(Poly)cation-induced protection of conventional and wireframe DNA origami nanostructures

Abstract

DNA nanostructures hold immense potential to be used for biological and medical applications. However, they are extremely vulnerable towards salt depletion and nucleases, which are common under physiological conditions. In this contribution, we used chitosan and linear polyethyleneimine for coating and long-term stabilization of several three-dimensional DNA origami nanostructures. The impact of the degree of polymerization and the charge density of the polymer together with the N/P charge ratio (ratio of the amines in polycations to the phosphates in DNA) on the stability of encapsulated DNA origami nanostructures in the presence of nucleases and in low-salt media was examined. The polycation shells were compatible with enzyme- and aptamer-based functionalization of the DNA nanostructures. Additionally, we showed that despite being highly vulnerable to salt depletion and nucleolytic digestion, self-assembled DNA nanostructures are stable in cell culture media up to a week. This was contrary to unassembled DNA scaffolds that degraded in one hour, showing that placing DNA strands into a spatially designed configuration crucially affect the structural integrity. The stability of naked DNA nanostructures in cell culture was shown to be mediated by growth media. DNA origami nanostructures kept in growth media were significantly more resistant towards low-salt denaturation, DNase I and serum-mediated digestion than when in a conventional buffer. Moreover, we confirmed that DNA origami nanostructures remain not only structurally intact but also fully functional after exposure to cell media. Agarose gel electrophoresis and negative stain transmission electron microscopy analysis revealed the hybridization of DNA origami nanostructures to their targets in the presence of serum proteins and nucleases. The structural integrity and functionality of DNA nanostructures in physiological fluids validate their use particularly for short-time biological applications in which the shape and structural details of DNA nanodevices are functionally critical.

The Growing Development of DNA Nanostructures for Potential Healthcare‐Related Applications

“…The composition, while ideal for the development of cells, may actually pose a hostile environment for DNA nanoconstructs, causing degradation and dehybridization of the constituent DNA molecules thereby rendering them incapable of fulfilling their purpose. [56a,59–63,67] Standard commercial 10% FBS includes a host of proteins for cell development but also DNA digesting components that can cleave DNA strands. The threat posed by serum nuclease activity to ssDNA and dsDNA molecules (e.g., short and plasmid forms) is well‐known, so it is essential to see if DNA nanostructures follow the same trend against serum‐based nucleases in physiologically relevant amounts.…”

Enhancing Biocompatible Stability of DNA Nanostructures Using Dendritic Oligonucleotides and Brick Motifs

“…[8][9][10][11] This is due to two main factors involved in degradation of DNA nanostructures upon exposure to biological conditions: i) denaturation caused by low divalent cation concentration (physiological salt concentration approximately 0.04-0.8 mm MgCl 2 ), and ii) digestion caused by the presence of nucleases. [8] Multiple strategies have been developed to chemically or physically prevent the DNA nanostructures from falling apart in cellular media (for example, 10 % FBS), [12][13][14][15][16][17][18][19][20] and in general, encapsulation of DNA nanostructures with different coating moieties prolongs the survival time the longest published. [12,14,[17][18][19] For example, while most bare DNA origami falls apart easily under physiological conditions, PEG-oligolysines, [12] lipid molecules, [17] or cationic polymers [18,19] can be applied as a coating material to extend the half-life of DNA origami by an order of up to approximately 100.…”

“…This finding also helps explain why most 3D DNA origami structures with average binding-domain lengths of 7-10 basepairs degrade when the global divalent salt concentration is low (0-0.8 mm MgCl 2 ). [12][13][14][15][16][17][18][19][20] DNA brick nanostructures that remain stable even in 1 PBS were still susceptible to nucleases. After confirming that DNA brick nanostructures were stable against low salt denaturation without the requirement of any additional stabilization techniques, the same 52 nt brick nanostructures were tested against nuclease digestion (Supporting Information, Figure S5).…”

Adenita: Interactive 3D modeling and visualization of DNA Nanostructures

“…The DNA nanostructures were prepared based on protocols already described in the work of Ahmadi et al [23,24]. These nanorods were initially designed with Cadnano 2.2.0 using the p8064 single stranded scaffold.…”

“…To showcase these editors and evaluate the precision of our data model, we designed cross-shaped nanostructures comprising two individual multilayer DNA origami nanorods. The nanorods consists of around 16,000 nucleotides, have an approximate size of 350nm × 8nm × 4nm (Figure 4a) and were originally designed for other applications [23].…”