full papers Cancer Detection
Distinct Rayleigh Scattering from Hot Spot Mutant p53 Proteins Reveals Cancer Cells Ho Joon Jun, Anh H. Nguyen, Yeul Hong Kim, Kyong Hwa Park, Doyoun Kim, Kyeong Kyu Kim,* and Sang Jun Sim*
The scattering of light redirects and resonances when an electromagnetic wave interacts with electrons orbits in the hot spot core protein and oscillated electron of the gold nanoparticles (AuNP). This report demonstrates convincingly that resonant Rayleigh scattering generated from hot spot mutant p53 proteins is correspondence to cancer cells. Hot spot mutants have unique local electron density changes that affect specificity of DNA binding affinity compared with wild types. Rayleigh scattering changes introduced by hot-spot mutations were monitored by localized surface plasmon resonance (LSPR) shift changes. The LSPR λmax shift for hot-spot mutants ranged from 1.7 to 4.2 nm for mouse samples and from 0.64 nm to 2.66 nm for human samples, compared to 9.6 nm and 15 nm for wild type and mouse and human proteins, respectively with a detection sensitivity of p53 concentration at 17.9 nM. It is interesting that hot-spot mutants, which affect only interaction with DNA, launches affinitive changes as considerable as wild types. These changes propose that hot-spot mutants p53 proteins can be easily detected by local electron density alterations that disturbs the specificity of DNA binding of p53 core domain on the surface of the DNA probed-nanoplasmonic sensor.
1. Introduction Induced dipole moment of periodic separation of charge is generated from oscillation or perturbation of the electron
H. J. Jun, A. H. Nguyen, Prof. S. J. Sim Department of Chemical and Biological Engineering Korea University Seoul 136-701, Korea E-mail: [email protected] Prof. Y. H. Kim, Prof. K. H. Park Medical Oncology Department of Internal Medicine Korea University College of Medicine Seoul 136-705, Korea Dr. D. Kim, Prof. K. K. Kim Department of Molecular Cell Biology Sungkyunkwan University School of Medicine Samsung Biomedical Research Institute Suwon 440-746, Korea E-mail: [email protected] DOI: 10.1002/smll.201400004
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cloud. This event transfers free electrons at the nanoparticle surface via a supporting insluting substrate for electrochemistry catalysis.[1a] Previous reports theorectically and experimentally proved electron transfer reactions at AuNP,[1b] quantitated electron transferate of electron transfer rate on AuNP during catalytic reactions,[1a,b] and interestingly desmonstrated a fact AuNPs can effectively interact with proteins and even interchange the electrons with some redox-proteins.[1c] Since electron movement surrounding AuNPs is directly associated to the plasma frequency of AuNP and in situ Rayleigh scattering profile permitted one to count a number of electrons during the catalytic reaction.[1a] Moreover, a correlative equation between experimental band shift in the Rayleigh wavelength profile with the electron quantity presents (electron idensity changes) in the biochemical reaction, and gives dormant tool for the precise distinction of electron transfer rates in redox heterogenous proteins on the AuNP surface.[1a-c] Using an electromagnetic wave, a particular light-matter interaction, is to resonance Rayleigh scattering of light at local dielectric environment of AuNP surface called localized surface Plasmon resonance (LSPR), and has been applied for
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biological and chemical sensing.[1d,e] LSPR is sensitive to altera- nesses such as having low sensitivity, low specificity, and high tions in the local dielectric environment generating wavelength cost, as well as being time-consuming.[11,12] Previously, we have developed a new nanoplasmonic shift responding to electromagnetic-field enhancement. Local dielectric environment is dependent on local electric field of sensor to detect the protein-DNA interaction.[13] In this binding hot spot core protein with specific DNA.[1f] The dielec- study, we further developed this sensor to identify cancer tric property inside a hot spot domain is a key chemo-physical cells though detecting resonance of local dielectric environfactor of the extent of electrostatic interactions of hot spot ment of hot-spot mutant p53 promoter on AuNP surface.[14] cores.[1f] The dielectric effect inside a hot spot domain in binding To monitor distinct Rayleigh scattering from hot-spot core p53 structure of protein/DNA determines electrostatic energies of proteins, we fabricated the LSPR-based platform from single hot spot mutants or wild type of a specific proteins.[1h] Elec- GADD45-conjugated AuNP. We found that the LSPR spectra trostatic interaction between protein and DNA are ubiqui- shift of resonance local dielectric of hot-spot mutant p53 protous, affected by protein structure, ligand binding, and electron tein changes upon amino acid substitution, deletion within transfer in hot-spot binding domain.[1f,h] In this work, we focus the hot-spot, which determine binding affinity of the p53 to on the resonance of local dielectric of hot-spot mutants p53 GADD45. These results demonstrated that the nanoplasmonic proteins on AuNP surface where the resonance occurs. sensor can detect mutant p53 proteins from wild types of real As proof-of-concept, the p53 is well-known a tumor sup- samples simply and directly, facilitating the efficient screening pressor protein that plays as an important role in preventing of many mutant proteins such as HER2 and BRCA1 and thus the development of cancer.[1g,2] It regulates the cell cycle by suggesting itself as a promising tool for use in cancer diagnosis. controlling the expression of various genes in response to DNA damage and cellular apoptosis.[3] With the loss of p53 function, usually the result of mutations within the DNA 2. Results and Discussion binding domain, unregulated cell growth can lead to tumors. Mutations in p53 protein are found in over half of all human AuNPs are ideal platforms for the quantification of biochemcancers,[4] in particular breast[5] and lung cancer,[4] where the ical species or the monitoring of dynamic processes inside mutation rate can reach 85%.[6] Almost all p53 mutant forms biological cells.[15] Colloidal AuNPs were synthesized by the have mutations at the hot-spot DNA binding domain, located method described in the experimental section,[16] with an at the residues 102-292,[7,8] which causes either hot-spot local average diameter of 30.7 ± 1.8 nm, as estimated by HR-TEM dielectric changes or reduction in its sequence-specific DNA images (Figure 1A). To reduce inter-particle coupling effects, binding affinity.[5] These hot-spot mutant variants reduce tumor suppressor function because p53 is less able to recognize the specific nucleotide sequence on promoters because a reduction occurs of chargecharge and charge-polar interaction of local dielectric environment of hot-spot mutant p53 protein, which leads to uncontrolled regulation during cellular processes.[5] Thus the binding affinity between mutant p53 variants with specific DNA sequence is characterized by a lower binding constant compared with the wild type.[9] In this context of local dielectric environment of hot-spot mutant domain, the evaluation of the binding affinity of mutant p53 proteins to the p53-binding element of GADD45 promoter can be a useful approach for early cancer detection. GADD45, which causes growth arrest and DNA damage, is one of the p53 regulatory proteins and contains a consensus p53-binding sequence in its promoter region.[10] The accurate detection of proteins related to disease is an important area of biomedical and biochemical research, offering great potential for highly reliable diagnosis and prognosis.[4] However, since most mutations occur at Figure 1. (A) HR-TEM images of spherical Au nanoparticles (∼30 nm) chemically synthesized a specific site on the DNA binding central through citrate mediated reduction of HAuCl4 solution. (B) Dark-field image of bare Au domain, current methods exhibit weak- nanoparticles (∼30 nm) at a magnification of 1000X. small 2014, 10, No. 14, 2954–2962
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Figure 2. UV-Vis absorption spectra of gold nanoparticles (AuNPs, 528 nm), OEGs-AuNPs, (533 nm) and DNA-conjugated AuNPs (538 nm).
