Direct real-time molecular scale visualisation of the degradation of condensed DNA complexes exposed to DNase I

Abstract

The need to protect DNA from in vivo degradation is one of the basic tenets of therapeutic gene delivery and a standard test for any proposed delivery vector. The currently employed in vitro tests, however, presently provide no direct link between the molecular structure of the vector complexes and their success in this role, thus hindering the rational design of successful gene delivery agents. Here we apply atomic force microscopy (AFM) in liquid to visualise at the molecular scale and in real time, the effect of DNase I on generation 4 polyamidoamine dendrimers (G4) complexed with DNA. These complexes are revealed to be dynamic in nature showing a degree of mobility, in some cases revealing the addition and loss of dendrimers to individual complexes. The formation of the G4-DNA complexes is observed to provide a degree of protection to the DNA. This protection is related to the structural morphology of the formed complex, which is itself shown to be dependent on the dendrimer loading and the time allowed for complex formation.

Figure 1

(a) Control AFM images of (left) aggregates of G4 adsorbed at the water–mica interface and acquired upon exposing a mica surface to a 0.1 µg ml^–1 aqueous solution of G4 (z scale is 10 nm) and (right) an example AFM image of naked DNA adsorbed onto a freshly cleaved mica surfaces, recorded in 1 mM PBS containing 2 mM MgCl₂ and 1 mM NiCl₂. Examples of the effect of DNase I enzyme on (b) naked DNA and G4–DNA complexes on mica at (c) 0.5:1, (d) 1:1 and (e) 5:1 ratios. In (e), no degradation is observed within the time frame of the experiment, hence the images show different areas of the sample to illustrate this before pH is raised to disassemble the complexes. Scale bar and z scale in nm, (b) 100, 7, (c) 100, 10, (d) 100, 7 and (e) 100, 7.

Figure 2

AFM image sequences for the 1:1 G4 to DNA ratio, in which DNA and dendrimer molecules were allowed to equilibrate in solution for (a) 15 min and (b) 2 h prior to deposition onto mica and subsequent exposure to the DNase I enzyme. Scale bar and z scale in nm, (a) 70 and 3, (b) 100 and 5.

Figure 3

(a) AFM image of a ‘flower-like’ complex formed at 20:1 G4:DNA ratio. Higher resolution images (centred on regions marked X and Y) of the substrate background show features consistent with a densely packed layer of G4. Scale bar and z scale in nm, 50 and 5, 15 and 5, 13 and 5. (b) Time-lapse images showing the effect of DNase I on G4–DNA complex, 1:1 ratio, incubated for 15 min before deposition and imaged in 1 mM PBS, containing 2 mM MnCl₂ and 1mM NiCl₂, pH 7.4. Scale bar, 100 nm and z scale, 7 nm.