Part:BBa_K3060009

RCA ssDNA for G-quadruplex

This part is a ssDNA, using the primer (BBa_K3060008) to form a circular template for the rolling circle amplification (RCA, or R). The complementary sequence of the G-quadruplex on the circular template can amplificate longer ssDNA with repetitive G-quadruplet by RCA process. Thus, the G-quadruplex can be formed.

Usage

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

Characterization

1. Exploration of the condition of the individual ssDNA G4/Hemin DNAenzyme activity

The measurements of ordinary parts has been uploaded to Part:BBa_K1614007 (https://parts.igem.org/Part:BBa_K1614007#Usage_and_Biology)

2. RCA process amplifies the signal

The materials and methods used to complete the experiment have been uploaded to the wiki of 2019_DUT_China_A (https://2019.igem.org/Team:DUT_China_A/Improvement#materials-methods)

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 2.), 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 3.), calculated that the concentration of the circular template prepared was 1.1394μM, and then diluted the G4 sequence to this concentration.

Figure 2. 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.

Figure 3. 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.

Figure 4. The absorbance compare between the individual short G4 ssDNA and the long G4 chain based on RCA

As shown in the figure (Figure 4,, 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 1. The absorbance compare between the individual short G4 ssDNA and the long G4 chain based on RCA

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.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]