the AuNP solution was diluted with ultrapure water to an optical density of 0.04 OD at 528 nm and immobilized on a glass slide, this created a particle to particle spacing of greater than 2.5 times the particle diameter, a spacing above which there are no coupling effects.[17,18] The AuNPs were functionalized with HS-C11-EG3-OH to prevent the non-specific adsorption of proteins.[19] AuNP surface was further conjugated with the GADD45 promoter, a 30-base pair element containing the consensus p53-binding sequence, for the detection of p53 protein. The functionalized products were confirmed by UV-VIS at 528 nm (Figure 2). Due to the plasmon resonance characteristics of AuNPs,[20] a damping of the surface plasmon band compared with bare AuNPs occurred as a red shift of 4.5 nm and 10.5 nm corresponding to capping by OEG3 and the GADD45 promoter, respectively. This was used as evidence that the AuNPs had been conjugated with OEG3 and the GADD45 promoter, indicating the successful attachment of the DNA onto the surface of the AuNPs. Initially, we used the purified DNA binding domain of mouse p53 (mp53 DBD) to test the correlation between DNA binding affinity of proteins and the Rayleigh light scattering. For this purpose, the wild-type and two mutant p53 proteins, I252F and R270H, were prepared and bound to GADD45 promoter conjugated to AuNPs. The particles were monitored by the Rayleigh light scattering. Ile252 and Arg270 mutants corresponding to Ile255 and Arg273 mutants found in human cancers are known to have weak and no binding affinity to the consensus p53-binding site, respectively.[21] The binding of the wild-type and the mutant mp53 DBDs on the promoter was monitored by the Rayleigh light scattering spectra generated by LSPR using the following steps. Under a 100W tungsten white light lamp, a dark-field microscope was used to view a single illuminated AuNP visible as a distinctly bright spot with extremely low background light from the glass substrate (Figure 1B). The scattered light of the individual AuNP was collected by a 100x objective lens. The scattered light spectra was then resolved in a grating spectrograph, and synchronized by a highly sensitive CCD camera.[19,22] Each AuNP was situated easily by adjusting the microslit and sample stage. At each measurement step, to eliminate noise, the background spectrum was normalized to zero at an area adjacent to the individual AuNPs. Finally, the
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spectral data were processed using the Lorentzian algorithm in Origin Pro 8.6. According to the Mie theory, the LSPR of noble metal nanoparticles is not only strongly dependent on their shape, particle type and size, but also local dielectric environment.[14,23] The local dielectric environment of AuNPs is extremely sensitive to perturbations caused by binding events on the AuNP surface. The refractive index of an AuNP depends on the number of p53 proteins binding to the GADD45-conjugated AuNP surface, and thus strongly depends on the binding affinity of p53 proteins with the GADD45 promoter. Figure 3 shows the typical LSPR light scattering spectra for an AuNP after mp53-DBD proteins bind to the GADD45 promoter. The experimental spectra were fitted to a Lorentzian calculation with a shift to the redder end of the spectrum. Compared with a bare AuNP, the maximum LSPR shift (LSPR Δλmax) from a single AuNP after coupling with the GADD45 promoter for the wild type (Figure 3A) and the two mutants (Figure 3B), I252F and R270H, was 9.6nm, 4.2 nm and 1.7 nm, respectively (Figure 4). Wild-type mp53-DBD yielded produced a higher Δλmax than the mutant proteins (Table 1), indicating that the mutant proteins have a low affinity for the GADD45 promoter.[5] This is consistent with the previous report[21] in which it has been proven that the R270H mutant has a weaker binding affinity with GADD45 than the I252F mutant (Table 1). In order to confirm LSPR Δλmax, we compared the LSPR data with electrophoretic mobility shift assays (EMSA), a common method of measuring DNA-protein binding.[24] Figure 5 presents binding between GADD45 promoter with three mp53-DBDs (WT, R270H and I252F). When a specific competitor was added, the binding affinity of mp53-DBD to GADD45 remained to be 100% in the wild type, ∼50% in I252F mutants and 0% in R270H mutant. The nanoplasmonic shift for each protein variant was fairly consistent with this EMSA measurement. These results reflected the reduction of binding affinity caused by mutation in DNA binding domain. Thus, LSPR was able to quickly and accurately discriminate mp53 forms present in real samples by their affinity for a specific promoter. Since it has been validated that the DNA binding affinity of p53 protein to GADD45 is proportional to Δλmax using the purified p53 proteins, we further apply our system to examine breast and lung cancer cell lines (Table 2 and 3) which have the mutant p53 proteins. p53 mutant (mutation at core binding domain) and p53 wild type (no mutation at core binding domain) cell lines are well known in the literature[25–27] and presented in Table 3. p53 concentrations have been quantified by ELISA (Figure 6) because the concentration of human p53 protein varies greatly between samples and p53 is present in low concentrations in breast and lung cancer compared with wild types.[24,28] LSPR spectra generated from the nanoplasmonic biosensor determined the binding strength of the p53 proteins in the cancer cells lines. Figure 7 shows the LSPR light scattering spectra Δλmax for single AuNPs after the binding of breast and lung cancer p53 proteins onto the GADD45 promoter. The experimental spectra were fitted to the Lorentzian calculation with a shift to the redder end of the spectrum. Individual mutant forms were measured with LSPR and the Δλmax for the samples
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MB-435s; lung cancer H522, H441, Calu-3, and H1703). Among the mutant types, the SKBR3 sample showed the lowest Δλmax (Figure 7A), which is possibly caused by the unique properties of the p53 protein of SKBR3. In these cancer cells, Arg175 of p53 protein is known to be a hot spot; it is the most essential residue for the function of p53 protein.[11,30] Thus, if a mutation occurs at a hot spot, the binding affinity is much lower than for another mutations.[31] One of advantage of LSPR sensor is direct measurement of a specific DNA/protein interaction without a need of purification. Therefore, p53 variants in crude protein extracts were directly measured and results of Δλmax values were shifted corresponding to the mutations at core domains for instance, the Δλmax values of BT474 (I282K) and MDA-MB-231 (R280K) were 1.0 nm and 2.66 nm, respectively. In fact, protein-specific DNA interaction is dependent on physical forces including electrostatic interaction, hydrogen bonding, hydrophobic interaction, and dispersion forces.[32] Therefore, chemo-physical properties and the composition of amino acids in the DNA binding domain have a role in determining binding affinity. For example, in BT474 cell lines, E282K mutation in p53 proteins abolishes the electrostatic interaction between protein and DNA. However, in the case of the MDA-MB-231 cells which have R280K mutation in p53 protein, the reduced binding affinity might be achieved by the loss of hydrogen bonding because of the substitution mutation of Arg to Lys. Similar to human breast cell samples, the Figure 3. (A) Representative Rayleigh light scattering spectra of single AuNP according to the crude extract from the lung cancer cell binding of the GADD45 promoter (SH-DNA) and the DNA binding domain of mouse p53 wild line (H460) containing the wild type p53 type. LSPR λmax shifted to the longer wavelengths after every step of the chemical binding of protein produced a higher Δλmax than adsorbates and target analytes. (B) Representative Rayleigh light scattering spectra of single those of other lung cancer cells that conAuNPs according to the binding of the GADD45 promoter (SH-DNA) and the DNA binding tain the mutant p53 proteins (Table 1). domain of mouse p53 (R270H). H522 sample, which has p53 proteins with a deletion mutation in the DNA binding are shown in Table 1. The specific Δλmax for the LSPR was domain, resulted in the smallest shift due to the minimal determined by the strength of p53-GADD45 interaction. DNA binding activity. Binding affinity of mutants is highly We used five humans breast cancer cell lines (T47D, BT474, reduced compared with wild type. For example, BT474 SKBR3, MDA-MB-231, and MDA-MB-435s) and four lung (E282K) and Calu-3(M237I) mutants presented low LSPR cancer cell lines (H522, H441, Calu-3, and H1703) (Table 3) Δλmax compared with wild type although we injected the to study binding affinity of mutant p53 proteins present in same p53 concentration (Figure 6). This is demonstrated a the applicable cell lines to the GADD45 promoter using the low LSPR Δλmax, the result of the reduction in binding affinity Δλmax detected by the plasmonic biosensor.[29] For compar- with the GADD45 promoter due to the mutations in DNA ison, ZR-75-1, MCF-7 and H460, which are known to have binding domain. To further validate our methods, it is essenthe wild-type p53 proteins, were introduced. As expected, tial to examine the nonspecific protein binding to GADD45 the cell extract from these three cell lines produced a higher promoter using the negative control. In Figure 7, SP6 RNA Δλmax than the samples from other cancer cell lines (breast polymerase was introduced as a negative control. Under the cancer T47D, BT474, SKBR3, MDA-MB-231, and MDA- same experimental conditions as the trials using p53 protein, small 2014, 10, No. 14, 2954–2962
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Table 1. Comparison of binding affinity between wild and mutant type p53 proteins through the ratio of Δλmax (wild /mutant type). Type of p53 protein
p53 mutation (cell line)
Δλmax (nm)
Comparison with Δλmax (wild type higher)
Mouse p53 protein
Wild type
9.6
–
Breast cancer Protein
Figure 4. LSPR peak shifts according to the binding of the hot-spot site of each mp53 protein variant for specific GADD45 DNA. Control is SP6 RNA polymerase, a unspecific protein.
