4.1. Brief introduction
Along with the development of the precision medicine, rapid and in-time protein function monitoring become a very important issue. Especially the direct sensing in the cellular extracts or even in cell. To achieve this kind of direct sensing, the designed probe or the sensing system should able to work under the complicated environments, either in vitro or in vivo. In addition, the system should better able to detect a variety of protein to provide the advantage of versatility. With the aids of this kinds of versatile and rebuts probe, the researchers are not only able to figure out what is the connection between the protein function and health states, and also able to alert the patient in the early stage of disease if the probe is packaged in assay or sensor.
More and more researches tackling on this issue point out that the activated protein detection is much more important than the total protein amount detection. It is because that most of the disease are dominated by the activated protein amount, instead of the presence amount. If latter one can easily misleading the clinical user to the false result.
Speers et al and Cravatt et al have successfully in developing the protein activity-target probes. Those probes can be used to detect a variety of enzyme functions (Speers et al., 2003, Cravatt et al., 2008). They are basically the structure-switch sensors, which deliver the signal out while binding-induced conformational changes occurs. Being different from the conformational concept, here we propose a novel type of dODN-dye complex probe that traduces the signal through a binding competition upon binding to specific transcription factor.
Among the thousands of DNA-binding proteins, the transcription factors is the most important group, since it mainly control the critical biological function in cell like cell
proliferation and apoptosis. To date, the protein detection methods for determining the activity of transcription factor have less efficiency and laborious (Jantz and Berg, 2002).
For example, the immunity-based techniques like ELISA and Western blot face the challenge of multistep, intensive reagent involvement (e.g. they usually require specific antibodies against each protein).
Fluorescence-labeled antibody is another commonly used method for detecting the intracellular transcription factor protein. It is useful under some circumstances.
However, the system is not able to give the correct signal while the binding activity of the antibody is interfered. The fluoresce is always bright even though the antibody binds to the non-target or nothing. This kind of property undoubtedly decreases the sensitivity of the system. Not to mention other in vitro methods like gel shift assay (Garner and Revzin, 1981) and fluorescence anisotropy. The usability is highly limited by purified protein as the standard, and usually time-costing and laborious (Jantz and Berg, 2002).
Being different from those aforementioned, Heyduk et al proposed a assays that detects the transcription factor and signal out through a binding-induced association of two DNA probe element (Heyduk et al., 2003). This system has achieved the convenient and versatile feature, since it is easy to use and able to target to various transcription factors.
However, the system relies on the covalent break between the DNA probe element, which might a limit in the responding time. In contrast, the aptamer-based transcription factor detection counts on the conformation change purely, but may not able to determine the activity of the transcription factor. Here, we utilise the feature of high binding affinity of the potency enhanced dODN to assemble a dye-intercalated complex that sense the transcription factor. Without the need of bonding break or the conformational change, the binding competition simply kick out the intercalated dye of
complex upon STAT3-binding. With different kinds of dODN and intercalated dye, this system is highly versatile for protein detection.
4.2. Experimental
• Assembly of doxorubicin intercalated hpdODN
The doxorubicin hydrochloride was obtained from the Sigma-Aldrich Taiwan. The
sequences of the hpdODN in this study was:
5’-CGTTTCTCATAAAGCGAAGCTTTA-TGAGAAACG-3’. It is in a structure of hairpin helix. With a simple mixture of doxorubicin and hpdODN (1 µg of each) and the remove of un-bonding reagent by Amicon Ultra centrifugal filter (10KDa), the doxorubicin-intercalated hpdODN, DOXON, was obtained. The doxorubicin used in this study was suitable for fluorescence related experiments.
• Circular dichroism and UV-Vis characterization
The circular dichroism (CD) spectropolarimeter (Aviv Biomedical Inc., Model 202) was used to characterise the secondary structures of the DOXON. A quartz cuvette with the 1 mm optical path was filled with 5 µM of sample solution in DPBS, and the CD spectra were obtained by taking the average of triplicates from 300 nm to 220 nm at 37 ̊C. The UV-Vis absorption spectra were measured on an Agilent UV-Vis spectrophotometer, Perkin Elmer (Lambda 25), with the use of 1.0 cm quartz cells. To determine the release profile, biotinylated DOXON was immobilised on the 24-well streptavidin coated plate.
With the addition of STAT3 or BSA, the doxorubicin in the supernatant solution was measured with the UV-Vis.
