

The present invention relates to a method for effectively analyzing the interaction between DNA and p53 protein using a single plasmonic nanosensor, and more particularly, to a method for analyzing the interaction between DNA and p53 protein using a plasmonic sensor having a single gold nanoparticle immobilized thereon, Lt; RTI ID = 0.0 > p53 < / RTI >

The p53 gene is a mutation gene that is commonly found in cancer, and is one of the genes that act as an important factor in cancer development. P53, which is a cancer-suppressing gene, is activated by damage to the intracellular DNA, inducing a base excision repair (BER) mechanism, delaying the cell cycle, inhibiting replication of an incomplete gene And is involved in the repair of damaged DNA. In addition, when p53 is so severe that the damage of the intracellular gene can not be restored, it induces a cell apoptosis mechanism and performs a function of removing a cell which is likely to be transformed into cancer. p53 is known to prevent the accumulation of damaged genes in the cell, thereby inhibiting the mechanism by which the damaged cells develop into cancer (Penka V. et al ., Proc . Natl . Acad . Sci ., 95: 14675. 1998; S. Collot-Teixeira et al., Reviews in Medical Virology , 14: 301, 2004; David Sidransky and Monica Hollstein, Annu . Rev. Med ., 47: 285,1996).

Mutant p53 protein is found in over 50% of cancers in the total cancers (H. Kitano, Science ., 295: 1662, 2002), with over 90% of them mutated in the DNA binding domain. If the p53 protein fails to bind to DNA as a mutation of the DNA binding domain and loses its function as a trans-acting promoter of transcription, it can not inhibit the cell division of the damaged cell, thereby increasing the possibility of cancer cell death of the damaged cell (Pavletich NP et al., Genes & Dev ., 7: 2557, 1993; Jean-Francois Millau, Mutation Research ., 681: 118, 2009; Wei Gu and Robert G. Roeder, Cell , 90: 59, 1997). Thus, monitoring the DNA binding capacity of normal p53 and mutant p53 proteins is of great value in the field of cancer cell research and diagnosis.

Recently, biosensor technology has focused on the development of non-labeling measurement technology that does not use labeling material. Typically, the method using surface plasmon resonance (RQ DUAN et al ., Neoplasma , 59 (3): 348, 2012; Wei Zhou meat al ., International Journal of Nanomedicine , 6: 381, 2011), a method for measuring the refractive index change of light due to the bending phenomenon of a three-dimensional microstructure, cantilever (Balle, MK. Et al, Ultramicroscopy, 82;. 1 , 2000), method of measuring the modified crystal resonant frequency change in accordance with the mass change of the biomolecule (Marx, KA, Biomacromolecules, 4 ;. 1099, 2003), based on the semiconductor process How to measure the electric field effect (Yuqing, M. et al ., Biotechnol . Adv . , 21; 527, 2003) and an electrochemical measurement method (Hanahan, G. et al . , J. Environ . Monit . , 6; 657, 2004; Sang Hee Han et al ., Analytica Chimica Acta , 665: 79, 2010).

Since the localized surface plasmon resonance (LSPR) method can quantitatively detect the reaction without complicated pre-purification and labeling processes, many studies on the interaction between biomolecules immobilized on the surface Has been used. In the LSPR method, when light having various wavelengths is irradiated to a material having a localized surface such as metal nanoparticles, polarization is generated on the surface of the metal nanoparticles, unlike bulk metals, and the characteristic of electric field is remarkable. These LSPR optical properties are sensitive to changes in the permittivity (refractive index) near the nanoparticles. This change in dielectric constant (refractive index) can be used to detect adsorption between biomolecules. For the past decades, the optical properties of LSPR have been based on biomaterial concentration, thickness, and binding reaction rate data for specific biochemical analytes including antigen / antibody, ligand / receptor, protein / protein reaction and DNA hybridization Various biomolecule interaction analyzes have been performed.

Conventional techniques for detecting mutant p53 using a surface plasmon resonance method include a method of detecting a mutant p53 protein by interaction of p53 protein and p53 protein-binding DNA using SPR (Sang Hee Han et al ., Analytica Chimica Acta , 665: 79, 2010), using mutagenic DNA probe interactions with p53 gene and p53 gene using a local surface plasmon resonance sensor (LSPR) with silver nanoparticles immobilized to detect mutant p53 gene How to do it (RQ DUAN et . al, Neoplasma, 59 (3 ): 348, 2012) and is the p53 protein bound to immobilized a p53 engageable antibody (anti-p53 antibody) on the nanoparticles, and then process the serum containing the p53 protein and antibodies A method for detecting the p53 protein contained in the serum by measuring the change in the LSPR wavelength meat al ., International Journal of Nanomedicine , 6: 381, 2011).