SP6 RNA polymerase showed the negligible LSPR spectra shift, suggesting that spectral change is caused by the specific binding of p53 proteins to GADD45 promoter. The findings of this study suggest the potential future use of individual AuNPs as label-free biosensors to identify the presence of various cancer-associated proteins over wide concentration ranges, and to detect different chemical or biological interactions. The specific interactions between target proteins and a promoter can be measured precisely and speedily by LSPR, and their importance for specific protein recognition can also be inferred.
3. Conclusion
Lung cancer Protein
(I252F)
4.2
2.3
(R270H)
1.7
5.6
(Wild type MCF-7)
9.3
–
(Mutant type T47D)
1.2
7.8
(Mutant type BT474)
1.0
9.3
(Mutant type SKBR3)
0.64
14.5
(Mutant type MDA-MB-231)
2.66
3.5
(Mutant type MDA-MB-435s)
2.1
4.4
(Wild type H460)
15
–
(Mutant type H522)
2.05
7.3
(Mutant type H441)
3.9
3.8
(Mutant type Calu-3)
2.9
5.2
(Mutant type H1703)
2.4
6.3
small molecules that can fold the mutant p53 to wild-type conformation in cancer therapy.
4. Experimental Section Materials: Sodium citrate, absolute ethanol, ethanolamine, N-hydroxysuccinimide (NHS), N-ethyl-N-(3-diethylaminopropyl) carbodiimide (EDC), and phosphate-buffered saline in foil pouches (PBS buffer, pH 7.4) were purchased from Sigma Aldrich (Korea), and nuclease-free water were purchased from Progmega Corporation. HS(CH2)11(OCH2CH2)3OH(OEG3-OH) was purchased
In summary, we demonstrated that the resonance Rayleigh scattering from hot-spot mutant p53 protein on AuNP surface was successfully used for distinguishing the mutant p53 proteins from the wild-type proteins by monitoring their binding affinity to DNA using the LSPR Δλmax shift. By applying this method, we were able to detect the purified mutant p53 proteins and mutant p53 proteins from the human breast and lung cancer cell lines. LSPR spectra reported the binding strength of p53 proteins to the GADD45 promoter by producing redder Δλmax values corresponding to wild and mutant types. The LSPR Δλmax shift for mutant p53 proteins was significantly lower than that for wild types. Using this method, we can detect known p53 protein mutants and their specific promoters in all bio-sample sources, and thus it is possible to distinguish between tumor and normal specimens at early stages of cancer development.[33] Moreover, Figure 5. Electrophoretic mobility shift assays (EMSA) used to qualitatively determine the Rayleigh scattering from hot-spot mutant difference in binding affinity between the GADD45 promoter labeled with “promoter” and the p53 protein is very essential method sug- DNA binding domain of mouse p53 labeled with “mP53”. The gel mobility of wild-type (WT), gests that it is possible platform to screen Arg270 mutant to His (R270H) and Ile252 mutant to Phe (I252F) was compared.
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Table 2. Information for the mouse p53 protein DNA binding domain involved in this study. 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
from Cos Biotech (Korea). A coverslip slide (22 × 40 × 0.1 mm) was purchased from Deckglaser (Germany). Ultra-pure water (18.2 mΩ·cm−1) was used to prepare all of the chemical solutions. A p53 human ELISA kit was obtained from abcam (England). Synthesis of Gold Nanoparticles: Spherical Au nanoparticles (AuNPs) were synthesized through a sodium citrate reduction of an aqueous HAuCl4 solution, as described in a previous study.[16] The glassware was thoroughly cleaned with aqua regia (3:1 HNO3 / HCl) and rinsed with ultrapure water (18.2 mΩ·cm−1). Next, 20 mL of 1.0 mM HAuCl4 was brought to the boil, and 2.0 mL of 1% sodium citrate was added, stirring vigorously. The solution was further boiled for 5 min to complete the citrate reduction of the gold ions. The solution was then stirred for the next 30 min, after which it was cooled to room temperature. The suspension was subsequently filtered with a 0.22-µm filter to remove any aggregated particles. The UV-vis absorption spectra of the gold solution were recorded using a life science UV-Vis spectrophotometer (DUR 730, Beckman). The size and morphology of the synthesized AuNPs were estimated via higher solution transmission electron microscopy (HRTEM, JEOL JEM-3011 operated at 300 kV) (Figure 1). Dark-Field Microspectroscopy: The Rayleigh light scattering spectra of individual gold nanoparticles were measured by darkfield microscopy and a spectrograph (Scheme 1A). To observe the scattered light of the single metal NPs, a transmission configuration for the resonant Rayleigh light scattering microspectroscopy was established using a dark-field microscope (Eclipse TE2000-U, Nikon, Japan). To measure the Rayleigh light scattering spectrum, the dark-field microscope was attached with a spectrograph (Microspec 2300i, Roper Scientifics) and a highly sensitive CCD camera (PIXIS: 400B, Princeton Instruments). A color camera (D50, Nikon) was mounted onto the front port of the microscope. A 100 W tungTable 3. Information for the human p53 protein DNA binding domain involved in this study. 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
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
a)Breast
b)Lung
cancer samples;
cancer samples.
