DNA origami nanostructures can be utilized as functional materials depending upon their arrangement into higher orders using molecular lithography process. DNA origami triangles and DNA origami 6-helix bundles (6HB) are synthesized under sodium and magnesium rich buffer solutions, adsorbed and desorbed on the negatively charged mica surface. The adsorption of origami depends upon Mg2+ that forms a salt bridge at the surface because of electrostatic attraction which further leads to a weak mobility of origami on surface and thereby a weaker distribution. The surface mobility of DNA origami is controlled by the addition of monovalent Na+ cations thus producing closely packed hexagonal, symmetrically arranged DNA triangles. But Na+ in excess results in replacement of the Mg2+ ions on the surface, leading to complete desorption. The effect of Na+ on 6HB adsorption indicates a better efficiency in the molecular combing process, which was investigated using Atomic Force Microscopy (AFM) imaging. The symmetry and degree of order of adsorbed DNA origami is depicted using 2D Fast Fourier Transform.
INTRODUCTION AND THEORETICAL BACKGROUND
After DNA origami technique[1,2] was introduced by Rothemund, many scientists and researchers have been working on DNA origami because of its ability to configure highly complex geometry with tiny nanometer precision[3]. Nanoscale devices and materials are been created using DNA origami technique which uses DNA as the base construction material. The properties of DNA origami have made it popular for designing and synthesizing 1D Nano wires and Nano tubes[4,5] as well as modern two- and three-dimensional DNA nanostructures i.e. (2D lattices & 3D crystals) with varying complexity[6]. The design of complex structures is highly sensible to the ratio of strands and requires purification whereas single-stranded DNA origami does not need purifications[1,2,7] as well as it avoids the problem of stichometry. DNA origami is the technique of folding long single stranded DNA scaffold into precise shape on a nanoscale by hybridization with various number of short synthesis oligonucleotides[8]. The shorter staple strands act as complementary thus bind the longer segments into various places which results in the formation of predefined shape upon hybridization[9].
We herein show that DNA triangles & 6-helix bundles can be arranged in a particular method by changing their surface mobility parameters by simply adding monovalent salts (NaCl). Design of the complex structures is observed by various methods including Atomic force microscopy. In our work, AFM investigations were done on strongly adsorbed DNA origami on negatively charged mica surfaces to facilitate stable imaging which is mediated by Mg2+ later to be replaced by Na+. Fast Fourier transform for each image is calculated using Gwyddion software to explain the effect caused by Na adsorption by DNA origami on mica surface.
DISCUSSION
To investigate the mechanism of how a cation species interacts with the DNA origami adsorption, DNA molecules were cultivated in a buffer solution containing monovalent (NaCl) and divalent (MgCl2) cations separately and then introduced on mica surface.
Adsorption of DNA origami triangles on mica surface
Molecular lithography mask has been fabricated from the triangular DNA origami design of Rothemund[1] by cation-mediated self-assembly. Mica is a layered mineral with a negative surface charge, and DNA is a negatively charged polymer. Mg2+ is a divalent cation that acts as a mediator between them by binding the negative charges on the DNA backbone and those on the mica by acting as a bridge[11]. AFM image for the adsorption of the DNA origami on mica surface due to Mg2+ ions. The presence of the Mg2+ in the buffer acts as salt bridge between mica surface and DNA origami which results in strong adsorption. DNA adsorption is high due to strong electrostatic interaction, which decreases the mobility of the DNA origami triangle and weakens the orientation at surface. Hence, a loosely fit arrangement occurs on the mica surface.
