In CTC-FAST project, we aim to improve the detection of circulating tumor cells (CTCs) by replacing the antibody based detection assay with DNA based one. Accordingly, we designed the folic acid (FA) conjugated DNA tetrahedrons to capture CTCs from the blood sample. To anchor the DNA tetrahedrons in our automatic device, we decided to apply the zinc finger fusion protein Zif268-PBSII, which will be coated on the main chamber of our device to interact with the corresponding motifs at the edge of DNA tetrahedron. To produce the tetrahedral ssDNA with highly complementary sequences in vivo, we construct the rolling circle replication (RCR) system in E. coli. Finally, to label the captured CTCs, we used the new fluorescence protein, mGreenLatern (mGL), which has a stronger brightness than canonical eGFP, to increase the sensitivity.

To examine whether the DNA tetrahedron could be assembled by single-stranded DNA (ssDNA) in a complementary manner, we designed four ssDNAs TD-1, TD-2, TD-3 and TD-4 (Sequences are shown in design) and performed the tetrahedron assembly according to the standard annealing protocol.

After annealing, the reaction mixture was subjected into native PAGE electrophoresis. The product size of annealing two ssDNAs (TD-1+TD-2 and TD-2+TD-3) was around 80 bp, while annealing three ssDNAs (TD-1+TD-3+TD-4 and TD-2+TD-3+TD-4) was around 120 bp. The product sizes of annealing two or three ssDNA were slightly smaller than anticipation, which may result from the partial complementation. Importantly, the product size of annealing four ssDNAs was 175 bp, suggesting a more completely complementation. However, we also observed the side product with size more than 175 bp, which may stand for the DNA polyhedrons.

Because the Mg2+ is important for DNA complementation, we speculated whether the increase of Mg2+ concentration would inhibit the unexpected polyhedron assembly. Accordingly, we increased the Mg2+ concentration from 5 mM to 25 mM. However, the native PAGE analysis showed no significant improvement of tetrahedron assembly.



Since the standard protocol cool the ssDNA mixture fastly, we guess this may cause unexpected interaction. Therefore, we try to gradually decrease the annealing temperature (3~4 °C/5 min), allowing the tetrahedron to properly assemble. However, the native PAGE analysis still shows no improvement significantly.



Together, we thought that the tetrahedron assembly from separated ssDNA may form side products like polyhedrons, which could be caused by the random complementation among the ssDNAs.

To rescue the random complementation, we decided to fuse the four ssDNAs into single tetrahedral ssDNA for tetrahedron assembly. Because of the reverse polarity of complementary DNA strands, one edge of the tetrahedron must form the “twin double helices” structure (See design for detail). We decided to generate the double-stranded DNA (dsDNA) encoding tetrahedral sequence as a template and performed in vitro rolling circle amplification (RCA) to produce the tetrahedral ssDNA. However, the serious problem is that the production of dsDNA templates with highly complementary sequences are rejected by most companies. To put the tetrahedral dsDNA template into order, we separated the dsDNA template into two parts (ssDNA-L and ssDNA-R) and added an extra stuffer to reduce the complementarity.

After getting the two parts of tetrahedral ssDNA (ssDNA-L and ssDNA-R), we performed the fusion PCR to fuse these two fragments into one tetrahedral ssDNA with a stuffer. The TA ligation and sequencing confirmed the inserted fragment was indeed the fusion of ssDNA-L and ssDNA-R.

-Engineering

The experiment went smoothly. We could remove the stuffer by EcoRI digestion and self-ligation. The dsDNA template for tetrahedral ssDNA could be excised form TA vector and relegated as RCA template. However, after discussing with the experts, we realized that the single tetrahedral ssDNA may also cause polyhedron side products.

According to the suggestion from experts, we decided to construct the RCR system in E.coli to generate circular tetrahedral ssDNAs from the dsDNA template and coexpress ssDNA binding protein (SSBP) to block the polyhedrons formation.

Accordingly, we builded two composite parts. One composite part (BBa_K4674011) is applied to express the RCR initiation proteins (RepA) and SSBP and the other one (BBa_K4674012) is the target dsDNA template cassette for RepA recognition (see design for detail). After cloning, the sequencing result confirmed that two composite parts were successfully cloned into the pET-15b expression vector.

We then induced the RepA and SSBP protein expression by IPTG. After 6 hours of IPTG induction at 37°C, we found that the RepA and SSBP were expressed. However, the expression of protein was found in the inclusion body (cell pellet).

-Future work

To confirm whether the RCR system is functional, we will perform the protein induction at lower temperature (e.g. 12~16°C), and examine the protein expression by SDS-PAGE analysis. After successfully inducing the protein expression, we will purify the his-tagged SSBP by Ni2+ beads, and perform PCR analysis to examine whether the RCR system successfully produces the circular tetrahedral ssDNA from the target cassette. Finally, the cis-autosplicing of purified circular tetrahedral ssDNA will be induced by Mg2+ ion, and the formation of tetrahedron will be examined by atomic force microscope (AFM).