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Figure 6. (A) Relative concentration of various breast cancer p53 proteins from ELISA experimental results. (B) Relative concentration of various lung cancer p53 proteins from ELISA experimental results.
sten lamp and a dark-field condenser (dry condenser, NA = 0.80– 0.95, Nikon) were used to illuminate the nanoparticles. The scattered light was collected with a 100×objective (CFI Plan Fluor ELWD, NA = 0.6) and directed to the spectrograph to be dispersed into monochromatic light. A CCD camera was used to measure the intensity of the monochromatic light, which was recorded as a function of light scattering intensity versus wavelength. Further details of this system have been previously reported.[16] Sample Preparation: To investigate the optical response of the individual AuNPs, every AuNP with a diameter of ∼30 nm was deposited onto an APTES-treated glass microscope slide (22 × 40 × 0.1 mm), with AuNP concentration and incubation time used to control interparticle spacing. In this case, the AuNPs were first immobilized by drop-coating 1µl of the diluted AuNP solution onto the center of a freshly coated APTES slide for 10 min at room temperature. This resulted in an interparticle distance large enough that the AuNPs were individually visible in the dark-field microscope. Subsequently, the coverslip slide glass was mounted onto an RC-30 closed-bath imaging chamber (Warner Instruments, USA) to view the AuNPs and to inject the adsorbates and reactant. Afterwards, the imaging chamber was inserted into the sample holder of the dark-field microscope and a fluidic flow was estab-
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Figure 7. (A) LSPR peak shifts according to the binding of the GADD45 promoter to each breast cancer p53 protein variant. (B) LSPR peak shifts according to the binding of the GADD45 promoter to each lung cancer p53 protein variant. Control is SP6 RNA polymerase, a unspecific protein.
lished by connecting the holder to a syringe pump (Harvard Apparatus) at 200 µL/min. Hybridization of Oligonucleotides: A 30-base-pair p53-binding DNA element found in the GADD45 promoter was prepare by annealing the forward and reverse oligonucleotides. The 5′-thiolmodified GADD45 promoter F (5′-GTACAGAACATGTCTAAGCATGCTGGGGAC-3′) and the 3′-thiol-modified GADD45 promoter R (5′-GTCCCCAGCATGCTTAGACATGTTCTGTAC-3′) were obtained from Bioneer Corporation (Daejeon, Korea). For the purpose of immobilization, the oligonucleotide was thiol-modified. To form a double-stranded DNA, the 5′-thiolated single stranded DNA with the forward sequence was mixed with the complementary DNA with the reverse sequence at a 1:1 molar ratio, in a final volume of 60 uL. The mixture was denatured by heating at 95 °C for 5 min, and the gradually cooled down to 4 °C. The quality of the doublestranded DNA was checked by gel electrophoresis. Preparation of Cancer Cells: Cancer cells were harvested and washed with 1X PBS and lysed with RIPA Lysis buffer (10 mM Tris-HCl pH8.0, 1mM EDTA, 150mM NaCl, 0.2% Triton X-100) containing Xpert protease inhibitor cocktail and Xpert phosphatase
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inhibitor cocktail (GenDEPOT, Seoul, Korea), and incubated on ice for 30 min. The cell lysate was then placed in a micro centrifuge at 12,000 rpm for 30 min at 4 °C. The total protein concentration was determined using the Bradford method with Sigma bovine serum albumin (BSA) as a standard. The exact p53 protein concentration was measured using ELISA experiments. Immobilization of Gold Nanoparticles (AuNPs) and Chamber Setting: Prior to immobilization, the glass slide (22 × 40 × 0.1 mm) was cleaned in a piranha solution (3:1 H2SO4/H2O2) by sonication for 30 min and rinsed thoroughly with distilled water. AuNPs diluted in distilled water were then immobilized electrostatically on the slide by drop-coating. The concentration of the AuNPs and incubation time play a significant role in reducing interparticle coupling effects by controlling interparticle spacing. The prepared slide glass was mounted on a closed confocal imaging chamber (RC-30, Warner Instrument Inc.), and inserted into the sample holder of a dark-field microscope. The absorbents and reactants were injected into the imaging chamber using syringe pump to functionalize AuNP surface. Fluidic flow inside the chamber was fixed at 100 µL min−1 by a syringe pump. Ethanol was injected in order to clean the surface of the AuNPs, and then rinsed with deionized water to remove all impurities. The flow of deionized water lasted 30 minutes to entirely remove non-immobilized AuNPs in the imaging chamber. LSPR Response of Individual AuNPs Sensors in the Presence of Adsorbates (dsDNA) and Target Analytes (p53 protein): In order to detect biomolecular interactions by monitoring the optical response of individual AuNPs, the target molecules, should be immobilized on the AuNP surface (Scheme 1B) The functionalization method used in this study to compare the binding between the GADD45 promoter and p53 proteins is as follows. First, to functionalize AuNP surface, a mixture of 200 µL of HS(CH2)11(OCH2CH2)3OH in ethanol was injected over the AuNPimmobilized glass slide and incubated for 10 h. Nuclease-free water containing the double stranded oligonucleotide (GADD45 promoter) was subsequently injected 200µl (2.5 µg/µl) in the imaging chamber and incubated for 12h. The surface of the AuNPs was then rinsed with deionized water to remove all unbounded molecules. Subsequently, a total volume of 200 µL of p53 protein (0.6 µg/µl) was injected into the chamber and incubated for 2 hours to allow binding between the GADD45 promoter and protein. After this reaction, the chamber was rinsed with deionized water to remove unreacted p53 proteins. The overall experimental process using the individual AuNPs sensors is described below (Scheme 1B). Electrophoretic Mobility Shift Assay (EMSA): The binding reactions of purified recombinant mouse p53 and its mutants carried out in a buffer containing 10 mM Tris-HCl pH 7.5, 50 mM NaCl, 1 mM MgCl2, 1 mM EDTA, and 4% glycerol. The 1 ng of labeled GADD45 promoter was used for each EMSA samples. To confirm the specific binding of mouse p53 and its mutants to DNA, we added excess amount of unlabeled GADD45 promoter (2500 ng). The reaction mixtures were incubated at 4? for 1 hour and loaded into 6% non-denature gel. The GADD45 promoter was labeled with T4 polynucleotide kinase (New England Biolabs) and [γ-P32] ATP. The labeled probes were further purified on Nick column (GE healthcare). Data Analysis: In dark-field image, we selected 5 regions each containing 4 AuNPs to characterize their spectra. The spectral
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Scheme 1. (A) Schematic process describing the system setup using a dark-field microscopy. (B) Overall procedure describing the experimental processes for binding affinity difference.
changes of the AuNPs in response to the binding of GADD45 promoter and p53 proteins were then recorded. These measurements were repeatedly applied for each protein mutant. To improve the precision of the measurement, the spectral noises were excluded by subtracting the background signal surrounding AuNPs. Finally, in order to determine λmax, the Lorentzian algorithm was applied to the experimental spectrum to the LSPR scattering wavelength shift (Δλmax) with a following formula: Δλmax = λmax (after chemical binding) – λmax (before chemical binding).
Acknowledgements This work was supported by a National Research Foundation of Korea (NRF) grant (no. NRF-2013R1A2A1A01015644/2010-0027955), University-Institute coopera-tion program (2012) of the National
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Research, and a Korea CCS R&D Center grant (grant no. 20110031997), both funded by the Korean government through the Ministry of Science, ICT & Future Planning. This work has been also supported by the Korea Institute of Energy Technology Evaluation and Planning and Ministry of Trade, Industry and Energy of Korea as a parts of the Project of “Process demonstration for bioconversion of CO2 to high-valued biomaterials using microalgae” (2012T100201516) in “Energy Efficiency & Resources Technology R&D” project.
