Controlling aggregation of cholesterol-modified DNA nanostructures

Abstract

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.

Binding of DNA origami to lipids: maximising yield and switching via strand-displacement

“…The addition of a 6 nt overhang on cholesterol-tagged DNA strands has been postulated to assist during nanostructure assembly by inhibiting strand aggregation (Ohmann et al, 2019). We included a 6 nt overhang next to the cholesterol group on our dsDNA strand (dsDNA-6 nt) and observed a significant decrease in binding only on DPhPC liposomes.…”

“…The oligonucleotide sequences were generated using NUPACK design software (Zadeh et al, 2011) and selected to prevent the formation of unwanted secondary structures. The 6 nt 'overhang' sequence introduced at the 5' end of oligos was chosen from Ohmann, et al, 2019. Oligos were modified at the 3' end with a tetraethylene glycol cholesterol moiety (TEG-cholesterol) and with a 5' Alexa 647 fluorescent group respectively.…”

“…We examined the effects of pH, ion concentration and membrane cholesterol content on the binding of cholesterol-modified DNA strands to liposomes. We investigated three types of DNA motif: a single stranded form, a duplex, and a duplex with a short ssDNA 'overhang' proximal to the cholesterol group, recently proposed by Ohmann et al to reduce aggregation during nanostructure assembly (Ohmann et al, 2019).…”

Nucleic Acid Hybrids as Advanced Antibacterial Nanocarriers

“…This often eliminates structural control, which is the primary advantage of DNA nanotechnology. Addressing the issue of aggregation with cholesterol modification, Ohmann and coworkers recently showed that aggregation of ssDNA was sequence-dependent, whereas, in the self-assembled constructs, the position of the cholesterol modification was the dominant factor [63]. More importantly, the authors outlined the significant influence of particular bases (AAA bases had no aggregation) preceding the cholesterol modification and overhangs (six nucleotide overhangs on the complementary sequence) in suppressing aggregation.…”

“…Cholesterol functionalization remains one of the most popular ways to anchor nucleic acid sequences with hydrophobic groups. These amphiphiles have the capacity to self-assemble into predictable morphologies (e.g., spherical micelles and vesicles) due to their tendency to microphase separate; these effects are dependent on the number of hydrophobic ligands, the type of residue and its position [ 61 , 62 , 63 ].…”