PT-ACRAMTU, a platinum-acridine anticancer agent, lengthens and aggregates, but does not stiffen or soften DNA

Abstract

We used atomic force microscopy (AFM) to study the dose-dependent change in conformational and mechanical properties of DNA treated with PT-ACRAMTU ([PtCl(en)(ACRAMTU-S)](NO3)2, (en = ethane-1,2-diamine, ACRAMTU = 1-[2-(acridin-9-ylamino)ethyl]-1,3-dimethylthiourea. PT-ACRAMTU is the parent drug of a family of non-classical platinum-based agents that show potent activity in non-small cell lung cancer in vitro and in vivo. Its acridine moiety intercalates between DNA bases, while the platinum group forms mono-adducts with DNA bases. AFM images show that PT-ACRAMTU causes some DNA looping and aggregation at drug-to-base pair ratio (r b) of 0.1 and higher. Very significant lengthening of the DNA was observed with increasing doses of PT-ACRAMTU, and reached saturation at an r b of 0.15. At r b of 0.1, lengthening was 0.6 nm per drug molecule, which is more than one fully stretched base pair stack can accommodate, indicating that ACRAMTU also disturbs the stacking of neighboring base pair stacks. Analysis of the AFM images based on the worm-like chain (WLC) model showed that PT-ACRAMTU did not change the flexibility of (non-aggregated) DNA, despite the extreme lengthening. The persistence length of untreated DNA and DNA treated with PT-ACRAMTU was in the range of 49-65 nm. Potential consequences of the perturbations caused by this agent for the recognition and processing of the DNA adducts it forms are discussed.

Fig 1. Chemical structure of PT-ACRAMTU

The acridine moiety intercalates between base pairs, whereas the metal forms a covalent monoadduct with an adjacent nucleobase. The molecule has an overall 2+ charge.

Fig 2. AFM images of 1154 bp DNA fragment

(A) Untreated DNA; DNA treated with different doses of PT-ACRAMTU (B) 0.02 r_b,(C) 0.05 r_b, (D) 0.1 r_b, (E) 0.2 r_b, (F) 0.3 r_b. (G–I) Zoomed images show DNA looping and double-strand breaks. The scale bar in all images is 200 nm.

Fig 3. Plot DNA aggregation as a function of increasing doses of PT-ACRAMTU (r_b = 0 to 0.3)

Aggregation was determined from the total length of free DNA and the total length of DNA aggregated DNA. There was negligible aggregation for r_b < 0.1. Aggregation increased with increasing PT-ACRAMTU for r_b values of 0.1 and above.

Fig 4. Contour length distribution of untreated DNA, and DNA treated with different doses of PT-ACRAMTU (r_b = 0 to 0.3)

The blue line represents the theoretical length of B-form DNA, 390 nm.

Fig 5. Plot of the increase in DNA contour length as a function of increasing doses of PT-ACRAMTU (r_b = 0 to 0.3)

The length increases sigmoidally, showing saturation at r_b ~ 0.15.

Figure 6. Plot of <θ² > between two segments vs. their separation distance

l. The analysis was performed for untreated DNA and DNA treated with different doses of PT-ACRAMTU (r_b = 0 to 0.3). The slope of this graph (fitted, dashed lines) is the inverse of the persistence length. The slope was evaluated for 3 nm < l < 150 nm to avoid end effects.