However, most of the p53 protein mutations occur mainly at specific sites located in the center of the DNA binding domain, so that conventional methods have low sensitivity and specificity, are costly and time-consuming (Alex N Bullock et al., Oncogene ., 19: 1245, 2000; Uma Chandrachud and Susannah, Technol Cancer Res Treat ., 8: 445, 2009).

The present inventors have previously developed a surface plasmon resonance sensor in which gold nanoparticles are immobilized and analyzed the interaction and binding activity between DNA and protein. And the activity of the promoter can be measured without a separate labeling procedure by observing the interaction of the protein (Min Sun Song et al . , Adv . Mater ., 25: 1265,2013).

As a result, the present inventors have made extensive efforts to develop a more accurate and sensitive mutant p53 protein detection method. As a result, the inventors of the present invention have found that a plasmonic sensor having a single gold nanoparticle immobilized thereon can be used as a GADD45 promoter having a base sequence specific to the p53 protein DNA binding domain And mutant p53 protein binding to the wild type and mutant p53 protein were analyzed, it was confirmed that mutant p53 protein at a low concentration can be detected more accurately and quickly than the conventional method, and the present invention has been completed.

It is an object of the present invention to provide a method for analyzing GADD45 promoter-p53 protein interaction and binding activity using a surface plasmon resonance sensor in which gold nanoparticles are immobilized.

In order to accomplish the above object, the present invention provides a method for inducing the binding of a GADD45 promoter to a large amount of p53 protein by contacting a wild-type or mutant p53 protein to a plasmonic sensor based on gold nanoparticles immobilized with the GADD45 promoter step; And (b) light scattering spectra were measured using a dark-field microscope and a Rayleigh scattering spectrum, and the maximum wavelength shift value (? _Max ) analysis obtained therefrom was used to analyze the GADD45 promoter And analyzing the interaction of a large amount of p53 protein with the GADD45 promoter-p53 protein.

(A) contacting wild-type or mutant p53 protein to a plasmonic sensor based on gold nanoparticles immobilized with the GADD45 promoter to induce the binding of the GADD45 promoter to the p53 protein; And (b) light scattering spectra were measured using a dark-field microscope and a Rayleigh scattering spectrum, and the maximum wavelength shift value (? _Max ) analysis obtained therefrom was used to analyze the GADD45 promoter And analyzing the binding activity of the p53 protein to the GADD45 promoter-p53 protein binding activity.

(A) contacting a plasmonic sensor based on gold nanoparticles immobilized with a GADD45 promoter with a sample containing a wild-type or mutant p53 protein to induce the binding of the GADD45 promoter to the p53 protein; And (b) light scattering spectra were measured using a dark-field microscopy and a Rayleigh scattering spectrum, and the maximum wavelength shift value (? _Max ) And detecting the presence or absence of the protein.

The present invention also relates to a method for detecting the binding of a GADD45 promoter to a plasmonic sensor based on gold nanoparticles immobilized with a GADD45 promoter, wherein the wild-type or GADD45 promoter binding site has an amino acid mutated p53 protein, ; And (b) light scattering spectra were measured using a dark-field microscopy and a Rayleigh scattering spectrum, and the maximum wavelength shift value (? _Max ) A method for analyzing the binding activity of GADD45 promoter-p53 protein according to p53 protein amino acid mutation comprising the step of analyzing the binding activity of GADD45 promoter and p53 protein according to amino acid mutation.

The analysis method using the single nanoplasmonic sensor of the present invention can directly analyze the interaction and binding force of the p53 protein binding to a specific promoter without a separate labeling procedure and compare the binding force of wild type and mutant p53 protein It is possible to apply the present invention to an analysis field for early diagnosis of a patient having a high probability of developing a tumor.

FIG. 1A is a schematic diagram illustrating an overall process of analyzing the interaction between DNA and p53 protein using a single nanoplasmonic sensor.
FIG. 1B shows an actual model image of a single nanoplasmonic sensor.
2a is HAuCl ₄ Is a gold particle TEM image of about 30 nm in size, which was made by reducing the solution to sodium citrate.
2B is a dark field image of gold nanoparticles (30 nm) at 1000 magnification.
Fig. 3 shows the results of UV absorption spectra of gold nanoparticles upon attachment of biomaterials.
4A shows the shift of the light scattering spectrum due to the binding of the promoter and the wild type p53 protein to the plasmonic sensor to which a single gold nanoparticle is immobilized.
FIG. 4B shows the shift of the light scattering spectrum due to the binding of the promoter and the mutant type p53 protein in the mouse to a plasmonic sensor immobilized with a single gold nanoparticle.
FIG. 5 is a graph comparing the binding rate of the promoter with the selective mutation of the p53 DNA binding domain of the mouse through the maximum wavelength shift of the gold nanoparticles.
FIG. 6A is a graph comparing the binding rate of various types of breast cancer p53 protein with the GADD45 promoter through the maximum wavelength shift of gold nanoparticles.
FIG. 6B is a graph comparing the binding rate of various kinds of lung cancer p53 protein with the GADD45 promoter through the maximum wavelength shift of gold nanoparticles.
FIG. 7A is a graph showing an ELISA (Enzyme-Linked Immunosorbent Assay) test result comparing the relative concentration values of various kinds of breast cancer p53 proteins.
FIG. 7B is a graph showing the results of an ELISA (Enzyme-Linked Immunosorbent Assay) test comparing relative concentrations of various kinds of lung cancer p53 proteins.
FIG. 8 is an image showing the results of an electrophoretic mobility shift assays (EMSA) analysis of the difference in binding force between the GADD45 promoter and the p53 DNA binding domain.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is well known and commonly used in the art.