• In vitro STAT3-triggered DOX release
Biotinylated complex is immobilised onto a streptavidin-coated 24-well plate and followed by adding the DPBS containing 0.5 µM of STAT3 or BSA protein. The fluorescence intensity of released dye was measured at 596 nm with an excitation wavelength of 480 nm through a micro-plate reader (Infinite M200 PRO, Tecan) with different time interval. The saturated fluorescence intensity is determined by measuring the signal with 50 µM STAT3 protein (30 min incubation) and considered as the maximum intensity (set as 100%). The signal obtained from the just-prepared complex is considered as the background (set as 0%).
• ELONA assays for KD estimation
ELONA assays were used for the estimation of the binding affinity (KD) of complex toward phosphorylated STAT3 and the BSA protein was used as the negative control (obtained from Cell Signalling Technology). For protein coating, 100 µL of 10 nM protein in DPBS was added to each well of 96-well polystyrene plate and incubated at 4 ̊C overnight. The wells were then washed four times with 0.05% Tween 20 in DPBS before being blocked with 1% BSA. A 100 µL of biotinylated complex in the DPBS was then added and incubated at room temperature for 2 hours. The wells were than washed four times with 0.05% Tween 20 in DPBS. The 100 µL of streptavidin–HRP solution was added and incubated at room temperature for 30 minutes. Afterward, the wells were washed with DPBS supplemented with 0.05% (v/v) Tween 20 and 0.1%
(w/v) BSA. Then the TMB substrate solution was used to quantify the binding complex.
The enzymatic reaction was stopped by adding 100 µL of 2 M H2SO4 and followed by absorbance measurement at 450 nm.
• MTT cell proliferation assays
The cell viability of complex-treated cells were assessed with Vybrant® MTT cell proliferation assay. 50 µL of 12 mM MTT solution was added to each well with 1 mL medium. After 2 hours of incubation, 750 µL of the medium was removed and 500 µL of DMSO was added into each well. After another 10 minutes of incubation, the samples were well mixed and measured for the absorbance at 540 nm.
4.3. Results and Discussion
The basic idea of fabricating a dye-intercalated decoy oligodeoxynucleotide system for detecting the oncogenic transcription factor, STAT3, is shown in the figure 4.1. This system contains the hairpin dODN (hpdODN) that designed by the author and the widely-used SYBR green fluorescent dye. For in vitro STAT3 detection, the biotinylated hpdODN is immobilised on the streptavidin-coated well plate. The SYBR green is intercalated into the double stranded structure and emits the fluorescence signal. At this state, the system is considered as the “on” state, since the SYBR green in a kind of fluorescent dye that emit high intensity signal when binding to the DNA.
Afterward, when the STAT3 protein binds to the hpdODN-SYBR system, the SYBR green is released into the bulk due to the binding competition. Therefore, the system goes to the “off” state upon the target binding. In this system, we are expect to see the high intensity background signal. Alone with the increasing concentration of the target STAT3 protein, the fluorescence signal decreases gradually due to the release of SYBR green. on the basis of this mechanism, the possible challenge will be the poor limit of detection (LOD). It is because the background signal (the as-prepared hpdODN-SYBR complex) is high. The change caused by the low amount of the target protein can be hardly observed through the fluorescence detection instrument. This issue will not only affect the performance of the in vitro detection, but also influence the sensitivity in cell imaging. It is because this system detects the STAT3 protein and output the signal in the form of fluorescence decrease. However, in the cell imaging application, it will be hard to identify whether the signal decrease comes from the target binding or the complex degradation, or even the SYBR green leakage caused by the non-specific binding of the protein/peptide in cell.
Figure 4.1 The schematic illustration of the SYBR green intercalated dODN system. In this system, the SYBR green is intercalated into the double stranded hpdODN, which is in the hairpin helix structure. In this illustration, the green star represents the intercalated SYBR green. It emits the fluorescence while in complex with the hpdODN.
This state is therefore considered as “on” state. After the binding of activated STAT3 protein to the hpdODN, the intercalated SYBR green in competed off from the sequence. The released dye then loses its fluorescence signal. This state is therefore considered as the “off” state. Through this mechanism, the concentration of the activated STAT3 protein is able to be determined by processing the fluorescence signal change.