To conjugate the folic acid to the adaptor ssDNA for CTC capture, we utilized the mechanism of SN2 reaction to prepare FA-NHS ester. Following the protocol of generating FA-NHS ester, we observed the expected pale yellow substance.

To confirm its identity, we utilized Fourier-transform infrared spectroscopy (FTIR) for qualitative analysis of the substance. The IR spectrum of FA-NHS ester indicated that the main component is FA, as compared to the standard IR spectrum of FA provided by spectral Database for Organic Compounds (SDBS). Importantly, three characteristic peaks (2700 ~ 3300 cm-1) in the IR spectrum of FA-NHS are also shown in the standard IR spectrum of NHS. The slight shift of wavelength of these peaks may be caused by the conjugation. Together, this result suggested the successful conjugation of NHS to FA.



Subsequently, we reacted the FA-NHS ester with NH2 modified adaptor ssDNA, resulting in the formation of FA-ssDNA. Since the FA compound show absorption around the 300 nm, we performed UV-vis spectroscopy to observe the absorption spectra, showing that the absorption spectra of FA-ssDNA and ssDNA almost completely overlap, with the former showing a slight elongation of the peak at approximately 305 nm.

To anchor the DNA tetrahedrons in our automatic device, we applied the specific interaction between zinc finger fusion protein (PBSII-Zif268) and its corresponding motifs at the edge of the tetrahedron (see designfor details). To express the PBSII-Zif268 fusion protein, we design the composite part and clone it into pET-15b plasmid. The sequencing result confirmed the correct insertion of BBa_K4674013 in the pET-15b vector.

After confirming the cloning, we induced the PBSII-Zif268 fusion protein expression by treating 0.25 mM IPTG at 37°C. The PAGE analysis showed that the fusion protein was located in the inclusion body (P lane). We then modified the induction temperature to 16°C, however, the PAGE analysis result still showed the inclusion body location of PBSII-Zif268 fusion protein.



We then try to extract the PBSII-Zif268 fusion protein from the inclusion body by 8 M Urea solution. The PAGE analysis showed that the treatment of 8 M Urea could solubilize the PBSII-Zif268 fusion protein (the Sup 2 lane).

-Future work

The 8 M Urea extracted PBSII-Zif268 fusion protein needs to be purified by Ni2+ bead and renature into the functional structure for docking DNA tetrahedron. We have found the protocol for protein renature on the Ni2+ beads or after elution. The corresponding experiments will be performed to optimize the PBSII-Zif268 fusion protein collection.

To examine whether the mGL-4A-C7 protein is functional in labeling CTCs as the modeling result, we builded the composite part (BBa_K4674010) for mGL-4A-C7 protein expression. As a control, we also builded the composite part (BBa_K4674009) for eGFP-4A-C7 protein expression.

After confirming the expression cassette insertion into pET-15b, we induced the mGL-4A-C7 and eGFP-4A-C7 protein expression by adding 1mM IPTG at 37°C. The Coomassie blue staining of PAGE analysis clearly showed that the induced mGL-4A-C7 and eGFP-4A-C7 protein was soluble and could be purified by Ni2+ beads.

To purify a large batch of mGL-4A-C7 and eGFP-4A-C7 protein for further examination and application, we performed the FPLC analysis to separate the non-specific binding protein from target protein. The absorption spectra at 280 nm indicated that there are two main proteins containing fractions. One is from 14th to 18th fraction, and the other is from 21st to 38th fractions.



Although the greens fluorescence clearly shown in the 21-38 fraction, we still performed the SDS-PAGE analysis, confirming the existence of mGL-4A-C7 protein.



After confirming the existence of target proteins, we freeze-dried the fractions containing mGL-4A-C7 or eGFP-4A-C7 proteins. The freeze drying protein was then rehydrated to the same concentration for examination.

We first examine whether the mGL exhibits a strong brightness when excited by 488 nm laser. The value of fluorescence brightness detected by standard eGFP filter indicated that, the mGL exhibits fluorescence at least twofold fluorescence brighter than eGFP at the same concentration, and protein could be stored at room temperature at least one week.



Next, we examine whether the mGL-4A-C7 protein could recognize the CTC mimics, the SKOV3 cells. Accordingly, we incubated SKOV3 cells with medium containing different concentrations of mGL-4A-C7 and eGFP-4A-C7 (50μg/ml, 100μg/ml, 200μg/ml) at 37 ℃ for 2 hours. The observation of fluorescence indicated that the 50 μg/mL of mGL-4A-C7 could label the SKOV3 cells, while the 50 μg/mL of eGFP-4A-C7 showed moderate labeling. Furthermore, the extension of incubating time to 4 hours did not significantly improve the labeling. Together, we confirmed that the mGL-4A-C7 could label the SKOV3 cells, suggesting mGL-4A-C7 protein as CTC labeling reagent.