(A) contacting wild-type or mutant p53 protein with a plasmonic sensor based on gold nanoparticles immobilized with a GADD45 promoter to induce the binding of the GADD45 promoter to the p53 protein; And (b) light scattering spectra were measured using a dark-field microscope and a Rayleigh scattering spectrum, and the maximum wavelength shift value (? _Max ) analysis obtained therefrom was used to analyze the GADD45 promoter And analyzing the interaction of the p53 protein with the GADD45 promoter-p53 protein.

In another aspect, the present invention provides a method for detecting a GADD45 promoter, comprising: (a) inducing a binding of a GADD45 promoter and a p53 protein by contacting a wild-type or mutant p53 protein to a gold nanoparticle-based plasmonic sensor immobilized with a GADD45 promoter; And (b) light scattering spectra were measured using a dark-field microscope and a Rayleigh scattering spectrum, and the maximum wavelength shift value (? _Max ) analysis obtained therefrom was used to analyze the GADD45 promoter And a method for analyzing the binding activity of p53 protein to GADD45 promoter-p53 protein binding activity.

In another aspect, the present invention relates to a method for inducing the binding of a GADD45 promoter to a p53 protein by contacting a plasmonic sensor based on gold nanoparticles immobilized with the GADD45 promoter with a sample containing wild-type or mutant p53 protein step; And (b) light scattering spectra were measured using a dark-field microscopy and a Rayleigh scattering spectrum, and the maximum wavelength shift value (? _Max ) And detecting the presence or absence of the protein.

In another aspect, the present invention provides a method for detecting a GADD45 promoter, comprising: (a) contacting a plasmonic sensor based on gold nanoparticles immobilized with the GADD45 promoter with a wild-type or p53 protein having an amino acid mutated at the GADD45 promoter binding site, Inducing binding; And (b) light scattering spectra were measured using a dark-field microscopy and a Rayleigh scattering spectrum, and the maximum wavelength shift value (? _Max ) The present invention relates to a method for analyzing the binding activity of GADD45 promoter-p53 protein according to the mutation of p53 protein amino acid, including the step of analyzing the binding activity of GADD45 promoter and p53 protein according to amino acid mutation.

The present invention relates to a method for analyzing the interaction between a GADD45 promoter and a p53 protein using a single nanoplasmonic sensor. More particularly, the present invention relates to a method for binding and immobilizing a GADD45 promoter using a single gold nanoparticle, The present invention provides a method for inducing the binding of p53 protein to analyze the difference in interaction or binding force depending on wild type and mutation.

The present invention uses a GADD45 promoter as a DNA specific for p53 protein in order to provide a method for analyzing the interaction between DNA and p53 protein using a single nanoplasmonic sensor. However, the present invention is not limited to this and is specific to p53 protein known in the art DNA or promoter can be used to measure the interaction or binding force with the p53 protein.

The p53 protein is known to bind to the GADD45 promoter to increase the expression level of GADD45 and induce the inhibition of the cyclin-CDK complex. The GADD45 promoter, which is a p53 protein binding domain, has a conserved sequence of about 30 bp, And can be used for early cancer diagnosis by analyzing the ability to bind to mutant p53 protein.

In addition, the present invention can analyze the mutation of the p53 protein, which plays an important role in the generation and inhibition of cancer development, by analyzing the difference of the binding force of the p53 protein. The difference of the binding force with the GADD45 promoter The comparison of wavelength shift values) allows the identification and analysis of important mutation positions in the DNA binding domain of the p53 protein. In particular, it is possible to directly analyze the interaction of p53 protein binding to a specific promoter without a separate labeling procedure, and it is possible to grasp the importance of each region according to the amino acid variation of the protein using the plasmonic sensor.

In the present invention, to analyze the interaction or binding force between the GADD45 promoter and the p53 protein, p53 protein having a DNA binding site mutation together with wild type p53 protein was used. To do this, we isolated wild-type and mutant p53 proteins from wild-type and mutant p53 proteins (Table 1), human breast cancer cell lines (Table 2) and lung cancer cell lines (Table 3) The differences were analyzed.

