Figure 1: Cross section of the DNA Origami StructureThe first challenge was to design an origami structure to meet the requirements for the project idea. For this purpose a structure based on William Shih’s O-Brick design was created. Figure 1 displays the cross section of the design. Based on the p7560 scaffold, the structure consists of three layers and leads to a cylinder with three inner rings. The structure's diameter measures 47 nm for the outer ring, 42 nm for the middle one and 39 nm for the inner ring.
With these three layers an even distribution of targeted insertions and deletions, which are responsible for the desired curvature, is possible.
Initially, the structure of the design was verified with TEM measurements, after folding along a 24h tempverature ramp and subsequent isolation in 2% agarose gel.
The optimal MgCl2 concentration for the folding process was determined to be 14mM (figure 2). After gel purification of the sample all bands were tested with TEM. At a MgCl2 concentration of 10 and 12 mM the origamis did show open ends and unfinished folding. At higher MgCl2 concentration stronger aggregation was observed. Therefore a MgCl2 of 14 mM was chosen for subsequent experiments for its high product to aggregation ratio.
Figure 2: Screening for the ideal Magnesium concentration. a) 2 % agarose gel after staining with EtBr. Lane (1): 1 kb DNA-Ladder, lane (2) empty, lane (3) scaffold, lane (4)-(9) folded DNA origami in TAE with increasing Mg2+ concentration from 10mM to 20 mM. b) TEM of the folded ring. The sample was taken out of the lower band at ca. 1.5 kb form lane 6. All other bands where imaged with TEM. The bands in lane 4 and 5 not correctly folded.
In the next step several approaches were tested in order to improve the folding yield. This included varying the mutual concentration of selected staple groups and changing the temperature ramp. Finally the output was increased through pre-assembly without the functionalized staple-strands and subsequent addition of the functionalized staples at room temperature (figure 3).
Figure 3: 2 % agarose gel of the yield optimization by subsequent addition of functionalized staples after EtBr staining. The depicted lanes contain the following samples: 1kb DNA Ladder (NEB), Scaffold (untreated), DNA origami with all staples added in the beginning of the folding process and folded with the temperature ramp opti65 (s. Labbook) (Standard), DNA origami folded after the same protocol but here the functional sugar staples where added after the folding process at room temperature (Z RT), DNA origami folded after the same protocol but here the functional fluorophore staples where added after the folding process at room temperature (F RT), DNA origami where bothe sets of functional staples where added after folding at room temperature (B RT).
When comparing the probe folded as before (Standard) and (B RT) the bands containing the folded origami in lane (B RT) are clearly more intense. One can also see that the amount of aggregation is drastically reduced under these conditions (material remaining in the gel pocket).
Functionalizing the origami
Carbohydrates were added onto the staples using click chemistry. Differences between modified and unmodified oligonucleotides were visualized on 20% native PAGE (figure 5).
Figure 4: 20% native PAGE gel of azide motived oligo nucleotides with and without sugar modification after Sybr Gold staining. The depicted lanes contain the following samples: Low MW DNA Ladder (NEB), azide modified oligoes without sugar modification in a 1:100 dilution (Control 1:100), azied modified oligoes with sugar modification in a 1:100 dilution (DNA 1:100).
For a preliminary test of the click reaction a modified DNA sequence and the carbohydrate 2-Azidoethyl-α-Mannopyranoside were used. A lower mobility of the clicked product in regard to the unclicked oligos would verify that the click reaction was successful and the sugar had attached to the DNA.
Lane (2) of figure 5 was a control sample containing only azide modified DNA. On the other hand the same oligonucleotide clicked with the carbohydrate was loaded into lane (3). The control sample is visible at a height lower than 50 bases as marked by the ladder whereas the molecules gathered in lane (3) can be seen between 50 and 75 bases. Moreover no unclicked oligonucleotides were visible in lane (3). Therefore quantitative transfer of the sugar to the oligonucleotide using click chemistry could be confirmed.
Furthermore fluorophores were covalently bound to 3’ Thymine using an enzymatic method (figure 6). In order to verify the incorporation of fluorophore staples into the structure, a 2% agarose gel was prepared. The gel was loaded with origami structures with fluorophore and no carbohydrate modification.
