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WP1 - DNA Nanocontainers

Trisoligonucleotides are branched oligonucleotides, consistent of 3 oligonucleotide strands with 15 nucleobases each, which are covalently linked with a C3h symmetric molecule. We have demonstrated the spontaneous self assembly of 20 trisoligonucleotides to form a dodecahedral DNA structure.To assess the potential of the dodecahedron as a scaffolding device, we assembled dodecahedra with one of six trisoligonucleotides derivatized with a DNA overhang sequence. This overhang could then be hybridized with hybrid molecules composed of a complementary sequence tag and modular function, for example, gold nanoparticles [1]. In the same way we could generate tetrahedral nanoconstructs this time by self-assembling 4 trisoligonucleotides. The DNA nanocontainers are therewith uniquely defined chemtainer constructs, capable to  address nano cargo sequence specifically via its overhang sequences.

Control uptake / release of nanoscale freight packages by physical signals

For the construction of DNA based nanoobjects, we found the necessity to optimize the synthesis- and purification-techniques for the trisoligonucleotide building blocks. Hence, the synthesis protocol for the phosphoramidite synthesis was optimized and a fluorous affinity purification method of trisoligonucleotides was developed. The trisoligonucleotides can be modified with fluorous tags as overhangs on two arms, designed as permanent or removable modifications, which allow the purification via fluorous-HPLC (Figure 1.1).

Figure 1.1: Fluorous-tag modifications in trisoligonucleotides, introduced as phosphoramidites on the 2nd and 3rd arm during DNA synthesis; three different phosphoramidites are used for functionalization and/or purification.

Furthermore, the introduction of fluorous-overhang sequences in trisoligonucleotides enabled us to create a fluorophilic cavity inside the tetrahedral DNA nanocontainer. We intended to cage fluorous nanoobjects and demonstrated the encapsulation of fluorous modified gold nanoobjects by TEM. With the fluorous technology we addressed the objective 1 concerning the nanoscale freight packages and partly its physically controlled uptake and release (objective 2).

Open and close dodecahedra by chemical signals

This objective was addressed by a strand displacement strategy. The chemical signal for the opening and closing of the dodecahedral or tetrahedral nanocontainer is therewith a counterstrand, which selectively addresses one trisoligonucleotide strand and its overhang sequence. We could demonstrate that the strand displacement can be used to separate trisoligonucleotides and to open a nanotetrahedron by the liberation of one trisoligonucleotide (Figure 1.2).


 

 

 

 

 

 

 

 

 

 

Figure 1.2: strand displacement experiments for trisoligonucleotides and a DNA-tetrahedron.

 

The strand displacement experiments were performed with fluorescent labelled trisoligonucleotides and analysed by agarose gel electrophoresis. This sequence dependent opening and closing strategy enables us to selectively address each trisoligonucleotide corner in the nanocontainer. In principle, we can use strand displacement events to selectively change addresses in the nanoconstruct by substitution of trisoligonucleotides and change of overhang sequences. These results demonstrate that we have reached the research goals stated under O.1.3.

Nanoscale synthesis inside containers

We have previously described the uptake of fluorous-modified gold nanoparticles by fluorous-labelled tetrahedral nanoconstructs. Although this method proved suitable by TEM studies, we decided to go for a more specific, sequence based recognition of nanoparticles. In order to achieve this goal, we have developed a novel phosphoramidite 6 (see Figure 1.3), with a high affinity towards gold particles. The synthesis of this novel phosphoramidite is sketched in Figure 1.3.

Figure 1.3: Synthesis of ligand amidite 6; We could successively incorporate phosphoramidite 6 in oligonucleotides via standard synthesis protocol. During cleavage from the solid support, the ester groups are saponified yielding a highly water soluble nona-carboxylic acid.

We have shown that reduction of gold-III precursors in the presence of ligand modified oligonucleotides (which we call nucleator strand) leads to the selective labelling of such strands with gold nano particles, which could be proved by PAGE gel electrophoresis.

It is expected that such ligand- or already gold-modified oligonucleotides can hybridize to overhang sequences of nanoconstructs. Fig. 1.4 shows the rationale of this approach, exemplified by the tetrahedral structure, we have described before.

Figure 1.4: Hybridisation of a nucleator strand inside tetrahedral chemtainer

Four overhang sequences, on the four corners of the object, are attracted together on the inside of the cavity, due to their fluorous modification on the 5’ end. One nucleator strand binds selectively and sequence specific to its complimentary overhang, thus locating the ligand (and therefore the point of nucleation) in the center of the cavity. Treatment with gold precursor (HAuCl4) and reducing agent Na(OAc)3BH­ results in nucleation and growth inside the cavity.

In WP1 we have evolved trisoligonucleotide based nanoconstructs towards selectively addressable chemical containers. We have developed a multitude of trisoligonucleotide functionalizations like fluorous-tags, photocleavable linkers, intercalating photoswitchable dyes and gold-ligands and introduced them to the DNA nanostructures. We have demonstrated the uptake of fluorous modified gold-nanoparticles and a controlled opening of a nanotetrahedron via strand displacement. For a directed freight uptake and for the nanoscale syntheses of gold-nanoparticles inside the container we also synthesised a DNA-gold-ligand, capable of binding to one overhang sequence inside the nano container. Therewith we have developed a multitude of tools leading to controlled uptake and release of cargo in the DNA nanocontainers. However we have not been able to synthesize a working system for demonstration combining all the tools in one, but we are very confident to make the system operational soon.

Collaborations

In collaboration with WP3 a transfer of synthetic knowledge led to a disulfide based DNA -lipid anchoring method. The collaboration with WP6 provided us with a wonderful tool, useful to illustrate and understand self constitution of complex architectures.

References

[1] Zimmermann, J.; Cebulla, M. P. J.; Monninghoff, S.; von Kiedrowski, G., Angew. Chem. Int. Ed. 2008, 47, 3626-3630.
[2] Gupta, A.; Will, S., U.S. Patent US 2008/0161548 A1, Jul 3, 2008.
[3] C. Beller, W. Bannwarth, Helv. Chim. Acta 2005, 88, 171.