Assembly of DNA-encapsulated lipid bilayers and their application to studies of GPCRs

Lipid bilayers and lipid-associated proteins play crucial roles in biology. As in vivo studies and manipulation are inherently difficult, membrane-mimetic systems are useful for the investigation of lipidic phases, lipid–protein interactions, membrane protein function and membrane structure in vitro. This dissertation describes a route to leverage the programmability of DNA nanotechnology to create DNA-encircled bilayers (DEBs), a novel nano-scaled membrane-mimetic system. DEBs are made of multiple copies of an alkylated oligonucleotide hybridized to a single-stranded minicircle, in which up to two alkyl chains per helical turn point to the inside of the toroidal DNA ring. When phospholipids are added, a bilayer is observed to self-assemble within the ring such that the alkyl chains of the oligonucleotides stabilize the hydrophobic rim of the bilayer to prevent formation of vesicles and support thermotropic lipid phase transitions. This straight-forward and robust route enables the rational design of DEBs so that their size, shape or functionalization can be adapted to the specific needs of biophysical investigations of lipidic phases and the properties of membrane proteins. Next, we optimized the DEB system to provide proper anchoring of a large variety of lipids by creating an improved DNA scaffold. This scaffold, called DNA double-decker, consists of two interconnected DNA minicircles stacked on top of each other. In comparison to the DNA minicircle in DEB system, this scaffold is two times thicker and contains two times more hydrophobic strands, which should increase the stability of the lipid bilayer rim. Finally, we explored the option of using DEBs in studies of GPCRs using CCR5 as a model protein. The CCR5 was labeled with DNA strands, purified and characterized. The strands on CCR5 are complementary to the protruding strands on the DNA minicircle in DEBs. This can allow the reconstitution of GPCRs inside DEBs with controlled orientation of the receptor