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Received: January 1, 2014 Revised: February 24, 2014 Published online: April 3, 2014
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Distinct Rayleigh Scattering from Hot Spot Mutant p53 Proteins Reveals Cancer Cells Ho Joon Jun, Anh H. Nguyen, Yeul Hong Kim, Kyong Hwa Park, Doyoun Kim, Kyeong Kyu Kim,* and Sang Jun Sim*
The scattering of light redirects and resonances when an electromagnetic wave interacts with electrons orbits in the hot spot core protein and oscillated electron of the gold nanoparticles (AuNP). This report demonstrates convincingly that resonant Rayleigh scattering generated from hot spot mutant p53 proteins is correspondence to cancer cells. Hot spot mutants have unique local electron density changes that affect specificity of DNA binding affinity compared with wild types. Rayleigh scattering changes introduced by hot-spot mutations were monitored by localized surface plasmon resonance (LSPR) shift changes. The LSPR λmax shift for hot-spot mutants ranged from 1.7 to 4.2 nm for mouse samples and from 0.64 nm to 2.66 nm for human samples, compared to 9.6 nm and 15 nm for wild type and mouse and human proteins, respectively with a detection sensitivity of p53 concentration at 17.9 nM. It is interesting that hot-spot mutants, which affect only interaction with DNA, launches affinitive changes as considerable as wild types. These changes propose that hot-spot mutants p53 proteins can be easily detected by local electron density alterations that disturbs the specificity of DNA binding of p53 core domain on the surface of the DNA probed-nanoplasmonic sensor.
1. Introduction Induced dipole moment of periodic separation of charge is generated from oscillation or perturbation of the electron
H. J. Jun, A. H. Nguyen, Prof. S. J. Sim Department of Chemical and Biological Engineering Korea University Seoul 136-701, Korea E-mail: [email protected] Prof. Y. H. Kim, Prof. K. H. Park Medical Oncology Department of Internal Medicine Korea University College of Medicine Seoul 136-705, Korea Dr. D. Kim, Prof. K. K. Kim Department of Molecular Cell Biology Sungkyunkwan University School of Medicine Samsung Biomedical Research Institute Suwon 440-746, Korea E-mail: [email protected] DOI: 10.1002/smll.201400004
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cloud. This event transfers free electrons at the nanoparticle surface via a supporting insluting substrate for electrochemistry catalysis.[1a] Previous reports theorectically and experimentally proved electron transfer reactions at AuNP,[1b] quantitated electron transferate of electron transfer rate on AuNP during catalytic reactions,[1a,b] and interestingly desmonstrated a fact AuNPs can effectively interact with proteins and even interchange the electrons with some redox-proteins.[1c] Since electron movement surrounding AuNPs is directly associated to the plasma frequency of AuNP and in situ Rayleigh scattering profile permitted one to count a number of electrons during the catalytic reaction.[1a] Moreover, a correlative equation between experimental band shift in the Rayleigh wavelength profile with the electron quantity presents (electron idensity changes) in the biochemical reaction, and gives dormant tool for the precise distinction of electron transfer rates in redox heterogenous proteins on the AuNP surface.[1a-c] Using an electromagnetic wave, a particular light-matter interaction, is to resonance Rayleigh scattering of light at local dielectric environment of AuNP surface called localized surface Plasmon resonance (LSPR), and has been applied for
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biological and chemical sensing.[1d,e] LSPR is sensitive to altera- nesses such as having low sensitivity, low specificity, and high tions in the local dielectric environment generating wavelength cost, as well as being time-consuming.[11,12] Previously, we have developed a new nanoplasmonic shift responding to electromagnetic-field enhancement. Local dielectric environment is dependent on local electric field of sensor to detect the protein-DNA interaction.[13] In this binding hot spot core protein with specific DNA.[1f] The dielec- study, we further developed this sensor to identify cancer tric property inside a hot spot domain is a key chemo-physical cells though detecting resonance of local dielectric environfactor of the extent of electrostatic interactions of hot spot ment of hot-spot mutant p53 promoter on AuNP surface.[14] cores.[1f] The dielectric effect inside a hot spot domain in binding To monitor distinct Rayleigh scattering from hot-spot core p53 structure of protein/DNA determines electrostatic energies of proteins, we fabricated the LSPR-based platform from single hot spot mutants or wild type of a specific proteins.[1h] Elec- GADD45-conjugated AuNP. We found that the LSPR spectra trostatic interaction between protein and DNA are ubiqui- shift of resonance local dielectric of hot-spot mutant p53 protous, affected by protein structure, ligand binding, and electron tein changes upon amino acid substitution, deletion within transfer in hot-spot binding domain.[1f,h] In this work, we focus the hot-spot, which determine binding affinity of the p53 to on the resonance of local dielectric of hot-spot mutants p53 GADD45. These results demonstrated that the nanoplasmonic proteins on AuNP surface where the resonance occurs. sensor can detect mutant p53 proteins from wild types of real As proof-of-concept, the p53 is well-known a tumor sup- samples simply and directly, facilitating the efficient screening pressor protein that plays as an important role in preventing of many mutant proteins such as HER2 and BRCA1 and thus the development of cancer.[1g,2] It regulates the cell cycle by suggesting itself as a promising tool for use in cancer diagnosis. controlling the expression of various genes in response to DNA damage and cellular apoptosis.[3] With the loss of p53 function, usually the result of mutations within the DNA 2. Results and Discussion binding domain, unregulated cell growth can lead to tumors. Mutations in p53 protein are found in over half of all human AuNPs are ideal platforms for the quantification of biochemcancers,[4] in particular breast[5] and lung cancer,[4] where the ical species or the monitoring of dynamic processes inside mutation rate can reach 85%.[6] Almost all p53 mutant forms biological cells.[15] Colloidal AuNPs were synthesized by the have mutations at the hot-spot DNA binding domain, located method described in the experimental section,[16] with an at the residues 102-292,[7,8] which causes either hot-spot local average diameter of 30.7 ± 1.8 nm, as estimated by HR-TEM dielectric changes or reduction in its sequence-specific DNA images (Figure 1A). To reduce inter-particle coupling effects, binding affinity.[5] These hot-spot mutant variants reduce tumor suppressor function because p53 is less able to recognize the specific nucleotide sequence on promoters because a reduction occurs of chargecharge and charge-polar interaction of local dielectric environment of hot-spot mutant p53 protein, which leads to uncontrolled regulation during cellular processes.[5] Thus the binding affinity between mutant p53 variants with specific DNA sequence is characterized by a lower binding constant compared with the wild type.[9] In this context of local dielectric environment of hot-spot mutant domain, the evaluation of the binding affinity of mutant p53 proteins to the p53-binding element of GADD45 promoter can be a useful approach for early cancer detection. GADD45, which causes growth arrest and DNA damage, is one of the p53 regulatory proteins and contains a consensus p53-binding sequence in its promoter region.[10] The accurate detection of proteins related to disease is an important area of biomedical and biochemical research, offering great potential for highly reliable diagnosis and prognosis.[4] However, since most mutations occur at Figure 1. (A) HR-TEM images of spherical Au nanoparticles (∼30 nm) chemically synthesized a specific site on the DNA binding central through citrate mediated reduction of HAuCl4 solution. (B) Dark-field image of bare Au domain, current methods exhibit weak- nanoparticles (∼30 nm) at a magnification of 1000X. small 2014, 10, No. 14, 2954–2962
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Figure 2. UV-Vis absorption spectra of gold nanoparticles (AuNPs, 528 nm), OEGs-AuNPs, (533 nm) and DNA-conjugated AuNPs (538 nm).
