Ultrasound-triggered release and activation of drugs and biomacromolecules from nucleic acid scaffolds
Zhao, Pengkun DOI:
10.33612/diss.168542653
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Publication date: 2021
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Zhao, P. (2021). Ultrasound-triggered release and activation of drugs and biomacromolecules from nucleic acid scaffolds. University of Groningen. https://doi.org/10.33612/diss.168542653
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5
Colorimetric assay to detect interactions
between proteins and nucleic acids
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Abstract
Protein–nucleic acid interactions play key roles in many biological processes. To develop a simple, sensitive, and specific method for detecting the association and dissociation of these entities is of great significance in medical diagnosis and analysis. Gold nanoparticles (AuNPs) with unique electronic, photonic and catalytic properties have been shown to be excellent scaffolds for the fabrication of novel chemical and biological sensors. In this work, positively charged AuNPs were exploited to electrostatically bind to negatively charged nucleic acid aptamers. As a consequence, the solution displays a purple or even blue color due to the aggregation of AuNPs. The binding of the target protein lysozyme with the aptamers reduces negative charge density of the aptamers, which stabilizes AuNPs thus resulting in remaining a red color. Once the complex is disturbed by ambient environment and disassociated, the color of solution turns back to blue/ purple again. This work provided a simple and visual approach to colorimetric detection of interactions between proteins and nucleic acids.
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5.1 Introduction
Protein–nucleic acid interactions are vital for all living organisms. Many important biological processes such as the transport and translation of RNA, packaging of DNA, genetic recombination, replication, and DNA repair are controlled by interactions of these two kinds of biomacromolecules.[1] Therefore, the study of protein–DNA interactions is important for exploring mechanisms as well as regulating the growth, development, differentiation, and evolution of living beings. As early as the late 19th century, scientists microscopically observed the association of proteins with DNA strands. Since then, a variety of in vitro and in vivo assays have been used by researchers to demonstrate that proteins interact with DNA and RNA influencing the structure and function of the corresponding nucleic acids.[2] These assays are based on molecular simulation,[3] electrochemical technology,[4] nanotechnology,[5] and protein microarray technology.[6]
Currently gold nanoparticles are widely exploited in optical bioassays because of their characteristics, such as good biocompatibility, excellent optical performance, special catalytic activity and convenience of controlled fabrication.[7] Of particular interest is that the assembly or disassembly of AuNPs with appropriate sizes can induce interparticle surface plasmon coupling and result in a visible color change from red to blue (assembly process) or from blue to red (disassembly process).[8] These unique
color changes of AuNPs provide a practical platform for absorption-based colorimetric sensing of many target analytes during the assembly–disassembly process. Since the first demonstration of the DNA sensing protocol based on target-induced color change of AuNPs by Mirkin’s group,[9] this colorimetric sensing platform has been expanded for the detection of small organic molecules,[10] ions,[11] proteins,[12] DNA[13] and even cells.[14] The current colorimetric assays mainly relying on AuNPs can be divided into two categories: cross-linking assembly-disassembly[15] or salt-induced assembly of AuNPs,[16] although the majority of colorimetric systems are belonging to the former class. However, the design of cross-linking induced assembly-disassembly systems is commonly time-consuming and intricate.
Here, we come up with a rapid and simple method to differentiate between association and disassociation of proteins and nucleic acids inspired by work on AuNPs with
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positive charges, which have been successfully tested in detecting antibiotic residues in raw milk by Ramezani et al.[17] The electrostatic interaction between positively charged AuNPs and polyanionic DNA (lysozyme binding aptamer (LBA)) leads to the aggregation of cationic AuNPs accompanied by a rapid red-to-blue color change. Negative charges of LBA would be diluted with the binding of lysozyme (Lys), resulting in the remaining of the red color of individual AuNPs. If the Lys-LBA complex is challenged by employing ultrasound, disassociation takes place and AuNPs again could interact with LBA electrostatically. In this case, the aggregation of AuNPs would take place and the red-to-blue color change would be observed again. A convenient and fast method is established to study the interaction between proteins and nucleic acids, relying on the changes of solution color and the shift of absorption spectra of AuNPs.
Scheme 1. Schematic representation of detection of the association and disassociation between
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5.2 Results and discussion
Figure 1. Colorimetric assay to detect the interaction between mLBA and Lys. (a) UV-vis
adsorption spectra for AuNPs-based detection before and after the addition of mLBA with different concentrations. (b) Plot of mLBA concentrations vs. absorbance ratio (A520/A670) for
the assay. The inset shows the photograph of AuNPs in the presence of different concentrations of mLBA (inset from left to right: from 0 to 150 nM). (c) UV-vis adsorption spectra for AuNPs-based detection before and after the addition of Lys-mLBA complex with different ratios (Lys:mLBA) (concentration of mLBA: 120 nM). (d) Plot of different ratios of Lys-mLBA complex vs. absorbance ratio (A520/A670) for the assay. The inset depicts the photograph of
AuNPs in the presence of different ratios of Lys-mLBA complex (Inset from left to right: from 0:1 to 4:1).