In the very beginning, we aims to verify the feasibility of using the hpdODN-SYBR system in detecting the STAT3 protein. The way to verify this idea is fabricating a in vitro assay just like what we have mentioned above and using it in detecting different concentration of the phosphorylated STAT3 protein (activated state). The detection result is recorded as a image and shown in the figure 4.2. According to this image, the well that has no STAT3 involvement shows a high intensity fluorescence. By adding 2.5 µM of STAT3 protein, the fluorescence signal is slightly decreased. With the aids of the image-processing software, we are able to determine that the signal decrease caused by 2.5 µM STAT3 protein is 9%. That is, when considering the fluoresce signal intensity of hpdODN-SYBR system without STAT3 presence as 100%, the one with 2.5 µM STAT3 protein is 91%. In the cases of higher concentration of the STAT3 protein (5 µM, 7.5 µM, and 10 µM), the signal intensity continuously decrease from 83% to 65%
then 49%. This preliminary test indicates that the decrease of fluorescence signal of the system is proportional to the concentration of STAT3 protein. Although this experiment cannot being used to determine the detection feature of the system, it proves the feasibility of using this system in detecting STAT3 protein quantitatively on the basis of this concentration dependent signal output. Besides, the detection is completed in 10 minutes, which is a quite short detection time, compared to many colorimetric assay (Kim et al., 2014). For example, the system generated by Kim et al spent more than 60 minutes to obtain a clear signal output. According this preliminary test, the feasibility of STAT3 detection and the potential fast (short incubation time requirement) detection feature is discovered. When it comes to the fluorescent dye intercalation, the pH condition and the ion strength both play an important role. To clarity the pH effect and salt effect on on the intercalation efficiency, the hpdODN-SYBR system is assembled under different conditions. Firstly the pH effect is evaluated. The concentration of the
proton H+ is high while in the low pH condition. The high proton concentration is expected therefore to influence the double strand stability of the sequence, since the hydrogen bonding between base pairs is verified to be sensitive to the proton (Vett et al., 2000). Here we measure the fluorescence intensity of the intercalated SYBR green as the index of pH effect. The dependence of SYBR green fluorescence is measured by adding phosphate buffer solution of different pH values to the complex prior to measurement. The observed fluorescence intensities are presented in the figure 4.3 (upper). The fluorescence intensities are highly comparable in the pH range between 5 and 7. In the contrast, a drop-decrease is observed at pH values lower than 4. Under the pH 2 condition, the fluorescence signal is nearly disappear (<3%). The other important factor is the salt concentration of the buffer solution. The ions concentration is high while the buffer solution contains high amount of salt, such as sodium chloride. The charged ions is expected therefore to influence the intercalator binding, since the electron transfer plays an important role in the intercalation. The redundant ion is probably undesired. The result of the salt dependence is shown in the figure 4.3 (lower).
It is obviously the fluorescence signal deceases alone with the increasing sodium chloride concentration. The signal intensity under the 10 mM and 500 mM (50 times difference) has a near 1.3 times difference between each other. On the basis of the pH effect evaluation and slat effect determination, it can be deduced that the high efficient dye intercalation of SYBR green may be achieved under the neutral condition (pH 7) and low salt condition (about 10 mM, or even lower). The alkaline condition is not in the consideration in this study because of many researches have point out the cytotoxicity of residual alkaline agent in the prepared material will harm the cell.
Overall, the neutral and low salt condition is preferred in the following experiment of hpdODN-SYBR system.
Figure 4.2 The preliminary experiment for verifying the concept of hpdODN-SYBR green detection system. The streptavidin-coated 96-well plate is immobilised with the biotinylated hpdODN (with SYBR green intercalation). From the left to the right, the increasing concentration (0 µM, 2.5 µM, 5 µM, 7.5 µM, 10 µM) of phosphorylated STAT3 are added into the well and incubated for 10 minutes. The obtained image is further processed with the image-J software. When considering the signal in the absence of phosphorylated STAT3 protein as the 100% of the intensity. The residual signal are 91%, 83%, 65%, and 49%, respectively corresponding to the different concentration of target.
Figure 4.3 The fluorescence intensity of hpdODN-SYBR complex under different pH and NaCl concentration. The same ratio of hpdODN and SYBR green are used in all experiments. The fluorescence intensity are determined under pH 2,3,4,5,6, and 7. The considered concentration of NaCl are 10 mM, 75 mM, 125 mM, 250 mM, and 500 mM.
To further understand the SYBR green intercalation in the hpdODN, the UV-Vis spectrometer and and circular dichroism is used to measure the feature of the intercalated SYBR green. The UV-Vis spectra allows the user to know the quantitative relationship between the fluorescence signal and concentration used in preparation. The circular dichroism provides the information about how SYBR green intercalation affects the structure of the double stranded helix hpdODN. The results are shown in the figure 4.4. With a increase concentration of SYBR green fluorescent dye, the absorbance around 485 nm continuously increase, which may suggest the increase of intercalated SYBR green dyes. On the other hand, the spectra obtained from the circular dichroism shows that the presence of SYBR green seems stabilise the hpdODN, and hence causes a higher ellipticity around 270 nm.
