Hairpins under tension: RNA versus DNA

Abstract

We use optical tweezers to control the folding and unfolding of individual DNA and RNA hairpins by force. Four hairpin molecules are studied in comparison: two DNA and two RNA ones. We observe that the conformational dynamics is slower for the RNA hairpins than for their DNA counterparts. Our results indicate that structures made of RNA are dynamically more stable. This difference might contribute to the fact that DNA and RNA play fundamentally different biological roles in spite of chemical similarity.

Figure 1.

The four studied hairpins. From left to right: DNA10, DNA18, RNA10 and RNA18. They exhibit a 13 base pair stem of the same sequence and loops of either 10 (DNA10 and RNA10) or 18 (DNA18 and RNA18) nucleotides. Apart from the replacement of DNA's Thymine by RNA's Uracil, the base sequences are the same for the DNA and RNA hairpins. The hairpins are presented with their 5′ ends at the bottom left side.

Figure 2.

Schematic representation of a molecular construct linked to functionalized beads. A DNA (resp. RNA) hairpin is inserted between two DNA/DNA (resp. RNA/DNA) handles. Each handle has a crystallographic length of about 0.5 μm (resp. 0.4 μm).

Figure 3.

Schematic representation of the simplified energy landscape used in our theoretical description. The state of the folded hairpin occurs at x = 0 and the state of the unfolded hairpin at x = L. The distances between the transition state and the folded and unfolded states are denoted by x_→ and x_←, respectively. By definition, we have L = x_→ + x_←.

Figure 4.

Force versus extension curves at 50 nm/s for the four different hairpins. The darker curves correspond to the stretching of the construct, whereas the lighter ones represent the release process. For both the DNA and RNA hairpins, the hysteresis between unfolding and folding increases with loop length. For the same hairpin sequence, the hysteresis is larger for the RNA construct than for the DNA one. The inset provides a closer look to the force flips observed with DNA10. In this inset, the unfolding and folding curves are shifted vertically for clarity.

Figure 5.

Force versus extension curves for DNA10 at 50, 150, 300 and 450 nm/s (from left to right). The darker curves correspond to the stretching of the construct, whereas the lighter ones represent the release process. The arrows indicate the force of the unfolding and folding events. The force flips observed with DNA10 at 50 nm/s vanishes at 150 nm/s and above. The hysteresis between unfolding and folding increases with the pulling velocity.

Figure 6.

Histograms of the hysteresis measured at 150 nm/s on the four different hairpins. The experimental distributions are fitted to calculated convolution of the unfolding and folding probability distributions (solid line), as explained in Materials and Methods and Supplementary Section S1 (Equations (4–6)). For each fitted histogram, the root mean squared error (rmse) is in the range (2.0–3.0) × 10⁻³. For DNA10 the data come from 46 stretch/release cycles on 12 molecules. DNA18: 95 cycles on 13 molecules. RNA10: 152 cycles on 44 molecules. RNA18: 150 cycles on 21 molecules.

Figure 7.

Spontaneous transitions between folded and unfolded hairpin states at constant extension. (Top panel) Force recorded on RNA10 (green) as a function of time, while the extension is constant apart from stepwise increases (black). Force flipping is observed for each extension value. (Second panel) Zoom on an extension where both states have a similar probability of occupation: the average dwell times are 1.77 s in the unfolded state and 3.1 s in the folded state. Presented time range: 80 s. (Third panel) Force recorded on DNA10 (orange) as a function of time, while the extension is constant apart from stepwise increases (black). Force flipping is observed for each extension value. (Bottom panel) Zoom on an extension where both states have a similar probability of occupation: the average dwell times are 0.11 s in the unfolded state and 0.25 s in the folded state. Presented time range: 10 s. The transition rate is much higher for DNA10 than for RNA10.