Team:DUT China A/Improvement

Improvement

We choose 2015 in components of the improvement in IGEM Heisenberg university team uploaded to the repository of couplet of G-quadruplex sequences (BBa_K1614007), by introducing the ssDNA sequences to linear ssDNA and transfer it into circular ssDNA template (BBa_K3060009), in the role of phi29DNA polymerase rolling circular amplification reached under the condition of a small amount of primers got a couple of G-quadruplex sequences, and then get more position combined with high hemin to achieve the purpose of signal amplification.

Principles

In the original element, G-quadruplex ssDNA can form a specific secondary topological structure after annealing, and formed DNAenzyme through Hoogsten bond with hemin. This kind of DNA mimic enzyme has the same activity of HRP which can catalyze hydrogen peroxide reaction form O2- ion. The reaction between ABTS solution and hydrogen peroxide was used as the background. After the addition of g-quadruplex/hemin mimic enzyme, ABTS quickly reacted with O2- ions to form ABTS- ions, which changed the color of the system from colorless to grandmother green (Figure 1). We introduced the single chain sequence of G-quadruplet into the circular template DNA, which was firstly combined with primer for incubation, then phi29 DNA polymerase and dNTPs were added into phi29 buffer, and hemin was added into the reaction system for binding and incubation after a period of time (specific time). After that, it was the same as the G-quadruplex ssDNA experiment that add the ABTS into the system. Detect the absorbance of the system after reaction was determined by ultraviolet spectrophotometry at the absorption wavelength of 418nm and the degree of reaction was compared.

Figure 1. Principle of G-quadruplet based on RCA

Materials and Methods

1.1 Materials and instruments

T4 DNA ligase, Exonuclease I (Exo I), and Exonuclease III (Exo III) were purchased from Takara (Beijing, China). Phi29 DNA polymerase was purchased from NEB (Ipswich, England). Hemin and 2,2'-azino-bis(3-ethylbenzthiazoline) 6-sulphonic acid (ABTS) were obtained from Aladdin (Shanghai, China). 30% hydrogen peroxide solution (H2O2) and dimethyl sulfoxide (DMSO) were purchased from Damao (Tianjin, China). Triton X-100 was purchased from Guangfu (Tianjin, China). Deoxynucleotide solution mixture (dNTPs) was purchased from Thermo Fisher Scientific (Beijing, China). DNA oligonucleotides were obtained from GENEWIZ (Suzhou, China). All DNA oligonucleotides were purified using high performance liquid chromatography. The absorbance signal was detected by the UV-vis spectrophotometer (Evolution 350, Waltham Mass, USA). The gel electrophoresis was performed on the PowerPac™ Basic electrophoresis analyzer (Bio-rad, USA) and imaged on a KETA G Imaging System (Wealtec, USA).

All buffer solutions were prepared using Millipore-Q water (≥18 MΩ, Milli-Q, Millipore). Hemin stocking solution (10 mM) was prepared in dimethyl DMSO and stored in the dark at −20 °C and was diluted to the required concentrations with 25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (pH 7.4) containing 100 mM KCl, 200 mM NaCl, 0.05% Triton X-100 and 1% DMSO prior to use.

1.2 Exploration of the best react condition for G4/hemin mimic DNAenzyme

G4ssDNA of 100uM was diluted to 20uM, annealed at 95°C and cooled to room temperature. The prepared G4ssDNA sequence was incubated with hemin of equal volume 15ul diluted to 20uM for 35min, and the four groups were parallel. Add 100ul 4mM ABTS solution and water for constant volume, add 3ul 30% hydrogen peroxide solution to the system after 15min, and measure the absorbance every two minutes. In the control group, only hemin of equal concentration was added in the system and no catalyst was added in the system. Then detect the absorbance of the system after reaction was determined by ultraviolet spectrophotometry at the absorption wavelength of 418nm and the degree of reaction was compared.