In the present invention, the gold nanoparticle-based plasmonic sensor having the GADD45 promoter immobilized therein may be (i) immobilized gold nanoparticles of 25 to 35 nm in size on a transparent support selected from glass or plastic ; And (ii) immobilizing the GADD45 promoter and the organic adsorbent HS (CH ₂ ) ₁₁ (OCH ₂ CH ₂ ) ₃ OH on the surface to which the gold nanoparticles are immobilized.

In the present invention, the step of inducing the binding of the GADD45 promoter and the p53 protein in step (a) is performed at 35 to 45 ° C for 20 to 40 minutes.

In an embodiment of the present invention, to fabricate a sensor for analyzing GADD45 promoter-p53 protein interaction based on surface plasmon resonance, gold nanoparticles of about 30 nm (Figs. 2A and 2B) (FIG. 2A) and fixed on a glass substrate. The glass substrate on which the gold nanoparticles were immobilized was mounted on an imaging chamber (RC-30, Warner Instrument Inc.), and the imaging chamber was mounted on a dark field microscope And inserted into a sample holder. The GADD45 promoter was then added to the gold nanoparticle surface by injecting a mixture of the GADD45 promoter (HS (CH ₂ ) ₁₁ (OCH ₂ CH ₂ ) ₃ OH) in a molar ratio of 1: 8 into the chamber for 12 hours Immobilized. The degree of surface modification of the gold nanoparticles was measured with a UV / VIS spectrophotometer (FIG. 3). The results are shown in FIG. 3. The gold nanoparticles were unmodified -> surface-modified gold nanoparticles -> gold nanoparticles .

In order to compare the degree of binding between the GADD45 promoter and the p53 protein immobilized on the gold nanoparticles in the nanoplasmonic sensor prepared by the method of the present invention, the wild-type p53 protein and the mutant p53 protein isolated from the mouse were injected into the chamber, To induce binding between DNA and protein. Light scattering spectra using a dark field resonance microscope were measured at each stage where immobilization was performed to determine the maximum wavelength value. As a result, it was confirmed that the maximum wavelength shifts to the right in each immobilization step (FIGS. 4A and 4B). This shift in wavelength is caused by a change in refractive index around single gold nano- It can be seen that the genetic environment is changed by attaching promoter and selectively binding proteins to the nanoparticles, and thus the wavelength value of the spectrum shifts to a long wavelength per adsorption step. In other words, by analyzing the maximum wavelength mobility, the interaction with the p53 protein that binds to the GADD45 promoter can be comparatively analyzed.

In order to accurately measure the interaction or binding force of various p53 proteins, spectrum analysis was performed by measuring background noise and mechanically generated noise signals of the dark field resonance microscope itself, and the peripheral refractive index change of single gold nanoparticles The maximum LSPR scattering wavelength shift value (? _Max ) obtained from the LSPR was calculated by the following equation (1).

[Equation 1]

Δλ _max = lambda _max (after binding) -? _max (before binding)

The mouse p53 protein, human breast cancer cell line, and p53 protein isolated from lung cancer cell line were bound to a nanoplasmonic sensor having a GADD45 promoter immobilized thereon. The spectrum was measured and the peripheral refractive index of a single gold nanoparticle The LSPR scattering maximum wavelength shift (λ _max ) obtained from the change was calculated.

As a result of comparing the maximum wavelength shift of the p53 protein in the mouse, it was confirmed that the maximum wavelength value of the spectrum of wild type p53 protein was shifted by 9.6 nm. In case of the mutant p53 protein (I252F and R270H), the maximum wavelength value of the spectrum was 4.2 nm, and 1.7 nm, respectively (FIGS. 5 and 4).

As a result of comparing the maximum wavelength shift values of the p53 protein of human breast cancer cell lines, it was confirmed that the maximum wavelength values of the spectrum of the wild type p53 protein isolated from the ZR-75-1 and MCF-7 cell lines were shifted by 8.6 nm and 9.3 nm, respectively . The mutant p53 protein isolated from T47D, BT474, SKBR3, MDA-MB-231 and MDA-MB-435s showed the highest peak wavelengths of 1.2 nm, 1.0 nm, 0.64 nm, 2.66 nm and 2.1 nm (Fig. 6A and Table 5). In addition, the maximum wavelength shift of the p53 protein in human lung cancer cell lines was found to be 15 m in the wild-type p53 protein isolated from the H460 cell line. The H522, H441, Calu-3 and H1703 3.9 nm, 2.9 nm, 2.66 nm, and 2.4 nm, respectively, in the case of the mutant p53 protein isolated from E. coli (FIG. 6B and Table 5).

Comparing the results, it can be seen that the shift values of the spectral wavelength are different according to the p53 protein amino acid mutation. This is caused by the difference in the binding ability of the GADD45 promoter, and the important mutation position of the DNA binding domain of the p53 protein is identified .

