Team:DUT China A/Validatity
Validatity
The aim of our project is to use DNA hydrogel to encapsulate the living CTCs, so we design a series of parts. The linear ss DNA(BBa_K3060005 and BBa_K3060002) are used to prepare the circular template. BBa_K3060001 is a ss DNA that we designed specially, once BBa_K3060001 successfully targeting CTCs, the ssDNA aptamers will expose the sticky end further for triggering the adhesion of sticky-end pairing ssDNA. The pairing ssDNA can induce rolling circle amplification, subsequent multi-primed chain amplification, with the help of primer 2(BBa_K3060005) and primer 3(BBa_K3060004), making the formation of CiC around CTCs.
The following is the characterization of the formation of hydrogel:
1. The formation of circular template
In order to examine the successful preparation of the circular template, different samples were characterized by polyacrylamide gel electrophoresis (PAGE) As shown in Fig. 1, ligation products of ssDNA (Lane 2) exhibited a series of dispersive bands with slower migration than that of ssDNA (lane 1), indicating the formation of circular templates and other byproducts. After the ligation products were treated with Exo I and Exo III, only one bright and well-defined band still existed, proving the complete digestion of ligation byproducts and successful preparation of the circular template (lane 3).
Fig. 1. PAGE image analysis of ssDNA (Lane 1), ligation products of ssDNA by reaction with Primer 3 and T4 DNA ligase (Lane 2), purified circular template by digesting above-mentioned ligation products with Exo I, and Exo III (Lane 3).
2. The formation of DNA Hydrogel
We use a variety of methods to characterize the formation of DNA hydrogel.
a. Agarose Gel Electrophoresis
Agarose gel electrophoresis was used to evaluate the formation of DNA hydrogel. DNA hydrogel is difficult to migrate through the agarose gel and remain the retention in home position.
Fig. 2. AGE image analysis of phi29(+) (Lane 1), phi29(-) (Lane 2).M1, DL2000 DNA size marker. M2, DL500 DNA size marker.
b. SEM
Scanning electron microscopy (SEM) was used to obtain the morphology of theDNA hydrogel. As shown in Fig. 3, there are a large number of hydrogel particles with a diameter of about 2 μm in the buffer.
Fig. 3. SEM images showing structures of DNA hydrogel particles.
c. DLS
We dynamic light scattering (DLS) analysis to get the size of the DNA hydrogel. As shown in Fig. 4, the size of hydrogel particles is between 100 nm and 3000 nm.
After centrifugation, we got a spherical DNA hydrogel at the bottom of the tube.
Fig. 4. DLS size distribution of DNA hydrogel particle. After centrifugation, we got a spherical DNA hydrogel at the bottom of the tube.
Fig.5 DNA hydrogel