1.3 Preparation of a circular template for RCA

Mix G4ssDNA (100 μM, 2 μL) with PrimerG4 (100 μM, 4 μL). In order to ensure that padlock probe can fully hybridize with primer, the mixture was heated to 95 °C and slowly cooled down to room temperature (RT). Then, add T4 DNA ligase (350 U/μL, 1 μL) and 10X T4 DNA ligase buffer (660 mM Tris-HCl (pH 7.6), 66 mM MgCl2, 100 mM DTT and 1 mM ATP). Ligation process was performed for 16 h at 16 °C to form a circular template by the ligation of the 5’-phosphate and 3’-hydroxyl ends of the ssDNA. Next, Exo I (5 U/μL, 2.5 μL) and Exo III (200 U/μL , 0.5 μL) were added and the mixture was incubated at 37 °C for 4 h to digest the leftover primer, linear ssDNA and other byproducts. The enzymes were denatured by incubating at 65 °C for 10 min. Finally, the prepared circular template was stored at 4 °C until use.

The circular template was characterized by polyacrylamide gel electrophoresis (PAGE), at the same time use DL500 as the marker, and then use ImageJ to analysis grayscale in order to calculate the concentration of the circular template.

1.4 RCA process

Add 10X phi29 DNA polymerase reaction buffer (50 mM Tris-HCl, 10 mM MgCl2, 10 mM(NH4)2SO4, and 0.1% Tween 20, 4 mM DTT), DNA polymerase (10 U/μL , 0.5 μL), dNTP (0.5 μL, Different experiments change the concentration) and BSA (2.5 μM, 0.5 μL, final concentration is 0.1 μM). The RCA reaction was performed at 37 °C for 1 h. The enzymes were denatured by incubating at 65 °C for 10 min.

1.5 Colorimetric assay

In order to ensure that the RCA reaction can fully form G-quadruplex structure, the mixture was heated to 95 °C and slowly cooled down to room temperature (RT). Dilute the RCA product sample into different multiples. Take 10 μL for each concentration and mix it with hemin solution (3 μM, 10 μL). Incubate it at RT for another 30 min to form G-quadruplex/hemin structure. Finally, the reaction mixture was added in 100 μL 4 mM ABTS2− in the 96-well plates and 4 μL of 30% H2O2 and replenish the solution to 200 μL. The absorption signal at 418 nm was measured using a UV–vis absorption spectrophotometer in the wave length range from 400 to 500 nm.

Results

2.1 Exploration of the condition of the individual ssDNA G4/Hemin DNAenzyme activity

2.1.1 Determination of reaction quantity and reaction time of hydrogen peroxide in the system

In the reaction process, both Hemin and G4/ Hemin DNAenzyme will carry out enzyme-catalyzed reaction with hydrogen peroxide as the substrate, which will reach the peak due to the constant decrease of substrate concentration during the reaction, and then attenuated due to the characteristics of the reaction. We used ssDNA G4 and Hemin solution with the same initial concentration of 1uM to determine the better conditions for the addition of hydrogen peroxide solution by measuring the reaction time of adding different amounts of hydrogen peroxide system.

Figure 2. The absorbance of the reaction system for different amount of hydrogen peroxide in different time

As shown in the figure (Figure 2), the time interval represented by the highest two points was selected as a good time for the reaction measurement. Under the condition of adding 1ul hydrogen peroxide, the scatter was dense and the reaction time had little influence. Under the condition of adding 3ul hydrogen peroxide, the best reaction time was between 2-5min. Under the condition of adding 5ul hydrogen peroxide, the best reaction time was between 5-7min. Under the condition of adding 7ul hydrogen peroxide, the optimum reaction time was over 10min.

2.1.2 The activity of reaction for G4/Hemin mimic DNA enzyme turn to time

Figure 3. the relationship between time and the G4/Hemin DNAenzyme.
Line green is the average of the four parallel experiments.

