Abstract
Equipping DNA with hydrophobic anchors enables targeted interaction with lipid bilayers for applications in biophysics, cell biology, and synthetic biology. Understanding DNA–membrane interactions is crucial for rationally designing functional DNA. Here we study the interactions of hydrophobically tagged DNA with synthetic and cell membranes using a combination of experiments and atomistic molecular dynamics (MD) simulations. The DNA duplexes are rendered hydrophobic by conjugation to a terminal cholesterol anchor or by chemical synthesis of a charge-neutralized alkyl-phosphorothioate (PPT) belt. Cholesterol-DNA tethers to lipid vesicles of different lipid compositions and charges, while PPT DNA binding strongly depends on alkyl length, belt position, and headgroup charge. Divalent cations in the buffer can also influence binding. Our MD simulations directly reveal the complex structure and energetics of PPT DNA within a lipid membrane, demonstrating that longer alkyl-PPT chains provide the most stable membrane anchoring but may disrupt DNA base paring in solution. When tested on cells, cholesterol-DNA is homogeneously distributed on the cell surface, while alkyl-PPT DNA accumulates in clustered structures on the plasma membrane. DNA tethered to the outside of the cell membrane is distinguished from DNA spanning the membrane by nuclease and sphingomyelinase digestion assays. The gained fundamental insight on DNA–bilayer interactions will guide the rational design of membrane-targeting nanostructures.
Highlights
The unique properties of DNA duplexes enable precise engineering of nanostructures with different shapes, geometries, and sizes.[1−3] Synthetic hydrophobic modifications expand the functional range of DNA nanostructures by facilitating specific interactions with lipid bilayers.[4−6] The modifications include conjugating DNA to hydrophobic molecules such as cholesterol[7−13] and porphyrin[14−16] or a string of ethylated phosphorothioate (PPT) groups to generate a charge-neutralized DNA backbone.[17−19] These hydrophobically tagged nanostructures advance the reorganization of membrane shape,[4,13,20−23] the molecular transport across membranes,[9,10,24] cell surface functionalization,[25,26] and cytotoxicity.[18,27]
Advancements in atomistic molecular dynamics (MD) simulations have proven to be extremely useful in predicting the interactions of DNA nanostructures with lipid bilayers
We examined the interactions of different hydrophobic anchors with giant unilamellar vesicle (GUV) membranes using confocal fluorescence microscopy
Summary
The unique properties of DNA duplexes enable precise engineering of nanostructures with different shapes, geometries, and sizes.[1−3] Synthetic hydrophobic modifications expand the functional range of DNA nanostructures by facilitating specific interactions with lipid bilayers.[4−6] The modifications include conjugating DNA to hydrophobic molecules such as cholesterol[7−13] and porphyrin[14−16] or a string of ethylated phosphorothioate (PPT) groups to generate a charge-neutralized DNA backbone.[17−19] These hydrophobically tagged nanostructures advance the reorganization of membrane shape,[4,13,20−23] the molecular transport across membranes,[9,10,24] cell surface functionalization,[25,26] and cytotoxicity.[18,27] to realize the potential of the structures for diagnostics, therapeutics, and synthetic biology, a fundamental understanding of the interaction between hydrophobic DNA with lipid membranes is imperative. DsDNA construct TChol with a terminal cholesterol lipid anchor and constructs TEt and THex and CEt and CHex carrying a terminal or central position alkyl-PPT belt. Incubation of GUVs with the hydrophobic duplexes in PBS or Opti-MEM cell culture medium followed by imaging yielded Cy3 fluorescent signal for constructs TChol, THex, and CHex (Figures 3A, S1), indicative of binding. Our findings highlight that the type and position of hydrophobic anchor, ionic buffer conditions, and membrane composition can influence DNA interactions with synthetic lipid bilayers. After a brief restrained equilibration, the systems were simulated without any restraints for 1 μs using the MD method
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