Unfolding DNA condensates produced by DNA-like charged depletants: A force spectroscopy study

Abstract

In this work, we have measured, by means of optical tweezers, forces acting on depletion-induced DNA condensates due to the presence of the DNA-like charged protein bovine serum albumin (BSA). The stretching and unfolding measurements performed on the semi-flexible DNA chain reveal (1) the softening of the uncondensed DNA contour length and (2) a mechanical behavior strikingly different from those previously observed: the force-extension curves of BSA-induced DNA condensates lack the "saw-tooth" pattern and applied external forces as high as ≈80 pN are unable to fully unfold the condensed DNA contour length. This last mechanical experimental finding is in agreement with force-induced "unpacking" detailed Langevin dynamics simulations recently performed by Cortini et al. on model rod-like shaped condensates. Furthermore, a simple thermodynamics analysis of the unfolding process has enabled us to estimate the free energy involved in the DNA condensation: the estimated depletion-induced interactions vary linearly with both the condensed DNA contour length and the BSA concentration, in agreement with the analytical and numerical analysis performed on model DNA condensates. We hope that future additional experiments can decide whether the rod-like morphology is the actual one we are dealing with (e.g. pulling experiments coupled with super-resolution fluorescence microscopy).

Doxorubicin hinders DNA condensation promoted by the protein bovine serum albumin (BSA)

“…In other words, the contour length decreases by ∼50% when using only BSA (without doxo). This is the lowest value found for this parameter under our experimental conditions, as shown in Ref …”

“…As known, a considerable energetic cost (due to enzyme action) is needed in vivo to open and unzip the double‐helix. In addition, it was previously demonstrated that the presence of BSA in the buffer suppresses DNA overstretching transition found at high forces (∼65 pN), thus stabilizing the double‐helix structure. Such result corroborates with the conclusion drawn above.…”

“…The force–extension curves obtained were fitted to the Marko–Siggia Worm‐Like Chain (WLC) model in order to obtain the persistence and contour lengths of the DNA–doxo complexes. In particular, when the DNA molecules are condensed one cannot determine the real contour length from the stretchings performed in the entropic regime, but instead the apparent contour length, since the small forces applied in this regime ( F < 3 pN) usually are not sufficient to fully stretch the condensed molecule, and therefore the measured (apparent) contour length will be smaller than the real one . For disperse DNA molecules, forces within the entropic regime are sufficient to almost fully stretch the complexes, and therefore the real contour length can be determined with high accuracy.…”

“…Such macromolecules can directly or indirectly interfere in the natural intracellular DNA‐related processes and in the DNA–drug interactions, by changing water activity on the double‐helix, or even by directly interacting with the biopolymer and/or with the drug used . Despite the relevance of such topic, few works have used force spectroscopy to investigate the DNA condensation process at a single molecule level and the interactions of condensed DNA with drugs. …”

“…It was recently shown that the BSA protein induces DNA condensation when added at a concentration of 20% in mass (wt/wt) in a buffer solution with [NaCl] = 125 mM, which is the condition chosen the perform the experiments here. Doxo is an anthracycline antibiotic much used in cancer chemotherapies, especially to treat some types of leukemias, sarcomas, lymphomas, myelomas, neuroblastomas, and so forth.…”