In this study we employed gold nanoparticles with the positive charge. AuNPs (~30 nm) (Figure S1) are red-colored due to their surface plasmon resonance (SPR) absorption located at around 520 nm. Addition of salt screens the electrostatic repulsion between negatively charged AuNPs, resulting in the aggregation of AuNPs that leads to a
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characteristic red-to-blue color change. We first challenged this colorimetric system with a lysozyme binding aptamer,[18] which we named it as mono-LBA (mLBA). As shown in Figure 1a, there was only one peak located at 520 nm for control (only buffer) and only Lys-added system, while the peak shifted to 530 nm and a new, broad absorption (550-750 nm) appeared for the mLBA-added system. Along with the increase of mLBA concentration, the absorbance at 520 nm gradually shifted and decreased, while the broad absorption at 550-750 nm gradually increased. The ratio between A520 and A670 is almost linear with mLBA concentrations within a range from 0 to 150 nM (Figure 1b). Besides, the color of AuNPs solutions changed from red to purple gradually. Having established the colorimetric assay based on mLBA and AuNPs interaction, we tested whether the association of lysozyme with mLBA can affect the electrostatic interactions between cationic AuNPs and mLBA. The UV-vis spectroscopic investigations (Figure 1c) demonstrated that the UV-vis absorption of AuNP solutions at 520 nm increased whereas the absorption at 670 nm decreased gradually with increasing Lys concentration. The evolution of the solution color can be easily detected by the naked eye (Figure 1d). With increasing Lys concentration (from 0 to 480 nM) incubated with mLBA, the color of AuNPs solutions turned from blue to red gradually. Lys-induced charge dilution of the negatively charged mLBA can potentially stabilize AuNPs and inhibit electrostatic assembly of AuNPs on mLBA and the color change of AuNPs.
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Figure 2. UV-vis adsorption spectra for specificity test of mLBA towards Lys. Inset picture
from left to right: mLBA+Lys+Au, mRan+Lys+Au, mLBA+Try+Au, mLBA+Au, mRan+Au and Try+Au respectively.
Trypsin (Try) treated as a control protein and a random DNA sequence (mRan) were introduced to explore the specificity of Lys-mLBA system by UV-vis spectra analysis and inspection by the naked eye. From Figure 2, it can be seen that the absorption spectra of AuNPs solution with DNA sequences (mRan and mLBA) or complexes (Lys-mRan and Try-mLBA) showed a characteristic red shift and broadening of the surface plasmon band of AuNPs with the intense plasmon resonance band at 670 nm, whereas a concomitant decrease in the intensity of the original plasmon resonance at 520 nm appeared. A significant color change of the solution from red to purple even blue could be observed for aforementioned samples. In the presence of both Lys and mLBA, the absorbance of AuNPs at 520 nm increased and the absorbance at 670 nm decreased. Besides, the color of AuNPs solution containing Lys-mLBA complex still remained red highlighting the selectivity of mLBA.
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Figure 3. Colorimetric assay to detect the interaction between pLBA and Lys. (a) UV-vis
adsorption spectra for AuNPs-based detection before and after the addition of Lys-pLBA complex with different ratios (Lys:pLBA) (inset picture from left to right: buffer only, Lys, pLBA, Lys-pLBA 0.25:1, Lys-pLBA 0.5:1, Lys-pLBA 1:1). (b) UV-vis adsorption spectra for specificity test of pLBA towards Lys (inset picture from left to right: Lys-pLBA, Lys-pRan, Try-pLBA, pLBA, pRan and Try only).