The second step is to weaken the DNA-mica interaction enough to allow the structures to diffuse on the surface. Incubation of the DNA origami triangles on freshly cleaved mica surfaces in the presence of 100 mM Mg2+ and low quantity of 1 M Na+ results in the formation of an ordered, densely packed DNA origami monolayer with hexagonal symmetry and the corresponding two-dimensional Fourier transform. The hexagonal symmetry arises from six triangular DNA origami tiles joining. The monovalent Na+ ions compete with divalent Mg²+ ions for the mica surface, but do not contribute to overcompensation of the negative surface charge and thus charge inversion of the surface[12]. Due to the addition of the Na+ ions, it replaces the Mg2+ ions on the mica surface, thereby reducing the adsorption capacity of the surface. This addition does not completely diffuse away the DNA molecules, only the mobility of these molecules increase on the surface. This results in self-assembly of the origami triangles in a hexagonal structure.
The 2D FFT indicates the degree of measure for its symmetry and ordering of the DNA origami triangles. Triangular origami tiles form a lattice with trigonal symmetry, which is clearly reflected in the FFT analysis[7]. It can be clearly observed that as the origami triangles arrange themselves into a hexagonal structure (highlighted region), the corresponding FFT gradually take the same shape as well.
Desorption of DNA origami triangles with 200 nM Na+ in buffer
By adding an excess amount of monovalent salts in the buffer, desorption characteristics of the adsorbed DNA molecules are determined. The modified solution is applied on an already adsorbed DNA layer on mica and observed.
We can clearly see that all the DNA origami triangles have diffused off the surface of mica in the 200 mM NaCl buffer. The kinetics for the DNA desorption involves the reduction of the binding force of DNA and ultimately complete desorption from the mica surface. Based on the Poisson-Boltzmann equation, the density of divalent cations can be drastically reduced by adding a salt containing monovalent cations, which directly weakens the binding strength of DNA molecules[11]. These molecules that are loosely hung onto the mica surface can be easily detached by action of an external force, a flow of MilliQ water in our case. The reduction of the Mg2+ ion concentration near the surface induces the DNA molecule to tears itself apart due to reduced intramolecular forces[13]. The coiled DNA molecules uncurl when high concentration Na+ is added, as the long-range repulsion force appears and instead of interacting with the Mg2+ ions, NaCl interactions increase.
Molecular combing of 6HBs
The DNA origami used in this work were six-helix bundles. AFM image of dried DNA origami nanotubes adsorbed to a mica surface. The adsorbed 6HBs are well dispersed and randomly oriented on the surface. Most of these strands are bent and folded over during adsorption and drying. This is due to the surface containing Mg2+ ions, and immediate adsorption occurs due to electrostatic force. The mechanism of adsorption goes same for the 6HBs as it was for the origami triangles. The angular distribution of the helix bundles on the mica surface. The random orientation angles in the histogram can be clearly attributed to the non-directional adsorption of the DNA molecules to the surface.
For molecular combing to occur, Na+ cations are introduced into the sample to loosen the origami strands adsorbed on the surface. Molecular combing is a widely used technique for the adsorption and alignment of DNA molecules on the surface with no modification required to the original molecule[14]. It involves three main steps: adsorption of the ends of the floating DNA molecule, exerting a stretching force due to the receding meniscus of fluid, deposition of DNA molecule on the substrate. As observed the presence of Na+ ions fulfils the condition for a stable combing process wherein many of the origami gets oriented unidirectional. Adding monovalent cations to the solution can be used as an additional mechanism to control the surface density of deposited DNA[15]. The addition of monovalent salts results in an increase in the adsorbed DNA molecules, reaching a maximum at 100 mM NaCl. Whereas 1M NaCl in solution results in a gradual reduction in the adsorption capacity due to the replacement of the Mg2+ ions. In many cases, this enables the DNA origami to partially hold onto the surface at one end whereas the other end is free. The free end is subjected to molecular combing. However, some of the origami is pinned to the surface at random spots due to the strong electrostatic forces from the Mg2+ ions still clinging on the surface.
The histogram for the AFM data of adsorbed 6HBs with Na+ in buffer indicates the angular distribution of molecules to be concentrated around 71° and 101° to the horizontal axis. The alignment yield obtained for the distribution of the highest number of molecules i.e. between 71° and 81° was found to be at 48%.
