We have designed and constructed a molecular scaffold made of DNA that can template both the number and relative order of protein subunits.
Our design consists of twelve short single-stranded pieces of DNA that come together into a small ring with twelve arms onto which proteins can be attached (Fig. 1). This design has two key advantages over alternative designs. It is more flexible than the larger DNA origami structures based on helix bundles making it much easier to bring together two proteins attached at different binding sites without the need for long and floppy linker molecules. In addition, it does not require an excess amount of the short, protein-modified DNA molecules, of which the majority is incorporated into the scaffold and which may have adverse effects in applications. We used simulations to verify that our designed scaffold is sufficiently flexible to bring all arms together. In experiments, we have shown that the scaffold can be formed with more than 85% overall yield (based on all DNA molecules), that it is indeed a circle with twelve parts, as designed, and that it has the correct overall mass. Moreover, we have shown that our design can easily be extended to 14, 16 or 18 functional sites.
In the second phase of this project we attached various pore-forming proteins and peptides to our DNA scaffold. In the approach we have taken here (Fig. 2), all building blocks are first modified separately with the selected peptide through either a permanent or a cleavable linker, and then purified and analysed. This ensures that we can actually scaffolds large numbers of proteins or peptides despite the moderate yield of the reaction between DNA and protein. We have shown that our purified peptide-modified DNA can scaffold any number of peptides up to a maximum of twelve (for the smallest scaffold we made) with a high yield.
We then studied the nanopores that were formed by the templated peptides and the effect of the DNA scaffold on these pores. Using electrical recording of a planar lipid bilayer, in which a single nanopore can be probed at a time, we established that our DNA scaffold can stabilise peptide nanopores that are otherwise unstable. Moreover, we have shown that the scaffold can be used as a docking site for a variety of molecules, enabling repeated detection or sequencing of an analyte at ultralow concentration, and the in situ assembly of protein complexes near a nanopore sensor. When we built scaffolds containing more peptides than required in the pores that occur in nature, we could not observe larger nanopores. Instead, the peptides usually formed the same pores as they would in their natural protein environment. Apparently, the peptides we have tested so far are not sufficiently accommodating to adapt to the template offered by our scaffold. Hopefully, alternative peptides that we plan to investigate in the future will prove more able to assemble into differently sized pores.
Our results have been disseminated within the scientific community on various occasions: several talks at seminars, posters at conference dedicated to Biointerface Science and a second conference on Physics of Life, and we are preparing a paper about our scaffolded nanopores.