the AuNP solution was diluted with ultrapure water to an optical density of 0.04 OD at 528 nm and immobilized on a glass slide, this created a particle to particle spacing of greater than 2.5 times the particle diameter, a spacing above which there are no coupling effects.[17,18] The AuNPs were functionalized with HS-C11-EG3-OH to prevent the non-specific adsorption of proteins.[19] AuNP surface was further conjugated with the GADD45 promoter, a 30-base pair element containing the consensus p53-binding sequence, for the detection of p53 protein. The functionalized products were confirmed by UV-VIS at 528 nm (Figure 2). Due to the plasmon resonance characteristics of AuNPs,[20] a damping of the surface plasmon band compared with bare AuNPs occurred as a red shift of 4.5 nm and 10.5 nm corresponding to capping by OEG3 and the GADD45 promoter, respectively. This was used as evidence that the AuNPs had been conjugated with OEG3 and the GADD45 promoter, indicating the successful attachment of the DNA onto the surface of the AuNPs. Initially, we used the purified DNA binding domain of mouse p53 (mp53 DBD) to test the correlation between DNA binding affinity of proteins and the Rayleigh light scattering. For this purpose, the wild-type and two mutant p53 proteins, I252F and R270H, were prepared and bound to GADD45 promoter conjugated to AuNPs. The particles were monitored by the Rayleigh light scattering. Ile252 and Arg270 mutants corresponding to Ile255 and Arg273 mutants found in human cancers are known to have weak and no binding affinity to the consensus p53-binding site, respectively.[21] The binding of the wild-type and the mutant mp53 DBDs on the promoter was monitored by the Rayleigh light scattering spectra generated by LSPR using the following steps. Under a 100W tungsten white light lamp, a dark-field microscope was used to view a single illuminated AuNP visible as a distinctly bright spot with extremely low background light from the glass substrate (Figure 1B). The scattered light of the individual AuNP was collected by a 100x objective lens. The scattered light spectra was then resolved in a grating spectrograph, and synchronized by a highly sensitive CCD camera.[19,22] Each AuNP was situated easily by adjusting the microslit and sample stage. At each measurement step, to eliminate noise, the background spectrum was normalized to zero at an area adjacent to the individual AuNPs. Finally, the
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spectral data were processed using the Lorentzian algorithm in Origin Pro 8.6. According to the Mie theory, the LSPR of noble metal nanoparticles is not only strongly dependent on their shape, particle type and size, but also local dielectric environment.[14,23] The local dielectric environment of AuNPs is extremely sensitive to perturbations caused by binding events on the AuNP surface. The refractive index of an AuNP depends on the number of p53 proteins binding to the GADD45-conjugated AuNP surface, and thus strongly depends on the binding affinity of p53 proteins with the GADD45 promoter. Figure 3 shows the typical LSPR light scattering spectra for an AuNP after mp53-DBD proteins bind to the GADD45 promoter. The experimental spectra were fitted to a Lorentzian calculation with a shift to the redder end of the spectrum. Compared with a bare AuNP, the maximum LSPR shift (LSPR Δλmax) from a single AuNP after coupling with the GADD45 promoter for the wild type (Figure 3A) and the two mutants (Figure 3B), I252F and R270H, was 9.6nm, 4.2 nm and 1.7 nm, respectively (Figure 4). Wild-type mp53-DBD yielded produced a higher Δλmax than the mutant proteins (Table 1), indicating that the mutant proteins have a low affinity for the GADD45 promoter.[5] This is consistent with the previous report[21] in which it has been proven that the R270H mutant has a weaker binding affinity with GADD45 than the I252F mutant (Table 1). In order to confirm LSPR Δλmax, we compared the LSPR data with electrophoretic mobility shift assays (EMSA), a common method of measuring DNA-protein binding.[24] Figure 5 presents binding between GADD45 promoter with three mp53-DBDs (WT, R270H and I252F). When a specific competitor was added, the binding affinity of mp53-DBD to GADD45 remained to be 100% in the wild type, ∼50% in I252F mutants and 0% in R270H mutant. The nanoplasmonic shift for each protein variant was fairly consistent with this EMSA measurement. These results reflected the reduction of binding affinity caused by mutation in DNA binding domain. Thus, LSPR was able to quickly and accurately discriminate mp53 forms present in real samples by their affinity for a specific promoter. Since it has been validated that the DNA binding affinity of p53 protein to GADD45 is proportional to Δλmax using the purified p53 proteins, we further apply our system to examine breast and lung cancer cell lines (Table 2 and 3) which have the mutant p53 proteins. p53 mutant (mutation at core binding domain) and p53 wild type (no mutation at core binding domain) cell lines are well known in the literature[25–27] and presented in Table 3. p53 concentrations have been quantified by ELISA (Figure 6) because the concentration of human p53 protein varies greatly between samples and p53 is present in low concentrations in breast and lung cancer compared with wild types.[24,28] LSPR spectra generated from the nanoplasmonic biosensor determined the binding strength of the p53 proteins in the cancer cells lines. Figure 7 shows the LSPR light scattering spectra Δλmax for single AuNPs after the binding of breast and lung cancer p53 proteins onto the GADD45 promoter. The experimental spectra were fitted to the Lorentzian calculation with a shift to the redder end of the spectrum. Individual mutant forms were measured with LSPR and the Δλmax for the samples
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MB-435s; lung cancer H522, H441, Calu-3, and H1703). Among the mutant types, the SKBR3 sample showed the lowest Δλmax (Figure 7A), which is possibly caused by the unique properties of the p53 protein of SKBR3. In these cancer cells, Arg175 of p53 protein is known to be a hot spot; it is the most essential residue for the function of p53 protein.[11,30] Thus, if a mutation occurs at a hot spot, the binding affinity is much lower than for another mutations.[31] One of advantage of LSPR sensor is direct measurement of a specific DNA/protein interaction without a need of purification. Therefore, p53 variants in crude protein extracts were directly measured and results of Δλmax values were shifted corresponding to the mutations at core domains for instance, the Δλmax values of BT474 (I282K) and MDA-MB-231 (R280K) were 1.0 nm and 2.66 nm, respectively. In fact, protein-specific DNA interaction is dependent on physical forces including electrostatic interaction, hydrogen bonding, hydrophobic interaction, and dispersion forces.[32] Therefore, chemo-physical properties and the composition of amino acids in the DNA binding domain have a role in determining binding affinity. For example, in BT474 cell lines, E282K mutation in p53 proteins abolishes the electrostatic interaction between protein and DNA. However, in the case of the MDA-MB-231 cells which have R280K mutation in p53 protein, the reduced binding affinity might be achieved by the loss of hydrogen bonding because of the substitution mutation of Arg to Lys. Similar to human breast cell samples, the Figure 3. (A) Representative Rayleigh light scattering spectra of single AuNP according to the crude extract from the lung cancer cell binding of the GADD45 promoter (SH-DNA) and the DNA binding domain of mouse p53 wild line (H460) containing the wild type p53 type. LSPR λmax shifted to the longer wavelengths after every step of the chemical binding of protein produced a higher Δλmax than adsorbates and target analytes. (B) Representative Rayleigh light scattering spectra of single those of other lung cancer cells that conAuNPs according to the binding of the GADD45 promoter (SH-DNA) and the DNA binding tain the mutant p53 proteins (Table 1). domain of mouse p53 (R270H). H522 sample, which has p53 proteins with a deletion mutation in the DNA binding are shown in Table 1. The specific Δλmax for the LSPR was domain, resulted in the smallest shift due to the minimal determined by the strength of p53-GADD45 interaction. DNA binding activity. Binding affinity of mutants is highly We used five humans breast cancer cell lines (T47D, BT474, reduced compared with wild type. For example, BT474 SKBR3, MDA-MB-231, and MDA-MB-435s) and four lung (E282K) and Calu-3(M237I) mutants presented low LSPR cancer cell lines (H522, H441, Calu-3, and H1703) (Table 3) Δλmax compared with wild type although we injected the to study binding affinity of mutant p53 proteins present in same p53 concentration (Figure 6). This is demonstrated a the applicable cell lines to the GADD45 promoter using the low LSPR Δλmax, the result of the reduction in binding affinity Δλmax detected by the plasmonic biosensor.[29] For compar- with the GADD45 promoter due to the mutations in DNA ison, ZR-75-1, MCF-7 and H460, which are known to have binding domain. To further validate our methods, it is essenthe wild-type p53 proteins, were introduced. As expected, tial to examine the nonspecific protein binding to GADD45 the cell extract from these three cell lines produced a higher promoter using the negative control. In Figure 7, SP6 RNA Δλmax than the samples from other cancer cell lines (breast polymerase was introduced as a negative control. Under the cancer T47D, BT474, SKBR3, MDA-MB-231, and MDA- same experimental conditions as the trials using p53 protein, small 2014, 10, No. 14, 2954–2962
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Table 1. Comparison of binding affinity between wild and mutant type p53 proteins through the ratio of Δλmax (wild /mutant type). Type of p53 protein
p53 mutation (cell line)
Δλmax (nm)
Comparison with Δλmax (wild type higher)
Mouse p53 protein
Wild type
9.6
–
Breast cancer Protein
Figure 4. LSPR peak shifts according to the binding of the hot-spot site of each mp53 protein variant for specific GADD45 DNA. Control is SP6 RNA polymerase, a unspecific protein.
