Magnesium-Free Immobilization of DNA Origami Nanostructures at Mica Surfaces for Atomic Force Microscopy

Abstract

DNA origami nanostructures (DONs) are promising substrates for the single-molecule investigation of biomolecular reactions and dynamics by in situ atomic force microscopy (AFM). For this, they are typically immobilized on mica substrates by adding millimolar concentrations of Mg2+ ions to the sample solution, which enable the adsorption of the negatively charged DONs at the like-charged mica surface. These non-physiological Mg2+ concentrations, however, present a serious limitation in such experiments as they may interfere with the reactions and processes under investigation. Therefore, we here evaluate three approaches to efficiently immobilize DONs at mica surfaces under essentially Mg2+-free conditions. These approaches rely on the pre-adsorption of different multivalent cations, i.e., Ni2+, poly-l-lysine (PLL), and spermidine (Spdn). DON adsorption is studied in phosphate-buffered saline (PBS) and pure water. In general, Ni2+ shows the worst performance with heavily deformed DONs. For 2D DON triangles, adsorption at PLL- and in particular Spdn-modified mica may outperform even Mg2+-mediated adsorption in terms of surface coverage, depending on the employed solution. For 3D six-helix bundles, less pronounced differences between the individual strategies are observed. Our results provide some general guidance for the immobilization of DONs at mica surfaces under Mg2+-free conditions and may aid future in situ AFM studies.

Environment‐Dependent Stability and Mechanical Properties of DNA Origami Six‐Helix Bundles with Different Crossover Spacings

“…The addition of Mg 2+ ions was necessary to ensure the efficient immobilization of the negatively charged DNA structures on the like‐charged mica surface. [ 32,52 ]…”

“…This, therefore, prohibits the addition of Mg 2+ ions to facilitate 6HB adsorption at the mica surface. Instead, we have modified the mica surface with a thin film of the polyamine spermidine, which enables DNA origami adsorption at mica even under essentially Mg 2+ ‐free conditions, [ 52 ] as can be seen in the AFM images in Figures S16–S20, Supporting Information. For all conditions chosen, the 6HBs are structurally intact and adsorbed at the spermidine‐modified mica surface in more or less comparable density, even though sample‐to‐sample and image‐to‐image variations in surface coverage can be rather pronounced.…”

“…The addition of Mg 2+ ions was necessary to ensure the efficient immobilization of the negatively charged DNA structures on the like-charged mica surface. [32,52] As can be seen in the resulting AFM images shown in Figure 3, the two designs behave rather differently in the different solutions. In low-Mg 2+ TAE buffer, the presence of EDTA (1 mm) leads to the removal of residual Mg 2+ ions from the DNA's backbone phosphates, which is a determining factor for the structural integrity of the 6HBs.…”

“…[68,73] On the other hand, the strong electrostatic interactions with the spermidine-modified mica surface during adsorption may also lead to the 6HBs assuming bent and kinked conformations that would not occur in bulk solution. [52] Even though any visibly damaged or broken 6HBs have been omitted in the analyses, bent and kinked 6HBs were included provided that the software could accurately fit their traces. Therefore, we assume that the experimentally determined persistence lengths are mostly governed by structural defects or non-native conformations, which provides an explanation not only for the lower values compared to the MD simulations but also the observation that the 42-bpCS design has a slightly larger persistence length in TAE/MgCl 2 buffer than 21-bpCS.…”

Time-Dependent DNA Origami Denaturation by Guanidinium Chloride, Guanidinium Sulfate, and Guanidinium Thiocyanate

“…For investigating the time dependence of DNA origami denaturation in the different Gdm salts, we exposed DNA origami triangles [ 40 ] to comparably low Gdm salt concentrations of 2 M. The Rothemund triangle is one of the most studied DNA origami nanostructures, particularly with regard to its stability under various conditions [ 1 , 2 , 5 , 10 , 11 , 12 , 14 , 16 , 17 , 20 , 24 , 25 , 41 , 42 , 43 , 44 , 45 ], including the presence of molar concentrations of Gdm salts [ 28 , 29 , 30 ]. It is composed of three trapezoids of parallel double helices that are connected to each other via one scaffold crossover and four bridging staples.…”