Kinetics and mechanism of the DNA double helix invasion by pseudocomplementary peptide nucleic acids

Abstract

If adenines and thymines in two mutually complementary mixed-base peptide nucleic acid (PNA) oligomers are substituted with diaminopurines and thiouracils, respectively, so-called pseudocomplementary PNAs (pcPNAs) are created. Pairs of pcPNAs have recently demonstrated an ability to highly selectively target essentially any designated site on double-stranded DNA (dsDNA) by forming very stable PNA-DNA strand-displacement complexes via double duplex invasion (helix invasion). These properties of pcPNAs make them unique and very promising ligands capable of denying the access of DNA-binding proteins to dsDNA. To elucidate the sequence-unrestricted mechanism of sequence-specific dsDNA recognition by pcPNAs, we have studied the kinetics of formation of corresponding PNA-DNA complexes at various temperatures by the gel-shift assay. In parallel, the conditions for possible self-hybridization of pcPNA oligomers have been assayed by mixing curve (Job plot) and thermal melting experiments. The data indicate that, at physiological temperatures ( approximately 37 degrees C), the equilibrium is shifted toward the pairing of corresponding pcPNAs with each other. This finding explains a linear concentration dependence, within the submicromolar range, of the pcPNA invasion rate into dsDNA at 37 degrees C. At elevated temperatures (>50 degrees C), the rather unstable pcPNA duplexes dissociate, yielding the expected quadratic dependence for the rate of pcPNA invasion on the PNA concentration. The polycationic character of pcPNA pairs, carrying the duplicated number of protonated terminal PNA residues commonly used to increase the PNA solubility and binding affinity, also explains the self-inhibition of pcPNA invasion observed at higher PNA concentrations. Melting of pcPNA duplexes occurs with the integral transition enthalpies ranged from -235 to -280 kJ.mol(-1), contributing to an anomalously high activation energy of approximately 150 kJ.mol(-1) found for the helix invasion of pcPNAs carrying four different nucleobases. A simplified kinetic model for pcPNAs helix invasion is proposed that interprets all unusual features of pcPNAs binding to dsDNA. Our findings have important implications for rational use of pcPNAs.

Figure 1

Schematics of dsDNA recognition by pcPNAs, which carry modified nucleobases, D instead of A and ^sU instead of T, along with the ordinary G and C. (a) Base pairing schemes showing that a steric clash departs D and ^sU from each other, hence significantly obstructing the complementary interactions between thus modified PNA nucleobases. Nonetheless, they can form stable pairs with normal DNA counterparts. (b) Outline of the double-duplex invasion process, which is based on the data presented in the this paper. A pair of pcPNAs can invade the dsDNA target site only in a free form after dissociation of pcPNA duplexes. As a result, the double-duplex invasion complex forms inside the DNA duplex via the strand displacement.

Figure 2

Polyacrylamide gel mobility shift assay of pcPNA–dsDNA complex formation. (a) Binding of PNA I to matched (pSD1; lanes 1–3) vs. mismatched (pSD1/m2; lanes 4–6) dsDNA targets (see Materials and Methods for description of target plasmids). Conditions: 20 mM sodium phosphate buffer (pH 7.0), 37°C, 10 h. PNA concentration varies from 0.15 μM (lanes 1 and 4) through 0.63 μM (lanes 2 and 5) to 2.5 μM (lanes 3 and 6). Sometimes, in experiments with this PNA, we observed the appearance of minor upper bands (like in lanes 2 and 3) corresponding to the secondary pcPNA–dsDNA complexes. Their formation could be due to the palindromic character of this particular recognition sequence allowing the formation of alternative strand-displacement structures. We did not observe such additional bands in case of the pcPNAs targeting to a nonpalindromic dsDNA site (10). (b) Kinetics of PNA I binding to pSD1 target as monitored by gel-shift assay. Conditions: 20 mM sodium phosphate buffer (pH 7.0), 40°C, 2 μM PNA.

Figure 3

Kinetic analysis of pcPNAs binding to the complementary dsDNA targets (C is the fraction of PNA–DNA complexes formed at a certain time). (a) Semilogarithmic plot of the kinetic data on binding of PNA I (curve 1; open circles) and PNAs II/III (curve 2; filled circles) to pSD1 and pSD2 targets, respectively. Conditions: 3.5 μM pcPNA I, 10 mM sodium phosphate buffer (pH 7.0), 37°C; 1.8 μM pcPNAs II/III, 20 mM sodium phosphate buffer (pH 7.0), 45°C. Note that the apparent upward curvature observed for two kinetic curves presented here should be ascribed to the experimental uncertainties in the measurements at each point: other similar curves (not shown) exhibited the random distribution of points near a straight line or, occasionally, somewhat downward curvature. (b) Double-logarithmic plot of the fraction of PNA–DNA complexes formed by pcPNAs II/III with pSD2 targets for 20 h at 37°C (curve 1; filled circles) or for 1 h at 50°C (curve 2; open circles) at different PNA concentrations. Conditions: 20 mM sodium phosphate buffer (pH 7.0), PNA concentration varies from 0.06 μM to 0.8 μM. The slope of linear approximations is equal to 1.1 (correlation factor: 0.98) and 1.7 (correlation factor: 0.99) at 37°C or 50°C, respectively. (Inset) Percentage of PNA–DNA complexes formed by pcPNAs II/III at 50°C in a wider range of PNA concentrations.

Figure 4

The Arrhenius plots of kinetic data for binding of 0.8 μM pcPNA I (filled circles) and 0.6 μM pcPNAs II/III (open circles) to the complementary dsDNA targets in 20 mM sodium phosphate buffer (pH 7.0). To draw these graphs, the pseudofirst-order rate constants, k_ps, were determined at different temperatures, T. The slopes of thus obtained Arrhenius plots yield the apparent activation energies of 150 kJ⋅mol⁻¹ for pcPNAs II/III(curve 2), and 156 or 92 kJ⋅mol⁻¹ for pcPNA I (curve 1) at lower and higher temperatures, respectively.

Figure 5

Formation and melting of pcPNA duplexes in 10 mM Tris⋅HCl buffer (pH 7.6) containing 10 mM NaCl. (a) Mixing curves for pcPNAs II/III obtained by variation of their molar ratios at 24°C (filled circles) and 48°C (open circles). (b) Temperature dependencies of the fraction of melted pcPNA duplexes obtained by the normalization of the absorbance vs. temperature profiles as described in Materials and Methods: PNA I, filled circles; equimolar ratio of PNAs II and III, open circles. (Inset) van't Hoff's plots obtained from the melting curves presented in the main figure (PNA I, right line; PNAs II/III, left line).