SP6 RNA polymerase showed the negligible LSPR spectra shift, suggesting that spectral change is caused by the specific binding of p53 proteins to GADD45 promoter. The findings of this study suggest the potential future use of individual AuNPs as label-free biosensors to identify the presence of various cancer-associated proteins over wide concentration ranges, and to detect different chemical or biological interactions. The specific interactions between target proteins and a promoter can be measured precisely and speedily by LSPR, and their importance for specific protein recognition can also be inferred.
3. Conclusion
Lung cancer Protein
(I252F)
4.2
2.3
(R270H)
1.7
5.6
(Wild type MCF-7)
9.3
–
(Mutant type T47D)
1.2
7.8
(Mutant type BT474)
1.0
9.3
(Mutant type SKBR3)
0.64
14.5
(Mutant type MDA-MB-231)
2.66
3.5
(Mutant type MDA-MB-435s)
2.1
4.4
(Wild type H460)
15
–
(Mutant type H522)
2.05
7.3
(Mutant type H441)
3.9
3.8
(Mutant type Calu-3)
2.9
5.2
(Mutant type H1703)
2.4
6.3
small molecules that can fold the mutant p53 to wild-type conformation in cancer therapy.
4. Experimental Section Materials: Sodium citrate, absolute ethanol, ethanolamine, N-hydroxysuccinimide (NHS), N-ethyl-N-(3-diethylaminopropyl) carbodiimide (EDC), and phosphate-buffered saline in foil pouches (PBS buffer, pH 7.4) were purchased from Sigma Aldrich (Korea), and nuclease-free water were purchased from Progmega Corporation. HS(CH2)11(OCH2CH2)3OH(OEG3-OH) was purchased
In summary, we demonstrated that the resonance Rayleigh scattering from hot-spot mutant p53 protein on AuNP surface was successfully used for distinguishing the mutant p53 proteins from the wild-type proteins by monitoring their binding affinity to DNA using the LSPR Δλmax shift. By applying this method, we were able to detect the purified mutant p53 proteins and mutant p53 proteins from the human breast and lung cancer cell lines. LSPR spectra reported the binding strength of p53 proteins to the GADD45 promoter by producing redder Δλmax values corresponding to wild and mutant types. The LSPR Δλmax shift for mutant p53 proteins was significantly lower than that for wild types. Using this method, we can detect known p53 protein mutants and their specific promoters in all bio-sample sources, and thus it is possible to distinguish between tumor and normal specimens at early stages of cancer development.[33] Moreover, Figure 5. Electrophoretic mobility shift assays (EMSA) used to qualitatively determine the Rayleigh scattering from hot-spot mutant difference in binding affinity between the GADD45 promoter labeled with “promoter” and the p53 protein is very essential method sug- DNA binding domain of mouse p53 labeled with “mP53”. The gel mobility of wild-type (WT), gests that it is possible platform to screen Arg270 mutant to His (R270H) and Ile252 mutant to Phe (I252F) was compared.
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Table 2. Information for the mouse p53 protein DNA binding domain involved in this study. 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
from Cos Biotech (Korea). A coverslip slide (22 × 40 × 0.1 mm) was purchased from Deckglaser (Germany). Ultra-pure water (18.2 mΩ·cm−1) was used to prepare all of the chemical solutions. A p53 human ELISA kit was obtained from abcam (England). Synthesis of Gold Nanoparticles: Spherical Au nanoparticles (AuNPs) were synthesized through a sodium citrate reduction of an aqueous HAuCl4 solution, as described in a previous study.[16] The glassware was thoroughly cleaned with aqua regia (3:1 HNO3 / HCl) and rinsed with ultrapure water (18.2 mΩ·cm−1). Next, 20 mL of 1.0 mM HAuCl4 was brought to the boil, and 2.0 mL of 1% sodium citrate was added, stirring vigorously. The solution was further boiled for 5 min to complete the citrate reduction of the gold ions. The solution was then stirred for the next 30 min, after which it was cooled to room temperature. The suspension was subsequently filtered with a 0.22-µm filter to remove any aggregated particles. The UV-vis absorption spectra of the gold solution were recorded using a life science UV-Vis spectrophotometer (DUR 730, Beckman). The size and morphology of the synthesized AuNPs were estimated via higher solution transmission electron microscopy (HRTEM, JEOL JEM-3011 operated at 300 kV) (Figure 1). Dark-Field Microspectroscopy: The Rayleigh light scattering spectra of individual gold nanoparticles were measured by darkfield microscopy and a spectrograph (Scheme 1A). To observe the scattered light of the single metal NPs, a transmission configuration for the resonant Rayleigh light scattering microspectroscopy was established using a dark-field microscope (Eclipse TE2000-U, Nikon, Japan). To measure the Rayleigh light scattering spectrum, the dark-field microscope was attached with a spectrograph (Microspec 2300i, Roper Scientifics) and a highly sensitive CCD camera (PIXIS: 400B, Princeton Instruments). A color camera (D50, Nikon) was mounted onto the front port of the microscope. A 100 W tungTable 3. Information for the human p53 protein DNA binding domain involved in this study. 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
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
a)Breast
b)Lung
cancer samples;
cancer samples.
