Inspired by immune system, our goal is to fabricate pore forming DNA nanostructure killing the cancer cell (termed Oligomeric Cell killer: OCK). The designed structure is shown below. Our design is characterized with four features: a broad plane-like domain to anchor on the cell surface, stick-domain to invade into the cell membrane, safety lock system for cell recognition, and connectable sites for oligomerization and pore formation.
Anchoring and penetration
OCKs anchor and penetrate lipid bilayer by their cholesterols.
-->Cholesterol sticking site, structure
Recognition (safety lock system)
The DNA aptamer of OCK recognizes cancer cell specific membrane protein and release the biotin-modified strand locked by the DNA aptamer.
-->Aptamer lock system
The activated biotins bind to the streptavidins of other monomers, resulting in the trimerization of OCKs
-->Trimerization by streptavidin-biotin complex
The physical constraint induced by oligomerization activates the azides and alkynes reactive groups on OCK, covalently bonding the stick-domain of each subunits of trimeric OCKs by click chemistry. Lipids are excluded from the gap made by stick-domain of OCKs. As a result, a part of membrane is punched, and pore formation is achieved
The detailed structure and scale of OCK are shown below. OCK is composed of two main domains. The plane-like domain (red) sticks to lipid bilayer, and the stick-domain (blue) penetrates into the lipid bilayer.
Recognizing specific trigger signal, OCK can trimerize, making a small hole (inner and outer diameter is 4 nm and 12 nm, respectively) to punch the cell.
OCK is equipped with functional sites for penetration and oligomerization.
1)Cholesterol sticking sites
It requires enough free energy to penetrate membranes. This is compensated by the free energy gained from biding of the DNA nanostructure bound cholesterols to the lipid bilayer. The more cholesterols are equipped to the structure, the more stabilized it stays near the membrane. We attached cholesterol to our OCK by two methods; hybridization and direct incorporation. Hybridization method is accomplished by hybridizing the scaffold strand and cholesterol-modified ssDNA strands. Regarding direct incorporation method, we supplemented cholesterol modified staples at the formation of OCK. For both methods, we design four cholesterol sites shown below.
2)Plane-domain and stick-domain
To ensure the penetration of DNA nanostructure (six double-helical DNA domains. diameter of 6 nm), previous research used 26 cholesterol binding sites (Martin Langecker, et al.) However, too much sites may cause the heterogeneity of the sample and lower yields. To resolve this points, we paid attention to character of DNA : non-specific binding to the liposome (Danilo D. Lasic, et al.), which we confirmed using Rectangle type DNA origami and POPC (see \Experiment\\Pilot study section). Although the non-specific binding is usually an undesirable feature, we thought that we could utilize this feature positively by means of the broad plane- domain. Integrating broad plane into our DNA nanostructure, we expect some free energy gain by the non-specific binding of the broad plane to the bilayer, which may stabilize the binding of our DNA structure to the bilayer. Combining the assistant of cholesterols and plane-domain, we thought that it is easy for the stick domain to penetrate into membrane.
The key feature of OCK is keeping monomeric state in solution and on normal cell, while oligomerization on cancer cell. To ensure this feature, recognition system (safety lock system) is required to prevent the non-specific oligomerization, while having ability to oligomerization.
As a recognition system, we use DNA aptamer. In general, DNA aptamer recognize specific ligand. And if some part of DNA aptamer is hybridized with complementary strand, the complementary strand is released from the DNA aptamer on the binding of ligands, because ligands take over the DNA strands of DNA aptamer from the complementally strands. We used this scheme as followings. Shown in the figure, the biotin modified aptamer complementary strands are adhering to OCK, therefore the streptavidins cannot access to the biotin. On recognition of cancer specific membrane protein, biotin modified strands are released and can bind to streptavidin. Therefore, the cancer cell recognition and OCK oligomerization are achieved simultaneously.
1)Necessity of precise control
When OCKs recognize cancer cell on the membrane, they are oligomerized (get trimer) and make pore. To achieve these steps, we designed the structure of prototype, which was simpler than the current design.
We choose biotin-streptavidin interaction as the oligomerization method, as the preliminary experiments using barrel structure (see \Experiment\\Pilot study section) showed that the hybridization method was not a suitable way to oligomerization because of the difficulty to prevent the non-specific interaction between monomers. We designed our prototype OCK as shown above, which has tow biotins on the left and right sides. However this simple design produce not only the desired trimer structure, but also the undesired oligomer shown below.
As a solution, we introduce the asymmetry into OCK, because symmetry of prototype produces the alternative form of oligomer. To do this, we built in biotin in one side (with recognition system to control the oligomerization), and streptavidin in other side. However, we found that simply attaching streptavidin in one side is not enough for our OCK, especially in the oligomerization process of OCK (see next section). We solved this problem by introducing a well in OCK and embedding the streptavidin in the well.
Precise arrangement of OCK subunit is necessary for proper oligomerization, especially for the tight connection of stick-domain, as the size of lipids are much small (-1 nm) compare to the size of OCK (-100nm). In this point of view, the flexibility of the position of streptavidin makes the situation difficult. To solve this problem easily, DNA well is equipped to OCK and the streptavidins were stored.
Thus far, the devices for regioselective oligomerization have been explained. However, there still remains a task; exclusion of lipids out from the gab made by trimer of OCKs. To do this, the linkers connecting each OCK subunits must be short enough to ensure the inter-subunit adhesion of OCKs's stick- domain. In that view of point, streptavidin is not suitable glue, because the diameter of streptavidins is approximately 5 nm. On the other hand, 1, 2, 3 - triazole (the product of Click chemistry) is adequate, because the scale of the product is in the sub- to a few nanometer range (In this section, click chemistry means the Huisgen cycloaddition reaction between alkyne and azide). Therefore, OCKs are equipped with alkyne and azide reactive groups.
Usually, this click reaction demands copper as catalyst. However, copper does not exist in human body. Therefore, copper free click chemistry was studied in this project. As explained in result, the reaction rate of copper free click chemistry is very slow in solution (association rate constant ~ 8.1 /M/s = 17h@2uM reagents), but is accelerated when the reaction groups (azide and alkyne) are forced to be close. In other words, azide and alkyne reactive groups do not react each other in solution, but easy to react each other after oligomerization. This character is very suitable to prevent non-specific oligomerization, while accelerating the specific oligomerization.
To detect whether click reaction happened, OCKs have two fluorescent dyes, Cy3 and Cy5. These fluorescent dyes are adhere to the azide-modified site and the alkyne-modified site respectively (dye - dye distance is roughly 1 - 2 nm), so FRET signal between Cy3 and Cy5 is observed if click reaction occurred.