Biomod/2013/Aarhus/Achievements And Future Work

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Achievements and future work

Origami

Two DNA origamis were designed: a two-layered base plate and a dome that could function as a lid. These were successfully folded and characterized using gel electrophoresis, AFM and TEM. However, connecting the two origamis was not achieved within the time frame of the BIOMOD competition.

Figure 71. The two components of the origami design. A: AFM image of the origami plate. B: TEM image of the origami dome.
Figure 71. The two components of the origami design. A: AFM image of the origami plate. B: TEM image of the origami dome.

Following the BIOMOD competition more experiments in connecting the plate to the dome will be performed, both using staple strands and the peptide lock. Furthermore, the complete structure with its peptide locks will need testing, to see if it can open in response to cleavage by MMP2, possibly by use of Förster resonance energy transfer (FRET).

Peptide lock

The synthesis of the peptide lock was attempted using a gradual buildup of DNA, but was unsuccesful. Coupling of one DNA strand of the lock to the peptide was achieved using click reactions, but the amide coupling of the amine-modified second DNA strand to the peptide was not achieved. After the BIOMOD competition, amide coupling with standard peptide reagents (HBTU, HOBt, etc.), have been planned to take place in order to obtain the desired molecule. Parallel a design using the succesfull click reaction will be investigated.

Chemical modifications

The photosensitizer was successfully synthesized in five steps from the extracted natural compound and subsequently conjugated to DNA. A cholesterol derivative with an NHS-ester handle was synthetized and subsequently coupled to amine modified DNA strands. Furthermore, to achieve a more direct way of labeling DNA with cholesterols, work will be continued in optimizing the triphosphate synthesis and the following purification. This can be used to synthesize a cholesterol modified nucleoside triphosphate which can be used in a cholesterol labeling-kit with TdT.

sisiRNA and CPPs

PEG polymers of two different sizes as well as the cell penetrating peptides GALA and melittin were conjugated to the 5’ ends of both segments of the sisiRNA passenger strand. Cells were transfected with all the different, singly modified duplexes and the activities were compared unmodified sisiRNA or siRNA. In these experiments, it was demonstrated that conjugating these large molecules to the sisiRNA generally did not decrease the activity of the constructs. A doubly modified construct with two melittins conjugated to the passenger strand of the sisiRNA was made, and this was tested in high concentrations without a transfection agent. From these experiments it was unclear if the CPPs enabled the sisiRNAs to cross the cell membrane, and it appeared that some component of the assay stimulated an increased expression of the luciferase protein. Experiments to investigate the cause of this effect have been planned after the end of the BIOMOD competition.

System in action

The ability of the photosensitizers to induce apoptosis in cells was investigated and it was shown that photosensitizer-DNA alone could not kill cells, but was functional in complex with a cholesterol-DNA. Future experiments will include expanding the cell analyses to anneal the photosensitizer-DNA conjugate into the origami plate and investigate the ability of this system to induce apoptosis. Cholesterol- and photosensitizer modified DNA strands were successfully incorporated into an origami plate. Cells were treated with the cholesterol-modified plates and visualized using confocal microscopy. As no origamis were visible in these images due to low concentrations of the sample, this experiment will be repeated. For the plate with attached sisiRNA-CPP conjugates, annealing of the constructs onto the plate was achieved with a doubly CPP-modified sisiRNA through staple strand overhangs. The plate was tested on cells, both with and without a transfection agent. It was not concluded if the CPPs enabled a cellular uptake, as the sisiRNA could not be liberated outside the cells without a functional peptide lock. However, the disulfide bridges in the sisiRNA design appeared to function as intended, and liberate the sisiRNA-CPP conjugates inside the cell, where it was shown to be functional.

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