Controlling aggregation of cholesterol-modified DNA nanostructures.

DNA nanotechnology allows for the design of programmable DNA-built nanodevices which controllably interact with biological membranes and even mimic the function of natural membrane proteins. Hydrophobic modifications, covalently linked to the DNA, are essential for targeted interfacing of DNA nanostructures with lipid membranes. However, these hydrophobic tags typically induce undesired aggregation eliminating structural control, the primary advantage of DNA nanotechnology. Here, we study the aggregation of cholesterol-modified DNA nanostructures using a combined approach of non-denaturing polyacrylamide gel electrophoresis, dynamic light scattering, confocal microscopy and atomistic molecular dynamics simulations. We show that the aggregation of cholesterol-tagged ssDNA is sequence-dependent, while for assembled DNA constructs, the number and position of the cholesterol tags are the dominating factors. Molecular dynamics simulations of cholesterol-modified ssDNA reveal that the nucleotides wrap around the hydrophobic moiety, shielding it from the environment. Utilizing this behavior, we demonstrate experimentally that the aggregation of cholesterol-modified DNA nanostructures can be controlled by the length of ssDNA overhangs positioned adjacent to the cholesterol. Our easy-to-implement method for tuning cholesterol-mediated aggregation allows for increased control and a closer structure-function relationship of membrane-interfacing DNA constructs - a fundamental prerequisite for employing DNA nanodevices in research and biomedicine.This work was supported by: European Research Council (ERC) consolidator grant [DesignerPores 647144 to U.F.K.]. Winton Programme for the Physics of Sustainability; Gates Cambridge; Oppenheimer Ph.D. studentship; and the European Union's Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grant agreement No 792270 [to K.G.]. Engineering and Physical Sciences Research Council (EPSRC); and the Cambridge Trust Vice Chancellor’s Award [to A.O.]. National Science Foundation (USA) [DMR-1827346]; National Institutes of Health [P41-GM104601]; the supercomputer time provided through XSEDE allocation grant [MCA05S028]; and the Blue Waters petascale supercomputer system (UIUC) [to A.A. and H.J.]. Winton Programme for the Physics of Sustainability; and Engineering and Physical Sciences Research Council (EPSRC) [to D.S.]. The FEI Tecnai G2-Spirit was funded by the Wellcome Trust [090932/Z/09/Z]. Funding for open access charge: ERC [DesignerPores 647144]