Project goals and motivation
Designing 100-nm scale structures with DNA origami is a rational approach with a very high success rate. However, at a smaller scale DNA falls short on structural plasticity and chemical versatility, features which in nature are provided by proteins. Hence it is very desirable to combine DNA structuring with functional protein units. Consequently, the first part of our project is devoted to two ideas on how to combine DNA origami with proteins such that the DNA origami 'carries' the protein.
Reading on DNA origami, we realized that several groups propose to use DNA structures to encapsulate other molecules small and large in order to deliver them to cells for pharmacological purposes. Again, nature has already developed powerful mechanisms to do so. Viruses are versatile nano-structures with a proven track record to deliver DNA or RNA to cells from unicellular organisms to humans. Compared to our above mentioned idea of proteins on DNA, we found this conceptually intriguing, because a virus is protein nano-structure which 'carries' a DNA. One should also not forget that DNA origami are based on a viral DNA (M13 phage) which in nature is delivered by proteins. Therefore, the second part of our project is devoted to the idea to attach proteins to a viral nano-structure.
The two parts of our project (part I: protein on DNA nano-structure; part II: protein on viral nano-structure) are linked by our attempt to use the same coupling principles. Our idea was to use peptide ligation to attach a protein either to a small peptide, which is azide modified for additional click chemistry, or directly to the virus shell. Conceptually, a peptide adapter can also be ligated to the virus capsid enabling the chemical coupling of the virus to DNA. We envisioned to use the enzyme sortase for peptide ligation, which mediates the linkage of a C-terminal amino-acid tag with the sequence ‘LPXTG’ to the N-terminal amino-acid tag G5 (penta Gly). The azide modified peptide is coupled to an alkyne modified DNA by a 'click' reaction (Huisgen cyclization). The respective oligonucleotide can be hybridized to our origami structures.
For the ‘protein on DNA nano structure’ part we also used chemically modified oligonucleotides which are recognized by monoclonal antibodies. We wanted to test how this can be used to generate two dimensional DNA origami patterns and to train our DNA origami and AFM skills. We used triangular origami presenting the modification either at the corners or at the center of the outer legs.
Project achievement overview
- We generated triangular DNA origami based on Rothemund’s design. We modified these DNA origami with A- and T-tail oligonucleotide pairs, which added biotin to the corner or the middle of the structure. Using a bivalent anti-biotin IgG antibody, we obtained two dimensional DNA origami patterns.
- We designed an alkyne modified oligonucleotide and an azide modified penta-Gly peptide and tested their cycloaddition.
- We cloned and produced in HEK 293 cells a recombinant AAV virus with a penta-Gly motif at the N-terminus of a capsid protein. Using this virus particle to deliver a gene coding for a cyan fluorescent protein, we demonstrated that the modified rAAV can still infect a human cell line.
- We cloned and expressed in E. coli a protein with an N-terminal LPXTG motif.