Team:DUT China A/Design
Outline
In our project, we designed a triggering mechanism based on specific combination of the circular template and specific targets on the cell membrane in order to assemble DNA hydrogel by rolling circle amplification (RCA, or R) and multi-primed chain amplification (MCA, or M) around the cell.
Figure 1. The formation of DNA Hydrogel by RCA and MCA.
Hydrogel self-assembling
Aiming at generating hydrogel in a constant system from a single raw material and with a high degree of crosslinking, we utilized and developed the combination of rolling circle amplification (RCA) and multi-primed chain amplification (MCA). They were carried out as follows:
Firstly, a circular template was prepared and added with primer 1(P1) corresponding to it. Phi29 DNA polymerase and dNTPs were used as raw materials for RCA, so as to obtain a long ssDNA chain with periodic circular template complementary sequence. Control the time of this stage (RCA), and then add primer 2(P2) corresponding to the ssDNA long chain and primer 3(P3) corresponding to the original circular template for a different site into the system to carry out MCA. In this stage (MCA), phi29DNA polymerase finished amplifying a cycle with all these three kinds of primers.
Figure 2. The process of RCA and MCA, for a single cycle with 3
different primer.
Although the kinds of primer were different, periodically amplifying the
sequence accesses that the roles of template chain and complementary chain
were continuously exchanged in the amplification, so as to complete the
generation of a large amount of ssDNA in a limited volume. These ssDNA
strands, which are in a large amount, continued to fold and weave, forming
nests of DNA monomers that pile up to assemble the hydrogel we need.
This design allows the formation of hydrogel in an environment where the process is always mild and the activity of the enzyme can be maintained for a certain period of time. Moreover, the raw material is simple and we can control the time of both RCA and MCA to manipulate the size and crosslinking degree of the hydrogel.
The preparation of the circular template
In this method, the preparation of single stranded circular DNA template is the focus of the whole system process. We used a general circular DNA preparation method to synthesize linear single strand DNA from known circular DNA template sequences, and designed both ends of the linear DNA as complementary fragments of P3, which can be combined to form all complementary sequences of P3. In the process of preparation, we added the designed linear single strand DNA and P3 to the system at the same time, theoretically forming a linear ssDNA that is complementary to P3 end to end in the system. At this time, linear single strand DNA transfered into the circular DNA with a nick and part of it is basepairing with P3. Next, T4 DNA ligase is added to the system to fill up the nick, and Exo I/III exonuclease is added to digest and prepare the circular template successfully.
Figure 3. The preparation of the circular template.
The choose of the receptor and aptamer
It is worth mentioning that this method based on ssDNA coiling and folding rather than constructing by whole base complementary, which enables us to make the gelling system work better on the cell surface without gelling off or misassembling. In that case, we need specific bind between this system and our target cell so our next goal is to transfer the template onto the cell surface. As for CTCs, we hope that capsulated cells can be used for living cells detection and research. Therefore, instead of the antigen and antibody molecular targets, we adopted the method of aptamer binding.EpCAM (epithelial cell adhesion molecule) was first found in colon cancer as a single transmembrane protein, which is involved in the regulation of cell adhesion, proliferation and differentiation. EpCAM abnormally expressed in tumor cells and expressed 100 times higher than normal level in primary breast cancer tissues, 810 times higher than normal level in lymph node metastasis. EpCAM was selected as the target to combine the circular template with the cell membrane through aptamer binding. We screened an EpCAM adaptor named SYL3C from the paper database and verified its binding activity.
Figure 4. The secondary structure of SYL3C.
The design of the triggering mechanism
Our project aims at capturing CTCs with DNA hydrogel and it is in need of the spcical initiation mechanism. This case is districted with two elements：Firstly, an aptamer which specifically recognizes receptors on the membrane of circulating tumor cells and we choose the EpCAM which is mentioned above as our molecular target . Secondly, special inhibitor designed by model which enables hydrogel to be specifically triggered by receptors on the cell membrane and assemble around CTCs. We mainly designed two triggering processes for hydrogel self-assembling.
The first design is based on the conformational transformation with hairpin structure. In terms of molecule level, our design includes three parts: aptamer, linker and primer. At the 5' end of the aptamer, we attached the FAM fluorescent radical group for labeling the aptamer binding to the cell surface, and at the 3' end of the aptamer we sequentially attached the linker and primer. The aptamer and primer are partially complementary to each other and that makes the primer silent in the absence of cells with EpCAM. When the cell binds to the aptamer, it competitively opens the complementary pairing between the primer and the aptamer, exposing the primer to participate in the formation of hydrogel. In this case, we selected 9 base complementary pairs and 10 base complementary pairs through modeling, and we named them as Aptamer 1 and Aptamer 2 respectively.
Figure 5. The conformational transformation with hairpin structure.
Figure 6. The sequence and structure of Aptamer 1 and Aptamer 2.
The second design is the inhibitor shedding. Structurally, our design
consists of three parts. The first part is the aptamer-linker with a
linker which has 23nt. The second part is the inhibitor used to mask the
aptamer and linker. The third part is the stem-loop structure containing
the primer, named H1. Among them, the inhibitor can mask the aptamer and
linker in the absence of cells. When the aptamer binds to the cell, the
inhibitor will be competed off to expose linker. As for the H1, the 5 'end
of the stem-loop sequence is the complementary linker. In order to make
the linker in the first part open the stem-loop structure, we designed
that the Gibbs free energy of the complementary pairing between Linker and
stem-loop sequence is less than that of the stem-loop structure. The 3
'end of the stem-loop is the primer for rolling circular amplification.
Figure 7. The inhibitor shedding.
Figure 8. The sequence of aptamer-linker, inhibitor, and H1.
Figure 9. The secondary structure of H1.