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Figure 6. (A) Relative concentration of various breast cancer p53 proteins from ELISA experimental results. (B) Relative concentration of various lung cancer p53 proteins from ELISA experimental results.
sten lamp and a dark-field condenser (dry condenser, NA = 0.80– 0.95, Nikon) were used to illuminate the nanoparticles. The scattered light was collected with a 100×objective (CFI Plan Fluor ELWD, NA = 0.6) and directed to the spectrograph to be dispersed into monochromatic light. A CCD camera was used to measure the intensity of the monochromatic light, which was recorded as a function of light scattering intensity versus wavelength. Further details of this system have been previously reported.[16] Sample Preparation: To investigate the optical response of the individual AuNPs, every AuNP with a diameter of ∼30 nm was deposited onto an APTES-treated glass microscope slide (22 × 40 × 0.1 mm), with AuNP concentration and incubation time used to control interparticle spacing. In this case, the AuNPs were first immobilized by drop-coating 1µl of the diluted AuNP solution onto the center of a freshly coated APTES slide for 10 min at room temperature. This resulted in an interparticle distance large enough that the AuNPs were individually visible in the dark-field microscope. Subsequently, the coverslip slide glass was mounted onto an RC-30 closed-bath imaging chamber (Warner Instruments, USA) to view the AuNPs and to inject the adsorbates and reactant. Afterwards, the imaging chamber was inserted into the sample holder of the dark-field microscope and a fluidic flow was estab-
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Figure 7. (A) LSPR peak shifts according to the binding of the GADD45 promoter to each breast cancer p53 protein variant. (B) LSPR peak shifts according to the binding of the GADD45 promoter to each lung cancer p53 protein variant. Control is SP6 RNA polymerase, a unspecific protein.
lished by connecting the holder to a syringe pump (Harvard Apparatus) at 200 µL/min. Hybridization of Oligonucleotides: A 30-base-pair p53-binding DNA element found in the GADD45 promoter was prepare by annealing the forward and reverse oligonucleotides. The 5′-thiolmodified GADD45 promoter F (5′-GTACAGAACATGTCTAAGCATGCTGGGGAC-3′) and the 3′-thiol-modified GADD45 promoter R (5′-GTCCCCAGCATGCTTAGACATGTTCTGTAC-3′) were obtained from Bioneer Corporation (Daejeon, Korea). For the purpose of immobilization, the oligonucleotide was thiol-modified. To form a double-stranded DNA, the 5′-thiolated single stranded DNA with the forward sequence was mixed with the complementary DNA with the reverse sequence at a 1:1 molar ratio, in a final volume of 60 uL. The mixture was denatured by heating at 95 °C for 5 min, and the gradually cooled down to 4 °C. The quality of the doublestranded DNA was checked by gel electrophoresis. Preparation of Cancer Cells: Cancer cells were harvested and washed with 1X PBS and lysed with RIPA Lysis buffer (10 mM Tris-HCl pH8.0, 1mM EDTA, 150mM NaCl, 0.2% Triton X-100) containing Xpert protease inhibitor cocktail and Xpert phosphatase
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inhibitor cocktail (GenDEPOT, Seoul, Korea), and incubated on ice for 30 min. The cell lysate was then placed in a micro centrifuge at 12,000 rpm for 30 min at 4 °C. The total protein concentration was determined using the Bradford method with Sigma bovine serum albumin (BSA) as a standard. The exact p53 protein concentration was measured using ELISA experiments. Immobilization of Gold Nanoparticles (AuNPs) and Chamber Setting: Prior to immobilization, the glass slide (22 × 40 × 0.1 mm) was cleaned in a piranha solution (3:1 H2SO4/H2O2) by sonication for 30 min and rinsed thoroughly with distilled water. AuNPs diluted in distilled water were then immobilized electrostatically on the slide by drop-coating. The concentration of the AuNPs and incubation time play a significant role in reducing interparticle coupling effects by controlling interparticle spacing. The prepared slide glass was mounted on a closed confocal imaging chamber (RC-30, Warner Instrument Inc.), and inserted into the sample holder of a dark-field microscope. The absorbents and reactants were injected into the imaging chamber using syringe pump to functionalize AuNP surface. Fluidic flow inside the chamber was fixed at 100 µL min−1 by a syringe pump. Ethanol was injected in order to clean the surface of the AuNPs, and then rinsed with deionized water to remove all impurities. The flow of deionized water lasted 30 minutes to entirely remove non-immobilized AuNPs in the imaging chamber. LSPR Response of Individual AuNPs Sensors in the Presence of Adsorbates (dsDNA) and Target Analytes (p53 protein): In order to detect biomolecular interactions by monitoring the optical response of individual AuNPs, the target molecules, should be immobilized on the AuNP surface (Scheme 1B) The functionalization method used in this study to compare the binding between the GADD45 promoter and p53 proteins is as follows. First, to functionalize AuNP surface, a mixture of 200 µL of HS(CH2)11(OCH2CH2)3OH in ethanol was injected over the AuNPimmobilized glass slide and incubated for 10 h. Nuclease-free water containing the double stranded oligonucleotide (GADD45 promoter) was subsequently injected 200µl (2.5 µg/µl) in the imaging chamber and incubated for 12h. The surface of the AuNPs was then rinsed with deionized water to remove all unbounded molecules. Subsequently, a total volume of 200 µL of p53 protein (0.6 µg/µl) was injected into the chamber and incubated for 2 hours to allow binding between the GADD45 promoter and protein. After this reaction, the chamber was rinsed with deionized water to remove unreacted p53 proteins. The overall experimental process using the individual AuNPs sensors is described below (Scheme 1B). Electrophoretic Mobility Shift Assay (EMSA): The binding reactions of purified recombinant mouse p53 and its mutants carried out in a buffer containing 10 mM Tris-HCl pH 7.5, 50 mM NaCl, 1 mM MgCl2, 1 mM EDTA, and 4% glycerol. The 1 ng of labeled GADD45 promoter was used for each EMSA samples. To confirm the specific binding of mouse p53 and its mutants to DNA, we added excess amount of unlabeled GADD45 promoter (2500 ng). The reaction mixtures were incubated at 4? for 1 hour and loaded into 6% non-denature gel. The GADD45 promoter was labeled with T4 polynucleotide kinase (New England Biolabs) and [γ-P32] ATP. The labeled probes were further purified on Nick column (GE healthcare). Data Analysis: In dark-field image, we selected 5 regions each containing 4 AuNPs to characterize their spectra. The spectral
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Distinct Rayleigh Scattering from Hot Spot Mutant p53 Proteins Reveals Cancer Cells
Scheme 1. (A) Schematic process describing the system setup using a dark-field microscopy. (B) Overall procedure describing the experimental processes for binding affinity difference.
changes of the AuNPs in response to the binding of GADD45 promoter and p53 proteins were then recorded. These measurements were repeatedly applied for each protein mutant. To improve the precision of the measurement, the spectral noises were excluded by subtracting the background signal surrounding AuNPs. Finally, in order to determine λmax, the Lorentzian algorithm was applied to the experimental spectrum to the LSPR scattering wavelength shift (Δλmax) with a following formula: Δλmax = λmax (after chemical binding) – λmax (before chemical binding).
Acknowledgements This work was supported by a National Research Foundation of Korea (NRF) grant (no. NRF-2013R1A2A1A01015644/2010-0027955), University-Institute coopera-tion program (2012) of the National
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Research, and a Korea CCS R&D Center grant (grant no. 20110031997), both funded by the Korean government through the Ministry of Science, ICT & Future Planning. This work has been also supported by the Korea Institute of Energy Technology Evaluation and Planning and Ministry of Trade, Industry and Energy of Korea as a parts of the Project of “Process demonstration for bioconversion of CO2 to high-valued biomaterials using microalgae” (2012T100201516) in “Energy Efficiency & Resources Technology R&D” project.
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Received: January 1, 2014 Revised: February 24, 2014 Published online: April 3, 2014
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