Among the mutated samples of the breast cancer p53 protein used in the present invention, the amino acid arginine was mutated to histidine at the DNA binding domain 175 position in the DNA binding domain 175 of SKBR3. This region is known to play a more important role in binding to the promoter than other sites of the p53 DNA binding domain. Mutations in the region with these important functions result in significantly less binding to the promoter than other mutant proteins, which can be confirmed as a result of spectral wavelength shift.

In the case of H522 of the lung cancer p53 protein mutation sample used in the present invention, p53 of the DNA binding domain 191 position (DNA binding domain 191 position) is knocked down due to gene deletion and there is no expression of p53 protein. Therefore, the binding capacity is significantly lower than that of other mutant proteins, and the lowest maximum shift value is observed.

That is, when comparing the maximum wavelength shift values of the mutant p53 protein and the wild type p53 protein (Compare with DELTA lambda _max ), it means that mutation occurred in a significant portion of the sample showing the highest value. It was confirmed that the important mutation position of p53 protein can be detected using a monic sensor.

In order to confirm that the difference in the maximum wavelength shift value according to the mutant p53 protein of the present invention was caused by the difference in the binding force, it was quantified by ELISA (Enzyme-Linked Immunosorbent Assay) 7a and 7b), no large difference in concentration was found for each sample, and the p53 concentration of the specific sample was high, so that the difference in the maximum wavelength value of the light scattering spectrum was not caused, but the difference Was observed.

Furthermore, the difference in the peak wavelength shift of light scattering between breast cancer p53 protein and lung cancer wild type p53 protein was confirmed by the fact that the concentration of wild type p53 protein in lung cancer was higher than that of breast cancer wild type p53 protein.

In order to confirm the validity of the difference of binding force of GADD45 promoter-p53 protein obtained by nanoplasmonic sensor, the electrophoretic mobility shift assays (EMSA) It was confirmed that the results agree with the results of light scattering analysis. This confirms that the nanoplasmonic sensor can more quickly and accurately analyze the difference in binding force between the promoter and the protein (wild type, mutant type).

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these examples are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples.

Wild type and mutant p53  Protein separation

The p53 protein used in this experiment was wild type and mutant type proteins obtained from rats, and wild type and mutant type p53 proteins obtained from human breast cancer and lung cancer cell lines, respectively, and the constituents thereof are shown in Tables 1 to 3.

Information on the DNA-binding domain of rat-derived p53 protein Name Conc.
(ug / ul) Pos. WT Mut AA Mut p53
wild type 12 - - - - - I252F 4 252 ATT TTT Ile Phe R270H 5 270 CGC CAC Arg His

Information on the DNA-binding domain of human-derived p53 protein - Breast cancer samples Name Conc.
(ug / ul) Pos. WT Mut AA Mut ZR-75-1 5.8 - - - - - MCF-7 6.2 - - - - - T47D 7.1 194 CTT TTT Leu Phe BT474 3.5 282 GAG AAG Glu Lys SKBR3 5.7 175 CGC CAC Arg His MDA-MB-231 5.7 280 AGA AAA Arg Lys MDA-MB-435s 8.3 266 GGA GAA Gly Glu

Information on the DNA-binding domain of human-derived p53 protein - Lung cancer samples Name Conc.
(ug / ul) Pos. WT Mut AA Mut H460 3.5 - - - - - H522 3.3 191 CCT del1a Pro Fs. H441 4.3 158 CGC CTC Arg Leu Calu-3 3.9 237 ATG ATT Met Ile H1703 5.1 285 GAG AAG Glu Lys

The p53 protein was isolated from the cell line using a method known in the art for protein separation.

Preparation of gold particles and gold nanoparticle immobilization and chamber setting on glass substrate

To fabricate a sensor for detecting the GADD45 promoter-p53 protein interaction based on the surface plasmon resonance phenomenon of the present invention, gold nanoparticles having a size of about 30 nm (FIGS. 2A and 2B) were synthesized by a method known in the art Min Sun Song et al . , Adv . Mater ., 25: 1265,2013). The prepared gold nanoparticles were immobilized on a cover slip slide (22 x 40 x 0.1 mm). Prior to this, the cover slip slide was vigorously washed with tertiary distilled water after sonication for 20 minutes in a piranha solution (3: 1 H ₂ SO ₄ / H ₂ O ₂ ).

The solution containing the spherical gold nanoparticles having a size of about 30 nm was diluted to 0.04 at an absorbance of 528 nm and physically adsorbed on a glass substrate (slide glass) through drop-coating. Here, the concentration of gold nanoparticles and the incubation time serve to reduce the coupling effect between particles by controlling the intergranular spacing.

Thereafter, the prepared glass substrate sample was mounted in a microfluidic imaging chamber (RC-30, Warner Instrument Inc.), and the imaging chamber was inserted into a sample holder of a dark field resonance microscope. The imaging chamber is connected to the syringe pump, and the velocity of the fluid flowing into the chamber is adjusted to 100 μl / min, as a means for injecting various chemicals such as adsorbates and reactants necessary for surface functionalization of gold nanoparticles.

