Dinuclear complex-induced DNA melting

Abstract

Dinuclear copper complexes have been designed for molecular recognition in order to selectively bind to two neighboring phosphate moieties in the backbone of double strand DNA. Associated biophysical, biochemical and cytotoxic effects on DNA were investigated in previous works, where atomic force microscopy (AFM) in ambient conditions turned out to be a particular valuable asset, since the complexes influence the macromechanical properties and configurations of the strands. To investigate and scrutinize these effects in more depth from a structural point of view, cutting-edge preparation methods and scanning force microscopy under ultra-high vacuum (UHV) conditions were employed to yield submolecular resolution images. DNA strand mechanics and interactions could be resolved on the single base pair level, including the amplified formation of melting bubbles. Even the interaction of singular complex molecules could be observed. To better assess the results, the appearance of treated DNA is also compared to the behavior of untreated DNA in UHV on different substrates. Finally, we present data from a statistical simulation reasoning about the nanomechanics of strand dissociation. This sort of quantitative experimental insights paralleled by statistical simulations impressively shade light on the rationale for strand dissociations of this novel DNA interaction process, that is an important nanomechanistic key and novel approach for the development of new chemotherapeutic agents.

Keywords: AFM; Biomolecules; DNA; Electrospray ionization; Molecular recognition.

Fig. 1

a Chemical structure of the [(Htom^Me){Cu(OAc)}₂]⁺ complexes. The descriptor for the complexes changes according to the chemical surroundings, for clarification see Additional file 1: Figure S1. b Model of binding modes of the complex molecules to adsorb to DNA and to each other. c UHV-AFM topographic image of the agglomeration of multiple strands (frequency shift setpoint df =− 2.5 Hz, oscillation amplitude A = 14.1 nm, resonance frequency f₀ = 259.0 Hz) and the dedicated interpretation d: two double strands (yellow, height 0.6 nm), one singular ds (orange, height 0.4 nm), melting bubble dissociation in two single strands (red, height 0.1 nm)

Fig. 2

a Dissociated melting bubbles along a double strand of λ-DNA (taken under UHV, df =− 2.0 Hz, A = 14.1 nm, f₀ = 259.0 Hz). b Closer look at one of the larger bubbles, including an area of supercoiled buildup. c Distribution of interval distances between the protrusion features along the bubbles, with a mean of 1.42 nm. d Distances and antiphasic patterning of features. The larger than average distance of 0.78 nm can be attributed to observations where complex coated ssDNA regions appear elongated [25]. e Overlay of a possible molecular model to match the observed topography [DNA model from [26] (1BNA)]

Fig. 3

λ-DNA subjected to UHV conditions on different substrates. a Untreated DNA on Au(111) (df =− 6.2 Hz, A = 12.6 nm, f₀ = 286.3 Hz). b [(Htom^Me)Cu₂]³⁺-treated DNA on Au(111) (df =− 2.0 Hz, A = 14.1 nm, f₀ = 259.0 Hz). The residues on the otherwise flat atomic planes are likely due to residual complex molecules. c DNA on potassium bromide (df =− 8.6 Hz, A = 4.7 nm, f₀ = 302.0 Hz, inset: df =− 20.2 Hz, A = 1.9 nm, f₀ = 302.0 Hz). d Distribution of groove distances, along with corresponding mean values for different substrates (Au, KBr)

Fig. 4

a Contour length percentage along a strand when summing up dissociated bubble segments from a certain threshold value (purple). Segments with consequential AT base paring along the λ-phage genome sequence (blue). The percentage of segments with odd complex adsorption pattern, derived from a statistical simulation (yellow). Percentage of areas where both conditions are met at the same time (green), fitting well to the actual observed bubble lengths. The dip at 1 bp accounts for the visual counting threshold. Inset: Distribution of melting bubble lengths. The relation between base pair and nm of 1 bp $\widehat{=}$ 0.71 nm was determined from the interval distance in Fig. 2c. b Explanation model for the bubble formation. Top: Zigzag adsorption pattern of complex molecules. Middle: Reduced phosphate distance by partial A-to-B transformation introduces tensile stress on the base pairing. Bottom: Dehybridization of weaker bound AT-base pairs