In the UV-Vis spectrum measurement, the concentration ratio is used to quantify the composition of hpdODN-SYBR system. However, the dye/base pair ratio is also an important index that dominates the properties of the dye-intercalated sequence.
Therefore, the fluorescence intensity under different dye/base pair ratio is quantified.
The result is shown in the figure 4.5. When the dye/base pair ratio is 0.1, which means every ten base pair may encounter with one SYBR green molecule, the intensity is near zero. This suggests that the SYBR green rarely intercalates into the sequence under this kind of condition. In the range between 0.2 to 2 of the dye/base pair ratio, the fluorescence intensity increases dramatically, which is nearly a exponential type of relation. In the region between 2 to 20 of the dye/base pair ratio, the fluorescence intensity keeps increasing, but with a relative-lower slope. Quantitatively, both the two region contains ten times difference in concentration (0.2 →2 and 2→20), but the fluorescence intensity differences are 7.1fold and 1.3 fold, respectively. The result indicates that generally the fluorescence intensity (which may be considered as the
amount of the intercalated dye) is proportional to the dye/base pair ratio. However, the intercalation is nearly saturated at the dye/base pair ratio of 20. The molecular weight of SYBR green is 509.7 g mol-1, and that of hpdODN is 10160.7 g mol-1. Therefore the dye/base pair ratio of 20 is actually indicating the weight ratio of 1. On the basis of this result, the complex ing the following experiments are composed in this ratio. For example, 1 µg/mL of both the SYBR green and hpdODN are used to prepare 2 µg/mL of hpdODN-SYBR green complex. Compare this result to the previous studies (Vos et al., 2000), it is obviously that the hpdODN needs more fluorescent dye for achieving the high signal intensity. This can be resulted from the preference of GC pair of SYBR green (Rane et al., 2000). The GC ratio of the hpdODN is 40% in this study, relative lower than the sequences used in the previous research (many of them are GC-rich aptamers).
Figure 4.4 The UV-Vis spectra and circular dichroism spectra of the SYBR green-intercalated hpdODN. The 10 µM hpdODN is titrated with the increasing concentration of SYBR green (1 µM to 25 µM). The absorbance at 524 nm is therefore increased gradually. The circular dichroism spectra is recorded from the hpdODN and the hpdODN-SYBR green complex under 37℃.
Figure 4.5 The fluorescence intensity recorded from the sample that composed of the hpdODN and SYBR green with different dye/base pair ratio. The ratios are 0.1, 0.2,0.5, 1, 2, 5, 10, and 20, respectively. For this experiment, the hpdODN is considered to have 15 base pairs. Since it has 33 bases but three of them are connectors for constructing the hairpin structure.
After secure the success intercalation of the SYBR green in the hpdODN, the basic feature of the detection performance of the hpdODN-SYBR system is delivered. The complex are immobilised on the well of plate and incubated with serial dilution of the phosphorylated STAT3 protein (1 µM as the beginning, and 10 times diluted in the following). It is worthwhile to emphasise again that the hpdODN-SYBR system is a kind of “signal off” detection system. That is, the information (presence or absence, concentration) of the target protein is output through the decrease of the fluorescence intensity. In this basic feature determination (figure 4.6), the region between 0.1 nM to 5 nM, and 100 nM to 1000 nM, both provide a insignificant signal change. Precisely, the 10 times difference can only induce 1~3% of signal change in these two region. In the contrast, under the concentration range from 5 nM to 100 nM, the signal change is significant. In the quantitative number, the 10 times difference causes ~40% of the signal decrease. Notably, the zero-signal is considered as 0 in this experiment, hence the
After secure the success intercalation of the SYBR green in the hpdODN, the basic feature of the detection performance of the hpdODN-SYBR system is delivered. The complex are immobilised on the well of plate and incubated with serial dilution of the phosphorylated STAT3 protein (1 µM as the beginning, and 10 times diluted in the following). It is worthwhile to emphasise again that the hpdODN-SYBR system is a kind of “signal off” detection system. That is, the information (presence or absence, concentration) of the target protein is output through the decrease of the fluorescence intensity. In this basic feature determination (figure 4.6), the region between 0.1 nM to 5 nM, and 100 nM to 1000 nM, both provide a insignificant signal change. Precisely, the 10 times difference can only induce 1~3% of signal change in these two region. In the contrast, under the concentration range from 5 nM to 100 nM, the signal change is significant. In the quantitative number, the 10 times difference causes ~40% of the signal decrease. Notably, the zero-signal is considered as 0 in this experiment, hence the