Mechanical characterization of base analogue modified nucleic acids by force spectroscopy

Abstract

We use mechanical unfolding of single DNA hairpins with modified bases to accurately assess intra- and intermolecular forces in nucleic acids. As expected, the modification stabilizes the hybridized hairpin, but we also observe intriguing stacking interactions in the unfolded hairpin. Our study highlights the benefit of using base-modified nucleic acids in force-spectroscopy.

Fig. 1. Experimental setup and stability of DNA hairpins. (A) Structure of tC; red represents additional part compared to cytosine. (B) Schematic representation of the optical tweezers. Inset shows the hairpins used with tC modification(s) in different positions in red. (C) Five force–distance cycles for an unmodified and a three-tC hairpin (3-tC) with unfolding (red) and folding curves (blue) and the corresponding rupture force histograms. The black horizontal lines indicate the median force.

Fig. 2. Mechanical and thermal stabilities of tC-modified DNA hairpins. Changes in unfolding (ΔF_u, red) and folding force (ΔF_f, blue), free energy (ΔΔG, purple) from mechanical (1 M NaCl) and melting temperature (ΔT_m, black, 5 mM NaCl) measurements comparing tC-modified and unmodified hairpins.

Fig. 3. Mechanical and thermal stabilities of tC-modified hairpins. (A) DNA hairpins 2-tC(opp) and 2-tC(stack) with tC modifications. (B and C) Changes in unfolding force (ΔF_u, red), folding force (ΔF_f, blue), free energy (ΔΔG, purple) at 1 M NaCl and melting temperature (ΔT_m, black) at 5 mM NaCl for 2-tC(opp) and 2-tC(stack) (B) and both experiments at 50 mM NaCl for 1-tC and 2-tC(stack) (C) compared to the unmodified hairpin. (D) Calculated change in free energy (ΔG) with temperature for unmodified, 1-tC and 2-tC(stack) hairpins. Vertical line represents the temperature where optical tweezers experiments were conducted and shaded region corresponds to the melting temperatures of the hairpins.