Figure 5: 2 % agarose gel of with origami with functionalized staples with Atto 488 attached before and after staining with EtBr under UV and laser light. The folding process was performed as described at yield optimization. Staples where factionalized enzymatically with Terminal Transferase (Roche) and Aminoallyl-dUTP –Atto488 and added in different excesses. The first three lanes show the gel before staining under laser light. Lanes 4-6 show the same gel under UV-light after staining with EtBr.
Several different excesses of fluorophore staples were tested: 10 times, 5 times and 3 times. The gel was first visualized at the excitation wavelength of the used fluorophore and then stained with the intercalating dye Etbr. After staining the gel was again visualized under UV-light to excite the Etbr. The same bands were observed in both pictures (figure 3). To eliminate the possibility of spectral crosstalk the unstained gel was also imaged with UV light. This verified that the fluorophores had incorporated into the structure properly. As can be seen in lanes (1-3) the flurophores incorporated better at a higher excesses. Therefore all subsequent experiments where preformed with at least a 10x execxes of all staples.
The next step was to provide a proof of concept for our idea by applying the completed origami structure onto cells. Therefore epifluorescence measurements with human basal epithelial cell line A549 and the origami structure performed.
Figure 7 depicts three overview scans taken by an epifluorescence microscope (Nikon) using a 100x objective. The origami structures were equipped with the fluorescence dye Cy5 with an excitation wavelength at 670 nm.
Figure 6: Epiflurecents overview scans at a magnification of 100x. a) Origami without sugar coating on A549 cells b) Origami coated with SLex on A549 cells c) Origamis coated with Tri-LN on A549 cells
Figure 6 a) shows the results of the excitation of the control sample, which contained origami structures without carbohydrate coating applied to cells.
Figure 6 b) is an overview scan of SLex coated origami structures with cells whereas figure 6 c) corresponds to Tri-LN coated origami structures and cells.
The fluorescence signal’s intensity was evaluated by measuring 6 proportionate areas within the corresponding scans (figure 7). This data does not show any difference in the fluorescence intensity between the negative sample and the SLex coated origami structure.
On the other hand that A549 cells exhibit a higher affinity to Tri-LN.
Figure 7: Comprising of the fluorescence intensity of the three probes. The fluorescence signal’s intensity was evaluated by measuring 6 proportionate areas within the corresponding scans. a) shows the a random area of the overview scan of the negative control. b) shows the a random area of the overview scan of the origami coated with SLex. c) shows the a random area of the overview scan of the origami coated with Tri-LN. d) shows a diagram of the measured intensities of all three probes.
In order to verify the binding of the origami structure on the cell membrane, the membrane was additionally stained CellMask Orange. Thereafter the experiment was repeated as described above. The following figure 8 shows a superposition of three images taken by epifluorescence microscope in different modes.
Figure 8: Superposition and single pictures of tow fluorescent images at different expiation wave length and a bright field image of A549 cells incubated with origami coated with Tri-LN. a) composite of the pictures b)-d). b) single picture taken with epifluorescence microscope at a the expiation wavelength of cell mask orange (500 nm, bandwidth 20 nm)a membrane stain. b) same spot at the expiation wavelength of Cy5 (628 nm bandwidth 40 nm), the dye attached to the origami structure. d) bright field image of the same spot. All pictures where taken with a Nikon Ti-E 531511 at a magnification of 100x.
Conclusion and outlook
By using the DNA origami technique it was possible to generate a structure on the nanometer scale that acts as a carrier for carbohydrates, taking into account special demands it faces arising from the aspirated cell applications.
We were able to successfully functionalize this nanocarrier with carbohydrates and fluorophores and subsequently completed the first experiments concerning glycan-cell interactions.
Based on our results and motivation there are many prospects both on the design and application side.
First of all, the design and the folding process can be improved to generate a higher yield. Higher concentration of origami structures will also ease the use of statistical methods like FACS to give a clear answer to the binding of the origami structure onto cells. Another major objective is to reveal positions and movement of carbohydrate receptors on the cell membrane. The focus can also be pointed at analyzing selected cell types concerning their binding affinity towards the differently sugar-coated origami structures.
Despite these outstanding issues, these results already reveal the high potential of the molecular design to answer central questions of biochemistry and medicine.
As the whole cell communication apparatus is based on the specific glycan-cell interaction the tool developed and investigated in this project is highly interesting to basic research and provides important knowledge for the development of novel therapeutic methods for a wide range of diseases such as diabetes, cancer and viral infections.