Subsequently, we studied whether the established colorimetric assay could be able to detect the binding between multiple units of LBA arranged along a polynucleic acid chain (pLBA) and Lys. pLBA was obtained by rolling circle amplification (RCA) technique with the template incorporating a complementary sequence of LBA (Table S1). The circular template was prepared by ligation of 5’-phosphorylated linear DNA with T4 DNA ligase and the successful synthesis was confirmed by agarose gel electrophoresis with the circularized template exhibiting reduced mobility compared to the linear template (Figure S2a-2b). Fabrication of the pLBA and pRan (containing a random sequence not binding Lys as a control) was verified by gel electrophoresis using a high molar mass RNA ladder (Figure S2c). As shown in Figure S3, similar to mLBA, the absorbance at 520 nm gradually shifted and decreased, while the broad absorption (550-750 nm) gradually increased along with increasing LBA concentration. Besides, the color of the solutions changed from red to blue gradually. We also studied the interaction of lysozyme with pLBA by UV-vis spectroscopy. The UV-vis spectroscopic investigations (Figure 3a) demonstrated that the UV-vis absorption of AuNPs solution at 520 nm increased and the absorption at 670 nm decreased gradually simultaneously with increasing Lys concentration. The evolution of the AuNPs solution color from blue
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to red can be easily detected by the naked eye (Figure 3a inset picture). When the binding ratio was 1:1, the color of AuNPs solution totally turned red. From Figure 3b, only Lys bound with pLBA showed a sharp band at 520 nm, and the AuNPs solution challenged with Lys-pLBA exhibited a red color, while other AuNPs solution containing complexes (Lys-pRan and Try-pLBA) or sequences (pRan and pLBA) all turned blue. These results proved that pLBA bound to Lys with high specificity.
Figure 4. Colorimetric assay to study the dissociation between pLBA and Lys. Inset picture
from left to right: Lys-pLBA, Lys-pLBA+US 1 min, Lys-pLBA+US 2 min and Lys-pLBA+US 3 min.
Finally, we tested the dissociation between pLBA and Lys by colorimetric assay. From Figure 4, Lys treated by US for 1 min can be dissociated with Lys-pLBA complex, thus resulting in recovery of negative charges of pLBA and further inducing a red shift of the characteristic band at 520 nm by electrostatic interaction between LBA and AuNPs. In addition to that, the color of AuNPs solution turned blue after treatment of US. Pure Lys sonicated for different times was investigated as a control experiment and did not show any distinctly reduced catalytic activity, demonstrating high tolerance of Lys to US (Figure S4 and Table S2).
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5.3 Conclusions
In summary, aforementioned results demonstrated our established colorimetric assay based on cationic 30 nm AuNPs solution provides a sensitive and simple method to study the association and disassociation between nucleic acids and proteins. The most important feature of this assay is direct visualization of the interactions of proteins with nucleic acids by the naked eye, which makes it more convenient than other methods that rely on advanced instruments and laborious characterization procedures.
5.4 Experimental sections
5.4.1 Chemicals and Materials
All chemical reagents were of analytical grade and were used without further purification. All oligonucleotide sequences were synthesized and HPLC-purified by Biomers Co. Ltd. (Germany). T4 DNA ligase (5 Weiss U/µL), phi29 DNA polymerase (10 U/µL) and dNTP Set (100 mM each) were purchased from Thermo Fisher Scientific. Lysozyme from chicken egg white, Lysozyme activity kit, Trypsin (lyophilized powder), chloroauric acid (HAuCl4), sodium citrate, sodium borohydride and cysteamine were ordered from Sigma. Roti®GelStain was received from Carl Roth (Germany). Milli-Q water was used throughout the experiments.
5.4.2 Characterization and equipment
Gel images were obtained by Bio-Rad gel imager (E-box, Vilber). Transmission electron microscopy (TEM) images were got by using a Libra 120 Transmission Electron Microscope (Carl Zeiss, Germany) with 120 kV accelerating voltage. Particle hydrodynamic diameter was measured on a Malvern Zetasizer Ultra (Malvern Instruments Inc, USA) with a He-Ne laser (633 nm) and a backscattering angle of 173º. Ultrasound experiment was performed via an ultrasonic processor (Q125 Sonicator, Power 125 W, Frequency 20 kHz). Optical absorption spectra were measured with a spectrophotometer (SpectraMax M3, Molecular Devices).
5.4.3 Preparation of positive charged 30 nm Au nanoparticles
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the presence of cysteamine according to the published protocol with slight modifications. Typically, a cysteamine solution (400 µL, 213 mM) was added to a 40 mL of 1.42 mM HAuCl4 solution. After stirring for 20 min at room temperature, 10 µL of 10 mM NaBH4 solution was added and the mixture was vigorously stirred for 30 min at room temperature in the dark. After further mild stirring, the resulting wine-red solution was stored in refrigerator and ready for use. As-prepared 30 nm AuNPs were characterized by both TEM and Zetasizer. The morphology of the AuNPs was examined using a Libra 120 Transmission Electron Microscope with 120 kV accelerating voltage. Particle hydrodynamic diameter was measured on a Malvern Zetasizer Ultra with a He-Ne laser (633 nm) and a backscattering angle of 173º.
