Specific versus nonspecific binding of cationic PNAs to duplex DNA

Abstract

Although peptide nucleic acids (PNAs) are neutral by themselves, they are usually appended with positively charged lysine residues to increase their solubility and binding affinity for nucleic acid targets. Thus obtained cationic PNAs very effectively interact with the designated duplex DNA targets in a sequence-specific manner forming strand-invasion complexes. We report on the study of the nonspecific effects in the kinetics of formation of sequence-specific PNA-DNA complexes. We find that in a typical range of salt concentrations used when working with strand-invading PNAs (10-20 mM NaCl) the PNA binding rates essentially do not depend on the presence of nontarget DNA in the reaction mixture. However, at lower salt concentrations (<10 mM NaCl), the rates of PNA binding to DNA targets are significantly slowed down by the excess of unrelated DNA. This effect of nontarget DNA arises from depleting the concentration of free PNA capable of interacting with DNA target due to adhesion of positively charged PNA molecules on the negatively charged DNA duplex. As expected, the nonspecific electrostatic effects are more pronounced for more charged PNAs. We propose a simple model quantitatively describing all major features of the observed phenomenon. This understanding is important for design of and manipulation with the DNA-binding polycationic ligands in general and PNA-based drugs in particular.

FIGURE 1

Proposed pathways for specific and nonspecific interaction of cationic pcPNAs with dsDNA. Reaction 1, reversible binding of pcPNAs to unrelated (nontarget) dsDNA; Reaction 2, formation of pcPNA duplexes via association of two matching pcPNAs; Reaction 3, reversible binding of highly charged pcPNA duplexes to DNA; and Reaction 4, irreversible binding of free unpaired pcPNAs with the dsDNA target site resulting in the formation of the sequence-specific double-duplex invasion complex. The pathway for bisPNA binding to dsDNA target site involves Reaction 1 followed by Reaction 4, since bisPNAs do not associate to form PNA pairs.

FIGURE 2

PNA-DNA association obeys pseudo-first-order kinetics but it is affected by the total concentration of nontarget DNA present in the reaction mixture. (A) Gel-electrophoresis shift assay is used to monitor time course of pcPNAs 1690 binding to DNA target site located within a 350-bp fragment of p8/PvuII digest. Both experiments were performed at 37°C in 10 mM TE (pH 7.4), which contains 2 mM Na⁺. The concentration of pcPNAs and DNA target sites was 625 nM and 23 nM, respectively. The amount of nonspecific DNA (in bp) in the reaction mixture was varied from 60 μM (upper panel) to 131 μM (lower panel) by addition of appropriate amounts of linearized pUC19. (B) The yield of the PNA/DNA complex, C, fits linear regression; total concentrations of nonspecific DNA (in bp): 60 μM (•) and 131 μM (○). Pseudo-first-order rate constants given by the slopes of linear regression are 0.0297 and 0.0100 min⁻¹, respectively.

FIGURE 3

Dependence of k_ps on the total concentration of nontarget DNA, [N]₀. Pseudo-first-order rate constants for pcPNA 1690 (circles) and bisPNA 522 (triangles) binding to corresponding DNA targets. Concentration of PNA was 625 nM in both cases. DNA concentration was varied by changing the amounts of the PCR-amplified, 265-bp fragment (⊙), PvuII restriction digest of corresponding plasmid (○ and ▵), and addition of nontarget linearized pUC19 (•). Experiments were performed in 10 mM TE, pH 7.4 (pcPNA) and pH 7.0 (bisPNA) buffer at 37°C. The lines are drawn to guide the eye.

FIGURE 4

The effect of the total charge carried by PNAs on the pcPNA-DNA association kinetics. PNA pairs 1914/1915 (○), 1914/1916 (•), and 1914/1917 (▵) target identical DNA sequences located within a 350-bp fragment of p10/PvuII, but differ in the number of lysine residues (and consequently charges) attached to their termini (see Table 1). Kinetic experiments were carried out in 10 mM TE, pH 7.4, at 37°C. The PNA concentration was maintained constant at 625 nM (each strand), whereas [N]₀ varied by changing the concentration of the p10/PvuII restriction digest. The solid line presents the nonlinear fit of the kinetic data for PNA pair 1914/1915 according to Eq. A15; regression parameters are K₂ = 0.4 ± 0.2 μM⁻¹ and Broken lines are drawn to guide the eye.

FIGURE 5

Salt-dependence of the pcPNA 1690 binding to DNA target sites. The extent of complex formation was assessed after 40-min incubation at 37°C in 10 mM TE, pH 7.4 buffer with addition of desirable amounts of NaCl. PNA concentration (571 nM) and DNA target concentration (17 nM) were kept constant. Total concentration of DNA (in bp) changed from 45 μM (•) to 90 μM (○) by addition of linearized pUC19.

FIGURE 6

Effect of pcPNA duplex formation on the kinetics of PNA-DNA complex association. (A) Normalized thermal denaturation profiles for PNA pairs 1914/1915 (•), 1914/1917 (○), and PNA 1690 (▵). (B) Double-logarithmic plots of −ln(1–C) after a 40-min (PNA pair 1914/1915 and PNA 1690) or 70-min (PNA pair 1914/1917) incubation of a target plasmid (p10/PvuII or p8/PvuII) in the presence of varying amounts of corresponding pcPNAs ([P]₀ increased from 67 nM to 533 nM). Straight lines present linear fits with slopes of 0.97, 0.94, and 1.17 for PNA pairs 1914/1915, 1914/1917, and PNA 1690, respectively. Incubations were performed in 10 mM TE pH 7.4 buffer at 37°C. Concentration of nontarget DNA was 60 μM of bp.