Surface charges effects on the 2D conformation of supercoiled DNA

We have adsorbed plasmid PuC19 DNA on a supported bilayer. The mobility of the lipids within the bilayer ensured a 2D equilibrium of the DNA molecule. By varying the fraction of cationic lipids in the membrane, we have tuned the surface charge. Plasmids conformations were imaged by Atomic Force Microscopy (AFM).We performed two sets of experiments: deposition from salt free solution on charged bilayers and deposition from salty solutions on neutral bilayers. Plasmids can be seen as rings, completely opened structures, or tightly supercoiled plectonemes, depending on the experimental conditions. The plectonemic conformation is observed either on charged surfaces (in the absence of salt) or at 30 mM salt concentration on a neutral bilayer. We demonstrate the equivalence of surface screening by mobile interfacial charges and bulk screening from salt ions.

💡 Research Summary

In this study the authors investigated how surface charge and bulk ionic strength influence the two‑dimensional conformation of supercoiled plasmid DNA. A supported lipid bilayer (SLB) composed of a neutral phospholipid (POPC) mixed with varying fractions of a cationic lipid (DOTAP) was used as a model membrane. Because the lipids remain fluid, the interfacial charges are mobile and can rearrange in response to the adsorbed DNA, creating a dynamic electrostatic environment that differs from conventional fixed‑charge surfaces.

Two complementary experimental series were performed. In the first series, plasmid pUC19 (2.7 kb, intrinsically supercoiled) was deposited from a salt‑free Tris buffer onto SLBs with different cationic lipid fractions (0 %–50 %). In the second series, DNA was deposited from solutions containing 0, 10, 30, or 100 mM NaCl onto a neutral SLB (0 % DOTAP). After a brief incubation, excess solution was gently rinsed away and the DNA molecules were imaged in liquid by non‑contact atomic force microscopy (AFM). The AFM images allowed a direct classification of each plasmid as a closed ring, a fully opened linear contour, or a tightly supercoiled plectoneme.

The results reveal a clear correspondence between the degree of electrostatic screening—whether provided by mobile surface charges or by dissolved ions—and the prevalence of the plectonemic conformation. On highly positively charged surfaces (≥30 % DOTAP) even in the complete absence of added salt, more than 80 % of the plasmids adopted a plectoneme. This demonstrates that the mobile interfacial charges generate an effective two‑dimensional Debye layer that screens the DNA’s negative backbone, reducing inter‑segment repulsion and allowing the molecule to collapse into its energetically favored supercoiled state.

Conversely, on a neutral SLB the same plectonemic population was only observed when the bulk solution contained ≈30 mM NaCl. At this ionic strength the Debye length shrinks to roughly 1 nm, providing bulk screening comparable to that achieved by the charged surface. At lower salt concentrations (≤10 mM) the DNA was predominantly observed as relaxed rings or fully opened linear molecules, indicating insufficient screening to drive supercoiling in two dimensions.

The authors therefore demonstrate an experimental equivalence between “surface screening” by mobile interfacial charges and “volume screening” by dissolved electrolytes. This equivalence validates the use of a mobile‑charge SLB as a controllable platform for mimicking bulk ionic conditions while keeping the system strictly two‑dimensional. It also underscores the importance of charge mobility: unlike fixed‑charge substrates, the fluid lipid matrix can rearrange to accommodate the DNA, leading to a more realistic representation of biological membranes where charge carriers (e.g., phosphatidylserine, glycolipids) are themselves mobile.

Beyond the fundamental biophysical insight, the work has practical implications for nanobiotechnology. By tuning either the fraction of cationic lipids or the bulk salt concentration, one can deliberately select the DNA conformation required for a given application—whether a relaxed ring for surface‑based enzymatic assays, a linear strand for nanowire templating, or a compact plectoneme for high‑density DNA storage or mechanical spring elements. The AFM‑based approach also provides a powerful method to directly visualize DNA conformations on surfaces, complementing traditional techniques such as electrophoretic mobility shift assays or fluorescence microscopy, which lack the spatial resolution to distinguish plectonemic structures.

In summary, the study establishes that mobile surface charges on a fluid bilayer can replace bulk salt in screening DNA electrostatics, leading to identical supercoiled (plectonemic) conformations. This finding bridges the gap between surface‑confined and solution‑phase DNA physics, offering a versatile tool for the design of membrane‑based DNA nanodevices and for probing the interplay of charge, mobility, and polymer conformation in biologically relevant environments.