Dynamic Interactions between Lipid-Tethered DNA and Phospholipid Membranes

Patrick M. Arnott, Himanshu Joshi, Aleksei Aksimentiev, and Stefan Howorka
Langmuir 34(49) 15084-15092 (2018)
DOI:10.1021/acs.langmuir.8b02271  BibTex

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Lipid-anchored DNA can attach functional cargo to bilayer membranes in DNA nanotechnology, synthetic biology, and cell biology research. To optimize DNA anchoring, an understanding of DNA−membrane interactions in terms of binding strength, extent, and structural dynamics is required. Here we use experiments and molecular dynamics (MD) simulations to determine how the membrane binding of cholesterol-modified DNA depends on electrostatic and steric factors involving the lipid headgroup charge, duplexed or single-stranded DNA, and the buffer composition. The experiments distinguish between free and membrane vesicle-bound DNA and thereby reveal the surface density of anchored DNA and its binding affinity, something which had previously not been known. The Kd values range from 8.5 ± 4.9 to 466 ± 134 μM whereby negatively charged headgroups led to weak binding due to the electrostatic repulsion with respect to the negatively charged DNA. Atomistic MD simulations explain the findings and elucidate the dynamic nature of anchored DNA such as the mushroom-like conformation of single-stranded DNA hovering over the bilayer surface in contrast to a straight-up conformation of double-stranded DNA. The biophysical insight into the binding strength to membranes as well as the molecular accessibility of DNA for hybridization to molecular cargo is expected to facilitate the  creation of biomimetic DNA versions of natural membrane nanopores and cytoskeletons for research and nanobiotechnology.

Abstract

Lipid-anchored DNA can attach functional cargo to bilayer membranes in DNA nanotechnology, synthetic biology, and cell biology research. To optimize DNA anchoring, an understanding of DNA−membrane interactions in terms of binding strength, extent, and structural dynamics is required. Here we use experiments and molecular dynamics (MD) simulations to determine how the membrane binding of cholesterol-modified DNA depends on electrostatic and steric factors involving the lipid headgroup charge, duplexed or single-stranded DNA, and the buffer composition. The experiments distinguish between free and membrane vesicle-bound DNA and thereby reveal the surface density of anchored DNA and its binding affinity, something which had previously not been known. The Kd values range from 8.5 ± 4.9 to 466 ± 134 μM whereby negatively charged headgroups led to weak binding due to the electrostatic repulsion with respect to the negatively charged DNA. Atomistic MD simulations explain the findings and elucidate the dynamic nature of anchored DNA such as the mushroom-like conformation of single-stranded DNA hovering over the bilayer surface in contrast to a straight-up conformation of double-stranded DNA. The biophysical insight into the binding strength to membranes as well as the molecular accessibility of DNA for hybridization to molecular cargo is expected to facilitate the  creation of biomimetic DNA versions of natural membrane nanopores and cytoskeletons for research and nanobiotechnology.

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All-atom molecular dynamics simulation of dsDNA molecules cholesterol-anchored to a POPE lipid bilayer membrane. The movie illustrates side view (left) and top view (right) of the system in a 300 ns equilibration trajectory. The DNA backbone (green) and bases (red) are shown using a cartoon representation. Each cholesterol molecule (red vdW spheres) is attached to the DNA backbone via a triethylene glycol linker (blue lines). Lipid molecules are shown as gray lines with the phosphorous atom of each headgroup highlighted as a vdW sphere. The lines define the simulation unit cell. For clarity, water, ions and DNA from the neighboring periodic cells are not shown.

All-atom molecular dynamics simulation of dsDNA molecules cholesterol-anchored to a lipid membrane composed of a 50/50 mixture of POPE and POPG lipids. The movie illustrates side view (left) and top view (right) of the system in a 300 ns equilibration trajectory. The DNA backbone (green) and bases (red) are shown using a cartoon representation. Each cholesterol molecule (red vdW spheres) is attached to the DNA backbone via a triethylene glycol linker (blue lines). Lipid molecules are shown as gray lines with the phosphorous atom of each PE and PG headgroup highlighted as a gray or orange vdW sphere, respectively. For clarity, water, ions and DNA from the neighbouring periodic cells are not shown.

All-atom molecular dynamics simulation of dsDNA molecules cholesterol-anchored to a lipid membrane composed of a 50/50 mixture of POPE and POPC lipids. The movie illustrates side view (left) and top view (right) of the system in a 300 ns equilibration trajectory. The DNA backbone (green) and bases (red) are shown using a cartoon representation. Each cholesterol molecule (red vdW spheres) is attached to the DNA backbone via a triethylene glycol linker (blue lines). Lipid molecules are shown as gray lines with the phosphorous atom of each PE and PC headgroup highlighted as a gray or green vdW sphere, respectively. For clarity, water, ions and DNA from the neighboring periodic cells are not shown.

All-atom molecular dynamics simulation of ssDNA molecules cholesterol-anchored to a POPE lipid bilayer membrane. The movie illustrates side view (left) and top view (right) of the system in a 300 ns equilibration trajectory. The DNA backbone (green) and bases (red) are shown using a cartoon representation. Each cholesterol molecule (red vdW spheres) is attached to the DNA backbone via a triethylene glycol linker (blue lines). Lipid molecules are shown as gray lines with the phosphorous atom of each headgroup highlighted as a vdW sphere. For clarity, water, ions and DNA from the neighboring periodic cells are not shown.

All-atom molecular dynamics simulation of ssDNA molecules cholesterol-anchored to a lipid membrane composed of a 50/50 mixture of POPE and POPG lipids. The movie illustrates side view (left) and top view (right) of the system in a 300 ns equilibration trajectory. The DNA backbone (green) and bases (red) are shown using a cartoon representation. Each cholesterol molecule (red vdW spheres) is attached to the DNA backbone via a triethylene glycol linker (blue lines). Lipid molecules are shown as gray lines with the phosphorous atom of each PE and PG headgroup highlighted as a gray or orange vdW sphere, respectively. For clarity, water, ions and DNA from the neighboring periodic cells are not shown.

All-atom molecular dynamics simulation of ssDNA molecules cholesterol-anchored to a lipid membrane composed of a 50/50 mixture of POPE and POPC lipids. The movie illustrates side view (left) and top view (right) of the system in a 300 ns equilibration trajectory. The DNA backbone (green) and bases (red) are shown using a cartoon representation. Each cholesterol molecule (red vdW spheres) is attached to the DNA backbone via a triethylene glycol linker (blue lines). Lipid molecules are shown as gray lines with the phosphorous atom of each PE and PC headgroup highlighted as a gray or green vdW sphere, respectively. For clarity, water, ions and DNA from the neighboring periodic cells are not shown.