Controlling aggregation of cholesterol-modified DNA nanostructures

Alexander Ohmann, Kerstin Göpfrich, Himanshu Joshi, Rebecca F. Thompson, Diana Sobota, Neil A. Ranson, Aleksei Aksimentiev, and Ulrich F. Keyser
Nucleic Acid Research 47(21) 11441-11451 (2019)
DOI:https://doi.org/10.1093/nar/gkz914  BibTex

Highlight

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. 

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.

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Movie S1.  All-atom MD simulation of cholesterol-modified ssDNA. The movie illustrates the 0.5 µs MD trajectory of a 30 thymidine (30T) ssDNA strand. Two independent simulation runs starting from the same initial configuration are shown (Run 1 and Run 2). The ssDNA wraps around the cholesterol group throughout both simulation runs. Water and ions are not shown for clarity.

Movie S2. All-atom MD simulation of a cholesterol-modified DNA duplex without overhang. The movie illustrates the 0.5 µs MD trajectory of a DNA duplex with one cholesterol tag but without an adjacent ssDNA overhang. Two independent simulation runs starting from the same initial configuration are shown (Run 1 and Run 2). Water and ions are not shown for clarity.

Movie S3. All-atom MD simulation of a cholesterol-modified DNA duplex with a 6 nt overhang. The movie illustrates the 0.5 µs MD trajectory of a DNA duplex with one cholesterol tag and an adjacent 6 nt overhang. Two independent simulation runs starting from the same initial configuration are shown (Run 1 and Run 2). Water and ions are not shown for clarity.