The surface of the gold nanoparticles was washed with an absolute ethanol solution, and then the third distilled water was injected to remove all impurities and unprocessed gold nanoparticles. At this time, since the gold nanoparticles may be electrically adsorbed on the glass substrate, distilled water was sufficiently injected for 30 minutes or more (FIGS. 1A and 1B).

certain DNA Wow p53  Nano for selective binding of protein Plasmonics  Sensor manufacturing

3-1: Nano Plasmonics  Sensor manufacturing

In order to compare the degree of binding between the p53 protein and the specific DNA, the GADD45 promoter, a specific DNA, was immobilized on the gold nanoparticles in the following manner.

GADD45 promoter

[SEQ ID NO: 1] GADD45 promoter UP

5'-GTACAGAACATGTCTAAGCATGCTGGGGAC-3 '

[SEQ ID NO: 2] GADD45 promoter LO

Gt;

First, the GADD45 promoter and the organic adsorbent (HS (CH ₂ ) ₁₁ (OCH ₂ CH ₂ ) ₃ OH) in which the thiol group was introduced into the RC-30 closed bath chamber immobilized with gold nanoparticles prepared in Example 1, 8 was injected and reacted for 12 hours to immobilize the DNA corresponding to the GADD45 promoter on the gold nanoparticle surface. After the reaction, unreacted impurities were removed by washing with third distilled water. The degree of surface modification of the gold nanoparticles was measured with a UV / VIS spectrophotometer (FIG. 3). The results are shown in FIG. 3. The gold nanoparticles were unmodified -> surface-modified gold nanoparticles -> gold nanoparticles .

3-2: Single Nano Plasmonics  Detection of p53 protein detection using sensor

In order to compare the degree of binding of the p53 protein to the GADD45 promoter immobilized on gold nanoparticles in the nanoplasmonic sensor prepared by the method of Example 2, the GADD45 promoter was immobilized on gold nanoparticles and washed in Example 2-2 Finally, 200 ㎕ of p53 protein selectively binding to GADD45 promoter DNA was injected into the chamber and reacted for 2 hours to induce binding between DNA and protein. After all the reactions were completed, unbound p53 protein was washed with distilled water and light scattering spectra were measured using a dark field resonance microscope at each step of immobilization.

As a result, it was confirmed that the maximum wavelength moved to the right in each immobilization step, and it was confirmed that the interaction with the p53 protein binding to the GADD45 promoter could be comparatively analyzed through the analysis of the maximum wavelength mobility (Fig. 4A And FIG.

In order to accurately measure the optical response of a single gold nanoparticle, the background spectrum and the mechanically generated noise signal of the dark field resonance microscope itself were measured and spectral analysis was performed.

(GADD45 promoter, wild-type p53 protein, or mutant p53 protein) was measured by randomly selecting 10 to 12 particles (Fig. 2B) of arbitrary single gold nanoparticles visible on the dark field After sequential flow, spectral changes were analyzed by measuring the spectrum of the same particles at each step. This process was repeated for each variation of each protein, and then the wavelet maximum wavelength value was accurately and effectively analyzed by applying the Lorentzian algorithm of the OriginPro 8.0 program to the spectrum obtained as a result of the experiment, for data processing. Finally, the LSPR scattering maximum wavelength shift (λ _max ) obtained from the change in the refractive index of the single gold nanoparticle was calculated by the following equation (1).

[Equation 1]

Δλ _max = λ _max (after binding) -λ _max (before binding)

Wild type Mutant type  Analysis and comparison of movement of light scattering spectrum by attachment of protein

In order to compare the difference in binding force between the GADD45 promoter and the p53 protein (wild type / mutant type) isolated in Example 1, the degree of migration of the light scattering spectrum using a nanoplasmonic sensor was analyzed.

In addition, SP6 RNA polymerase (Promega Corporation), which was not associated with the GADD45 promoter, was used as a control for reliable analysis of whether the GADD45 promoter and the p53 protein (wild type / mutant type) were selectively bound.

4-1: Rats p53  Protein (wild type / Mutant type )

After binding the GADD45 promoter to a single gold nanoparticle, the mouse p53 protein selectively binding to the GADD45 promoter was bound (Table 1), and the spectrum was measured. From the change in the peripheral refractive index of a single gold nanoparticle The obtained LSPR scattering maximum wavelength shift value (? _Max ) was calculated.

The maximum wavelength shift value of mouse p53 protein Type of p53 protein Name of cell lines Δλ _max (nm) Compare with Δλ _max
(wild type higher)  Mouse p53 protein Wild type 9.6 - I252F 4.2 2.3 R270H 1.7 5.6

As a result, it was confirmed that the maximum wavelength value of the spectrum of wild-type p53 protein was shifted by 9.6 nm, and that of the mutant p53 (I252F and R270H) protein was shifted by 4.2 nm and 1.7 nm, respectively (Fig. 5 and Table 4).

