Electrostatic Binding and Hydrophobic Collapse of Peptide–Nucleic Acid Aggregates Quantified Using Force Spectroscopy

Abstract

Knowledge of the mechanisms of interaction between self-aggregating peptides and nucleic acids or other polyanions is key to the understanding of many aggregation processes underlying several human diseases (e.g. Alzheimer's and Parkinson's diseases). Determining the affinity and kinetic steps of such interactions is challenging due to the competition between hydrophobic self-aggregating forces and electrostatic binding forces. Kahalalide F (KF) is an anticancer hydrophobic peptide which contains a single positive charge that confers strong aggregative properties with polyanions. This makes KF an ideal model to elucidate the mechanisms by which self-aggregation competes with binding to a strongly charged polyelectrolyte such as DNA. We use optical tweezers to apply mechanical forces to single DNA molecules and show that KF and DNA interact in a two-step kinetic process promoted by the electrostatic binding of DNA to the aggregate surface followed by the stabilization of the complex due to hydrophobic interactions. From the measured pulling curves we determine the spectrum of binding affinities, kinetic barriers and lengths of DNA segments sequestered within the KF-DNA complex. We find there is a capture distance beyond which the complex collapses into compact aggregates stabilized by strong hydrophobic forces, and discuss how the bending rigidity of the nucleic acid affects such process. We hypothesize that within an in vivo context, the enhanced electrostatic interaction of KF due to its aggregation might mediate the binding to other polyanions. The proposed methodology should be useful to quantitatively characterize other compounds or proteins in which the formation of aggregates is relevant.

Single-molecule kinetics and footprinting of DNA bis-intercalation: the paradigmatic case of Thiocoraline

“…The synthesis protocols for the 24-kb DNA molecule for stretching experiments, and the 6.8-kb DNA hairpin for unzipping experiments, are described in detail in (28). The 480-bp hairpin for DNA footprinting experiments is synthesized from a plasmid that contains the sequence of interest embedded between the restriction sites of Tsp45I and TspRI.…”

“…This region of the plasmid is polymerase chain reaction amplified and the product is digested with the two enzymes. A set of oligonucleotides is designed to create the handles structure on the TspRI-digested end following the same approach as explained in (28). Similarly, an oligonucleotide complementary to the Tsp45I end that folds into the end-loop structure is annealed and ligated to create the final hairpin structure.…”

“…The term x ssDNA ( n, f exp ) is the only term that depends on the number n of unzipped base pairs, and is modeled with an FJC of 2 n bases with the aforementioned elastic parameters. The other terms do not depend on n and are modeled as x b = f exp / k trap (where k trap is the stiffness of the optical trap) and x h using a WLC (Equation (1)) with the generally accepted parameters for double-stranded DNA (dsDNA) (28). …”

“…In this work we use optical and magnetic tweezers to study the anticancer bis-intercalator Thiocoraline (Figure 1a) (27), fully characterizing the thermodynamics, kinetics and selectivity of binding using a combination of DNA stretching and unzipping assays (28). A clear understanding of these properties was lacking due to the low solubility of this drug (29,30), making measurements difficult and leading to limited and conflicting results (31,32).…”

Interactions of an anticancer drug Formestane with single and double stranded DNA at physiological conditions

“…The aforementioned results indicated that the main interaction mode of FMT molecules with ssDNA is electrostatic attraction via negative phosphate groups of ssDNA with the protonated drug, but the hydrophobic contact can not be excluded. In this context Camunas-Soler et al reported the electrostatic binding and hydrophobic collapse of peptide-nucleic Acid[46].Comparision of the interaction of FMT upon addition of dsDNA and ssDNA at two physiological pH values (7.4 and 4.7) indicated that the decrease in peak current (∆I) decreases more sharply with the addition of ssDNA than in the presence of dsDNA as shown inFig. 10.…”