In the CTC-FAST project, we apply synthetic biology to produce DNA tetrahedrons for the capture of circulating tumor cells (CTCs) and expressed mGreenLantern (mGL) green fluorescence protein fused with C7 peptide for CTCs labeling. To prove that the design concept of CTC capture and labeling works well, we set the following milestones:

For CTC capture:

1. Successful conjugation of folic acid (FA) to adaptor single-stranded DNA (ssDNA)
2. Successful assembly of tetrahedrons from four complementary ssDNAs
3. Successful expression of RepA protein and ssDNA binding protein (SBP) in E. coli for RCR

For CTC labeling:

1. Protein expression of mGL-4A-C7 protein
2. Labeling of mGL-4A-C7 on SKOV3 cells (folate receptor alpha positive, mimicking the CTCs)

Successful conjugation of FA to adaptor ssDNA

According to the design of the connection between FA and ssDNA, we need to verify the successful connection of FA-ssDNA. We used UV-vis spectroscopy to observe and graph the absorption spectra of both ssDNA-NH2 and FA-ssDNA. As depicted in Figure 1, it is noticeable that the absorption spectra of FA-ssDNA and ssDNA almost completely overlap, with the former showing a slight elongation of the peak at approximately 305nm, albeit with an extremely minimal shift. We infer that this may be due to the very low content of FA connected to ssDNA, resulting in an indistinct spectral shift.

Successful assembly of tetrahedrons from four complementary ssDNAs

According to the design of four complementary ssDNAs (TD-1, TD-2, TD-3, and TD-4), the mixture of 2 or 3 ssDNAs should only form partially complementary dsDNAs. The native PAGE result indicated that the size of the mixture of TD-1+2, or TD-2+3 is around 80 bp, while the size of the mixture of TD-1+2+3 or TD-2+3+4 is around 120 bp. This result suggested the partial complementation of 2 or 3 ssDNAs. Importantly, the mixture of 4 ssDNAs shows a size of around 175 bp, which corresponds to the sum of four ssDNA.

Successful expression of RepA and ssDNA binding protein (SBP) expression.

To express tetrahedral ssDNA in E. coli by RCR, we cloned the RepA and SBP genes into pET15b expression vector, and induced the protein through add IPTG.

Protein expression and purification of mGL-4A-C7 protein

After modeling, we chose to link mGL protein with C7 peptide by using 4 alanines. The biobrick was cloned into pET-15b vector for protein expression in BL21. The SDS-PAGE analysis indicated that mGL-4A-C7 protein could be expressed (lane-Input) and purified by Ni beads (lane-Elution 1).



We then performed the FPLC assay to purify the mGL-4a-C7 protein. The absorption curve at 280 nm from FPLC analysis indicated the fractions with eluted protein. Accordingly, we found two major peaks, The first one locates from 14th to 18th fractions and the secondary peak locates from the 21st to 38th fractions.



We then performed SDS-PAGE analysis to examine which peak stands for the eluted mGL-4A-C7 protein. The result shows that the mGL-4A-C7 protein (~30kDa) is in the secondary peak (21st to 38th)

Labeling of mGL-4A-C7 protein on SKOV3 cells

The 21st-38th fractions are collected and freeze drying to recover the mGL-4A-C7 protein. To examined whether mGL-4A-C7 protein is functional, we treated the FRa-positive SKOV3 cells by adding 50ug/ml, 100ug/ml, and 200ug/ml mGL-4a-C7 protein in the culture medium and incubating for 2 hours. Fluorescence microscope examination showed that the mGL-4a-C7 protein could efficiently label SKOV3 cells.