CONCLUSION
AFM imaging proves to be a very useful tool in the study of the ion interactions with DNA origami at various charged surfaces. The present study indicates the salt dependency of the DNA origami molecules for the adsorption characteristics on a surface. We have shown that adsorption strength can be easily maintained by the addition of monovalent cations, which readily compete with the divalent cations for the negative charge neutralisation of DNA and mica surface. Low concentration of divalent cations on the mica surface leads to a very loose attachment of the DNA, which in turn varies the surface mobility. Na+ ions cause the origami triangles to shift to a more favourable, tightly packed configuration by replacing the Mg2+ ions on the surface. Na+ also assists in molecular combing of 6HBs by breaking the strong electrostatic attraction of Mg2+, obtaining an alignment yield of 48% in 1M Na+ buffer when compared to the random orientation in Mg2+. Future experiments can also be carried out by varying the molar concentration of the Na+ ions and its effect on the combing process.
REFERENCES
Paul W. K. Rothemund, “Folding DNA to create nanoscale shapes and patterns”. Nature, 2006, 440(7082), 297-302.
Shen B., Linko V., Tapio K., Kostiainen M.A., Toppari J.J., “Custom-shaped metal nanostructures based on DNA origami silhouettes”. Nanoscale, 2015, 7, 11267.
Seeman N.C., “DNA in Material World”. Nature, 2003, 421, 427-431.
Teshome B., Facsko S., Keller A., “Topography-controlled alignment of DNA origami nanotubes on nanopatterned surfaces”. Nanoscale, 2014, 6, 1790-1791.
Gu Q. et al. “Fabrication & characterization of gold nano-wires templated on virus-like arrays of tobacco mosaic virus coat proteins”. T. Nanotechnology, 2006, 17, R14.
Fan Hong, Fei Zhang, Yan Liu, et al. “DNA origami: Scaffolds for creating higher order structures”. Chemical Reviews, 2017, 117 (20), 12584-12640.
Ali Aghebat Rafat, Tobias Pirzer, Max B. Scheible, Anna Kostina, Friedrich C. Simmel, “Surface-Assisted Large-Scale Ordering of DNA Origami Tiles”. Angew. Chem. Int. Ed., 2014, 53 (29), 7665−7668.
Zadegan R.M, Norton M., “Structural DNA Nanotechnology from design to applications”. Int. J. Mol. Sci., 2012, 13(6), 7149–7162.
[9] Douglas Shawn M., Dietz Hendrik, et al. “Self-Assembly of DNA into Nano scale three dimensional shapes” Nature, May 2009, 459 (7245): 414–418.
Yang Y, Han D, Nangreave J, Liu Y, et al. “DNA origami with double-stranded DNA as a unified scaffold”. ACS Nano. 2012, 6(9), 8209-15.
Pastré, D. et al. “Adsorption of DNA to mica mediated by divalent counterions: a theoretical and experimental study”. Biophys. J., 2003, 85, 2507–2518.
Albrechts, B., Hautzinger, D. S., Krüger, M., Elwenspoek, M. C., Müller, K. M., & Korvink, J. G., “Adsorption studies of DNA origami on silicon dioxide”. In 21st Micromechanics and Micro Systems Europe workshop, 2010, 1-4.
Yajing Kan, Qiyan Tan, Gensheng Wu, Wei Si & Yunfei Chen, “Study of DNA adsorption on mica surfaces using a surface force apparatus”. Scientific Reports, 2015, 5: 8442.
Zeinab Esmail Nazari, Leonid Gurevich, “Molecular Combing of DNA: Methods and Applications”. Journal of Self-Assembly and Molecular Electronics, 2013, Vol. 1, 125–148.
Annegret Benke, Michael Mertig and Wolfgang Pompe, “PH and salt dependent molecular combing of DNA: experiments and phenomenological model”. Nanotechnology, 2010, Vol. 22, 3.