Figure S1. Characterization of 30 nm (+)AuNPs. (a) Dynamic light scattering (DLS) of
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5.4.4 Preparation of the circular DNA template
Table S1. DNA sequences used in this work.
The circular template was formed using a linear lysozyme aptamer or random DNA template, a primer and T4 DNA ligase. Briefly, 30 µL of 5’-phosphorylated templates (100 µM) was hybridized with 45 µL of primer (100 µM) in 1x T4 ligase reaction buffer (40 mM Tris-HCl, 10 mM MgCl2, 10 mM DTT, 0.5 mM ATP, pH 7.8), followed by heating at 95 °C for 5 min then slowly cooling down to 25°C for 35 min. Subsequently, 15 µL of T4 DNA ligase was added to above solution and the mixture was incubated at 22°C overnight. The ligase was denatured by heating at 70°C for 10 min. Agarose gel characterization is shown in Figure S2a and S2b.
Figure S2. LBA template ligation and pLBA formation. (a) Agarose gel (3%)
characterization of LBA template ligation process. Lane M: ultra-low range DNA marker; lane 1: LBA RCA primer; lane 2: LBA RCA template; lane 3: hybridized template DNA without
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T4 ligase; lane 4: ligated template DNA with T4 DNA ligase. (b) Agarose gel (3%) characterization of random (Ran) DNA template ligation process (control). Lane M: ultra-low range DNA marker; lane 1: Ran RCA primer; lane 2: Ran RCA template; lane 3: hybridized Ran template DNA without T4 ligase; lane 4: ligated Ran template DNA with T4 DNA ligase. (c) Agarose gel (1%) characterization of LBA and Ran DNA RCA chain. Lane M: 1 kb plus marker; lane 1: LBA RCA 10 min; lane 2: Ran DNA RCA 10 min; lane 3: LBA RCA 60 min; lane 4: Ran DNA RCA 60 min.
5.4.5 Synthesis of pLBA DNA and pRan DNA
The pLBA DNA and pRan DNA were synthesized through rolling circle amplification (RCA) technique. Ligated lysozyme aptamer or random sequence templates (0.4 µM) were incubated with phi29 DNA polymerase (0.2 U/µL) and dNTPs (0.5 mM each) at 30°C for 10 min or 1 h in the reaction buffer (33 mM Tris-acetate, 10 mM Mg-acetate, 66 mM K-acetate, 1% (v/v) Tween20, 1 mM DTT, pH 7.9). The reaction was terminated by heating at 65°C for 10 min. The used sequences are summarized in Table S1. Agarose gel characterization is shown in Figure S2c.
400 500 600 700 800 0.0 0.2 0.4 0.6 0.8 1.0 Absor ba nce (a .u. ) Wavelength (nm) Buffer+Au pLBA 40 nM+Au pLBA 60 nM+Au pLBA 80 nM+Au
Figure S3. UV-vis adsorption spectra for AuNPs-based detection before and after the addition
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5.4.6 Influence of US on the activity of pure lysozyme
Figure S4. Influence of US on the activity of lysozyme with different time of treatment.
Lysozyme activity results in the lysis of the Micrococcus lysodeikticus cells. 1 mg of the cell powder was dissolved in 10 mL reaction buffer to achieve a final concentration of 0.01% (w/v) as the kit recommends. 30 μl of reaction buffer or 30 μl of Lys solution (30 μM) treated by US for 0 s, 30 s, 1 min, 2 min and 3 min respectively were introduced to 800 μl of aforementioned cell suspension respectively and mixed thoroughly. During incubation of the Lys sample and substrate, the reaction is followed by monitoring the decrease in absorbance at 450 nm. The maximum linear rates were calculated for each sample. The linear rate of Lys solution without treatment of US was used as 100%.
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5.4.7 Dissociation of lysozyme from pLBA by ultrasonication
To investigate the release of lysozyme from Lys-pLBA complex, the complex was treated by sonication with 1 min, 2 min and 3 min respectively. The sonication treatments were performed using a 3 mm diameter probe with an ice-water bath. The input power level was adjusted around 3W/cm2 with a constant frequency (20 kHz) and amplitude (50%).
5.4.8 Colorimetric assay to detect association and dissociation of
lysozyme
Lys at appropriate concentration was incubated with the mLBA solution (120 nM) or pLBA solution (80 nM) at room temperature for 30 min, and 10 μl of the mixture before and after US for different time was introduced to 200 μl AuNPs solution (5 nM). The photographs of the AuNPs solution were taken after incubation for 5 min. The UV-Vis spectra were collected in the range of 400-800 nm at room temperature.
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