In addition, in the case of the control group, the maximum wavelength shift value of the spectrum hardly occurred. As a result, the results of the experiment using the nanoplasmonic sensor are valid and reliable, and it is confirmed that only the p53 protein can be selectively discriminated.

4-2: Protein pretreatment process in human cell line

Trypsinize the cancer cells in a normal growth state to obtain a cell pellet and then mix the cell pellet with a lysis buffer, And incubated in ice for 30 minutes. After centrifugation, only the supernatant was transferred to a new tube, and the protein concentration present in the supernatant was quantitated using Bradford quantitation.

4-3: Comparison of the binding capacity of p53 protein (wild type / mutant type) in human breast cancer cell line

After binding the GADD45 promoter to a single gold nanoparticle, the p53 protein of a human breast cancer cell line (Table 2) binding selectively to the GADD45 promoter was bound and the spectrum was measured to determine the perimeter of single gold nanoparticles The LSPR scattering maximum wavelength shift (λ _max ) obtained from the refractive index change was calculated.

The maximum wavelength shift of p53 protein in human breast cancer cell lines Type of p53 protein Name of cell lines Δλ _max (nm) Compare with Δλ _max
(wild type higher) Breast cancer
Protein ZR-75-1 (wild type) 8.6 × MCF-7 (wild type) 9.3 - T47D 1.2 7.8 BT474 1.0 9.3 SKBR3 0.64 14.5 MDA-MB-231 2.66 3.5 MDA-MB-435s 2.1 4.4

(Comparison of maximum wavelength shift values of wild type and mutant type is based on MCF-7 value)

As a result, it was confirmed that the maximum wavelength values of the spectrum of wild-type p53 protein isolated from the ZR-75-1 and MCF-7 cell lines were shifted by 8.6 m and 9.3 m, respectively. T47D, BT474, SKBR3, MDA-MB-231 And MDA-MB-435s (Fig. 6A and Table 5), respectively. In the case of the mutant p53 protein isolated from MDA-MB-435s, the maximum wavelength values of the spectrum were shifted by 1.2 m, 1.0 m, 0.64 m, 2.66 m and 2.1 m, respectively.

4-4: Comparison of the binding capacity of p53 protein (wild type / mutant type) of human lung cancer cell line

After binding the GADD45 promoter to a single gold nanoparticle, the p53 protein of a human lung cancer cell line (Table 3) binding selectively to the GADD45 promoter was bound and the spectrum was measured to determine the perimeter of single gold nanoparticles The LSPR scattering maximum wavelength shift (Δλ _max ) obtained from the refractive index change was calculated.

The maximum wavelength shift of p53 protein in human lung cancer cell lines Type of p53 protein Name of cell lines Δλ _max (nm) Compare with Δλ _max
(wild type higher) Lung cancer
Protein H460 (wild type) 15 - H522 2.05 7.3 H441 3.9 3.8 Calu-3 2.9 5.2 H1703 2.4 6.3

As a result, the wild-type p53 protein isolated from the H460 cell line showed a shift of the maximum wavelength of the spectrum by 15 m. In the case of the mutant p53 protein isolated from H522, H441, Calu-3 and H1703, 2.05m, 3.9m, 2.9m, 2.66m, and 2.4m (Fig. 6b and Table 5).

4-5: Comparison of the importance of mutant proteins according to amino acid variation of protein

Using the nanoplasmonic sensor of the present invention, the wavelength shift values of the light scattering spectra were compared and analyzed in order to confirm the difference in binding force with the GADD45 promoter according to the amino acid modification of the p53 protein.

Among the mutated samples of the breast cancer p53 protein used in the present invention, the amino acid arginine was changed to histidine at the DNA binding domain 175 position in the case of SKBR3, and as shown in Table 5, Mutation occurred.

In addition, among the mutant samples of lung cancer p53 protein used in the present invention, H522 exhibited the lowest peak shift value (Table 6) as the gene was knocked down by p53 deletion at the DNA binding domain 191 position (Table 6) It can be confirmed that the bonding force is significantly lowered.

That is, when comparing the maximum wavelength shift values of the mutant p53 protein and the wild type p53 protein (Compare with DELTA lambda _max ), it means that the sample showing the highest value mutated at an important part, It was confirmed that the important mutation position of p53 protein can be detected using a monic sensor.

Various kinds of p53  Pure from protein p53  Comparison of relative concentration values of proteins only

The cell line samples of human breast cancer and lung cancer used in the single nanoplasmonic sensor of the present invention contain different concentrations of p53 protein. Although the total protein concentration was used after being quantitatively determined, the concentration of only the pure p53 protein contained therein was quantitated by ELISA (Enzyme-Linked Immunosorbent Assay) experiment because it exists at a low concentration between the wild type and the mutant type protein 7A and 7B).

