On the stability of peptide nucleic acid duplexes in the presence of organic solvents

Abstract

Nucleic acid double helices are stabilized by hydrogen bonding and stacking forces (a combination of hydrophobic, dispersive and electrostatic forces) of the base pairs in the helix. One would predict the hydrogen bonding contributions to increase and the stacking contributions to decrease as the water activity in the medium decreases. Study of nucleobase paired duplexes in the absence of water and ultimately in pure aprotic, non-polar organic solvents is not possible with natural phosphodiester nucleic acids due to the ionic phosphate groups and the associated cations, but could be possible with non-ionic nucleic acid analogues or mimics such as peptide nucleic acids. We now report that peptide nucleic acid (PNA) (in contrast to DNA) duplexes show almost unaffected stability in up to 70% dimethylformamide (DMF) or dioxane, and extrapolation of the data to conditions of 100% organic solvents indicates only minor (or no) destabilization of the PNA duplexes. Our data indicate that stacking forces contribute little if at all to the duplex stability under these conditions. The differences in behaviour between the PNA and the DNA duplexes are attributed to the differences in hydration and counter ion release rather than to the differences in nucleobase interaction. These results support the possibility of having stable nucleobase paired double helices in organic solvents.

Figure 1.

Plots of (A) T_m and (B) ΔG⁰ of PNA1·PNA2 (solid diamond), PNA1·DNA2 (solid square), DNA1·DNA2 (open square) and DNA3·DNA4 (solid triangle) as a function of the amount of DMF in the medium. (C) Plots of T_m of PNA1·PNA2 (open triangle) and DNA3·DNA4 (open square), as a function of the amount of formamide in the medium, compared to that of PNA1·PNA2 (dashed line) and DNA3·DNA4 (dotted line), as a function of the amount of DMF in the medium (DMF data taken from Figure 1A). The aqueous buffer was 10 mM phosphate buffer containing 100 mM NaCl and 0.1 mM EDTA, pH 7.2 ± 0.01 (data in Tables 1, S2–S5).

Figure 2.

Plots of T_m of self-complementary (hairpin) PNAs PNA3 (solid inverted triangle) and PNA4 (solid circle) as a function of the amount of (A) DMF and (B) dioxane in the medium. Plots of T_m of hairpin DNA control DNA5 as a function of the amount of (C) DMF (open triangle) and (D) dioxane (open square) in the medium. The aqueous buffer was 10 mM phosphate buffer containing 100 mM NaCl and 0.1 mM EDTA, pH 7.2 ± 0.01 (PNA3: H-AGAG-(eg1)₃-CTCT-Lys-NH₂, PNA4: H-ACAG-(eg1)₃-CTGT-Lys-NH₂, DNA5: 5′-AGA GTT TTC TCT-3′) (data in Tables S6–S9).

Figure 3.

Plots of T_m of PNA5 · PNA6 (open inverted triangle) containing tricyclic thymine (tT) and its control PNA6 · PNA7 (open circle), as a function of the amount of DMF in the medium (data in Table S10). The aqueous buffer was 10 mM phosphate buffer containing 100 mM NaCl and 0.1 mM EDTA, pH 7.2 ± 0.01 (PNA5: H-tT-GTA GAT CAC T-NH₂, PNA6: H-AGT GAT CTA C-NH₂, PNA7: H-GTA GAT CAC T-NH₂).

Figure 4.

Thermal stabilities of T·T, A·A and C·T mismatched PNA duplexes in DMF (data in Table S11). Plots of (A) T_m (solid diamond) and (B) ▵G⁰ (open diamond) of PNA1·PNA8 as a function of the amount of DMF in the medium. Plots of (C) T_m (solid triangle) and (D) ▵G⁰ (open triangle) of PNA2·PNA9 as a function of the amount of DMF in the medium. Plots of (E) T_m (solid square) and (F)▵G⁰ (open square) of PNA2·PNA10 as a function of the amount of DMF in the medium. The aqueous buffer was 10 mM phosphate buffer containing 100 mM NaCl and 0.1 mM EDTA, pH 7.2 ± 0.01.