It can be seen that there is a peak value in the curve obtained (Figure 3). When 3ul 30% hydrogen peroxide is added in the pre-experiment, the peak value can be obtained to be 2-5min, so the results are consistent with the pre-experiment. Hemin itself has the catalytic effect of hydrogen peroxide, but when the G4 conjugated sequence is introduced and incubated for a period of time, the simulated enzyme formed by the combination of the two has higher enzyme activity and reaches the reaction peak at 2-5min. The results were used as a reference for the selection of reaction conditions.

Figure 4. PAGE image analysis of G4ssDNA (Lane 1), circular template (Lane 2-6), ssDNA:primer = 2:0.5 (Lane 2), 2:1 (Lane 3),2:1.5 (Lane 4), 2:2 (Lane 5), 2:3(Lane 6). M, DL500 DNA size marker.

2.2 RCA process amplifies the signal

In order to prove that our system could amplify the signal of G4/Hemin DNAenzyme, we adopted the same concentration of circular template as G4 for rolling ring amplification, and then compared the absorbance of the amplified results of the G4 in circular template. As shown in the figure (Figure 4), PAGE result is circular template synthesized under different proportions of ssDNA and primer. It can be seen that the bigger proportion is, the more amount of circular template. We first used ImageJ to conduct grayscale analysis of PAGE results (Figure 5), calculated that the concentration of the circular template prepared was 1.1394μM, and then diluted the G4 sequence to this concentration.

Figure 5. PAGE image analysis of G4 circular template (Lane 1-2), 2 μl loading quantity (Lane 1), 4 μl loading quantity (Lane 2). M, DL500 DNA size marker (6 μl loading quantity).

We can control the length of the long chain containing G4 ssDNA sequences in cycle, which is amplified by the rolling circular amplification (RCA) in the process of RCA. We added 5μl dNTPs with a concentration of 25mM and 12.5mM to the system and respectively, through calculation, the corresponding amplification reaches 50 times and 100 times at most. At first, we measured that the absorbance at 418nm was approximately equal to the blank when 10μl long chain solution prepared by RCA was directly incubated with 10μl hemin (3μM), which was due to the crossing of the long ssDNA chain at too high concentration influences the formation of G-quadruplex stucture. According to this phenomenon, we dilluted the RCA products by 10, 25, 100, 200, 500 times, correspongdingly the G4 sequence by the same amount.

Table 1. The absorbance compare between the individual short G4 ssDNA and the long G4 chain based on RCA

Figure 6. The absorbance compare between the individual short G4 ssDNA and the long G4 chain based on RCA

As shown in the figure (Figure 6, Table 1), the absorbance of the long G4 chain based on RCA is significantly greater than that of the individual short G4 ssDNA structure in the region with dilution of more than 100 times. When dilluted 200 times, the absorbance of the 25 nM dNTPs is corresponding to the short ssDNA G4 sequence structure with 36 times dilution. It can be calculated that the signal of this group was amplified 5.56 times and the amplification efficiency of RCA was 5.56%; the absorbance of the 12.5 nM dNTPs is corresponding to the short ssDNAG4 sequence structure with 68 times dilution. It can be calculated that the signal of this group was amplified 2.94 times and the amplification efficiency of RCA was 5.88% (Table 2). It can be seen that the concentration is an important condition influencing the RCA process and our design can respectively amplify the signal of reaction process with G-quardruplex.

Table 2. The times of signal amplification and RCA efficiency to the two different concentrations of dNTPs.

Citation:

[1] Xu L, Jiang Z, Mu Y, et al. Colorimetric assay of rare disseminated tumor cells in real sample by aptamer-induced rolling circle amplification on cell surface[J]. Sensors and Actuators B: Chemical, 2018, 259: 596-603.
[2] Li R, Xiong C, Xiao Z, et al. Colorimetric detection of cholesterol with G-quadruplex-based DNAzymes and ABTS2−[J]. Analytica chimica acta, 2012, 724: 80-85.