As a result, p53 protein concentration was measured in the range of 0.57 ~ 1.20 ng / ul, and it was detected up to 17.90nM. In addition, no large difference in concentration was found for each sample, and as shown in the relative protein concentration value shown in FIG. 7, the p53 concentration of the specific sample was high, so that the difference in the maximum wavelength value of the light scattering spectrum was not caused, It was confirmed that there is a difference in bonding force depending on the presence or absence of the bond.

The result of the difference between the binding force of protein and DNA obtained from single nanoplasmonic sensor and the analysis result of electrophoretic migration

In order to confirm the validity of the difference in binding force between protein and DNA obtained through the nanoplasmonic sensor of the present invention, the difference in binding force was compared through an electrophoretic mobility shift assay (EMSA) experiment.

EMSA experiments for amino acid mutations of murine p53 protein were individually measured under the same conditions as for nanoplasmonic sensor experiments.

As a result of comparing the binding strengths of wild-type p53 protein, I252F sample and R270H sample, the results showed about 100%, about 50%, and about 0%, respectively (FIG. 8). The results of the light scattering analysis of the nanoplasmonic sensor of FIG. Respectively.

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereby. something to do. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

<110> Korea University Research and Business Foundation <120> Analysis Method for DNA Interaction with p53 Protein Using Single          Plasmonic Nanosensor <130> P13-B326 <160> 2 <170> Kopatentin 2.0 <210> 1 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> GADD45 promoter UP <400> 1 gtacagaaca tgtctaagca tgctggggac 30 <210> 2 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> GADD45 promoter LO <400> 2 gtccccagca tgcttagaca tgttctgtac 30

Claims (7)

A method for analyzing the interaction of a GADD45 promoter-p53 protein comprising the steps of:
(a) contacting wild-type or mutant p53 protein to a plasmonic sensor based on gold nanoparticles immobilized with GADD45 promoter to induce the binding of GADD45 promoter and p53 protein; And
(b) The light scattering spectrum was measured using a dark-field microscope and a Rayleigh scattering spectrum, and the maximum wavelength shift value (Δλ _max ) obtained from the measurement was used to analyze the GADD45 promoter analyzing the interaction of p53 protein.
A method for assaying GADD45 promoter-p53 protein binding activity comprising the steps of:
(a) contacting wild-type or mutant p53 protein to a plasmonic sensor based on gold nanoparticles immobilized with GADD45 promoter to induce the binding of GADD45 promoter and p53 protein; And
(b) The light scattering spectrum was measured using a dark-field microscope and a Rayleigh scattering spectrum, and the maximum wavelength shift value (Δλ _max ) obtained from the measurement was used to analyze the GADD45 promoter analyzing the binding activity of p53 protein.
A mutant p53 protein detection method comprising the steps of:
(a) contacting a plasmonic sensor based on gold nanoparticles immobilized with the GADD45 promoter with a sample containing a wild-type or mutant p53 protein to induce the binding of the GADD45 promoter to the p53 protein; And
(b) Light scattering spectra were measured using a dark-field microscope and a Rayleigh scattering spectrum, and the maximum wavelength shift value (? _max ) analysis obtained therefrom was used to analyze the mutant p53 protein And
Analysis of the binding activity of the GADD45 promoter-p53 protein according to the p53 protein amino acid variation including the following steps:
(a) inducing the binding of GADD45 promoter and p53 protein to a plasmonic sensor based on gold nanoparticles immobilized with GADD45 promoter by contacting the wild type or p53 protein having amino acid mutation at GADD45 promoter binding site; And
(b) Light scattering spectra were measured using a dark-field microscope and a Rayleigh scattering spectrum, and the maximum wavelength shift value (? _max ) analysis obtained therefrom was used to analyze the p53 protein amino acid Analysis of the binding activity of GADD45 promoter and p53 protein according to mutation.
The plasmonic sensor according to any one of claims 1 to 4, wherein the gold nanoparticle-based plasmonic sensor immobilized with the GADD45 promoter of step (a) comprises (i) a transparent support selected from glass or plastic Immobilizing gold nanoparticles having a size of about 35 nm to about 35 nm; And (ii) immobilizing the GADD45 promoter and the organic adsorbent HS (CH ₂ ) ₁₁ (OCH ₂ CH ₂ ) ₃ OH on the surface to which the gold nanoparticles are immobilized.
5. The method according to any one of claims 1 to 4, wherein the step of inducing the binding of the GADD45 promoter and the p53 protein in step (a) is carried out at 35 to 45 DEG C for 20 to 40 minutes Way.
5. The method according to any one of claims 1 to 4, wherein the wild-type or mutant p53 protein in step (a) is isolated from cancer cells and treated at the same concentration.

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