Periodic Melting of Oligonucleotides by Oscillating Salt Concentrations Triggered by Microscale Water Cycles Inside Heated Rock Pores

Abstract

To understand the emergence of life, a better understanding of the physical chemistry of primordial non-equilibrium conditions is essential. Significant salt concentrations are required for the catalytic function of RNA. The separation of oligonucleotides into single strands is a difficult problem as the hydrolysis of RNA becomes a limiting factor at high temperatures. Salt concentrations modulate the melting of DNA or RNA, and its periodic modulation would enable melting and annealing cycles at low temperatures. In our experiments, a moderate temperature difference created a miniaturized water cycle, resulting in fluctuations in salt concentration, leading to melting of oligonucleotides at temperatures 20 °C below the melting temperature. This would enable the reshuffling of duplex oligonucleotides, necessary for ligation chain replication. The findings suggest an autonomous route to overcome the strand-separation problem of non-enzymatic replication in early evolution.

Keywords: DNA; denaturation; finite element simulation; nonequilibrium processes; water cycle.

Figure 1. Water cycle at the microscale.

a) A microscale water cycle between water and gas Is driven by a thermal gradient In a porous rock. b) The process can be compared to the global-scale hydrological cycle on Earth that is driven by solar radiation. c) Experimental geometry with the phase changes of water. A temperature difference between 67°C and 55°C is applied across a 500 μm gap that is filled with water and air. The black arrows indicate the direction of the microfluidic water cycle: water evaporates at the warm side, condenses into droplets at the cold side where they fall back into the water by gravity. d) Photo of the microfluidic setup. The temperature difference was created by a transparent, heated sapphire here shown on top of a cooled silicon back plate. The chamber volume (30 mm × 14 mm) was cut out of a 500 μm thin spacer foil made from Teflon. e) A bright field image through the sapphire revealed the droplets of pure condensed water on the cold wall above the gas-water interface. The bottom remained dark. f) The fluorescence image showed the labeled DNA right after a condensed water droplet fell into the solution. The vortexes are created by diffusion and convection. Also seen is the characteristic accumulation of DNA at the gas-water interface caused by the evaporation dynamics studied recently.^[8] At the top part, the condensed droplets are not seen since DNA did not evaporate in the water cycle

Figure 2. DNA denaturation at the gas-liquid interface by droplet precipitation.

a) Melting curve of 51mer dsDNA in 50 mM NaCl measured by FRET inside the reaction chamber. The dashed line is a sigmoidal fit. b) Series of images showing the microfluidic precipitation of a pure water droplet at the gas-water interface. Top: DNA fluorescence (acceptor fluorescence), bottom: FRET signal. The white square indicates the region where the FRET signal was averaged. The uncertainty of the FRET signal was estimated to 0.08. c) Averaged FRET signal at the gas-water interface over time. Experimental conditions were: 50 mM NaCl, 10 mM Tris, 1 mM EDTA, pH 7.5, 5 μM DNA, temperature gradient of 12°C (hot and cold temperatures 67°C and 55°C, respectively), average T = 61 °C, P_atm = 0.2±0.1 bar.

Figure 3. DNA melting by NaCl concentration variation.

a) Denaturation curves of the 5lmer DNA at different NaCl concentrations measured by FRET in the thermocycler. Buffer concentrations were: 10 mM Tris and 1 mM EDTA for 500, 250, 100, 50 mM NaCl with DNA at 5 μM. At 10 mM NaCl, we used 1 mM Tris and 0.1 mM EDTA. No salt buffer was used for the pure water condition (Milli-Q water). b) Melting temperature T_m versus NaCl concentration. Dashed lines are fitted curves using a hybridization model. c) An experimental FRET time trace was compared to a simulation of the droplet dissolution. d) Snapshots of the simulation at different times are shown, demonstrating how convection and diffusion leads to fast melting dynamics of the DNA. The full simulation is provided as Movie 3 in the Supporting Information.

Figure 4. Investigation of different oligonucleotides and NaCl concentrations

a) Representative snapshots (DNA or RNA fluorescence and FRET) for various NaCl concentration, nucleic acid type, length, and temperatures. The roman numbers link to Table 1. White squares indicate the region where the FRET signal was averaged. If not otherwise reported, the buffer contained 10 mM Tris, 1 mM EDTA, at pH 7.5. Oligonucleotide concentration was 10 μM for I and II, 5 μM for Ill, and 2 μM for IV. Atmospheric pressure in all experiments was 0.2 bar to enhance the probability of observing spikes in the experiment. b) FRET time traces, simulations, and experiment. The NaCl dependency of water density (ρ) plays a significant role in our microfluidic denaturation system. This is confirmed by the agreement between our experiment (points) and the simulation (solid line). When the NaCl dependency of water density was not accounted (broken line), the reannealing time scale reduced and the agreement between simulation and experiment diminished. The conditions studied here correspond to the 24mer dsDNA, 5 μM in 500 mM NaCl (T_m 43°C) in the following temperature gradient: 15°C (cold side) and 24°C (warm side).

Figure 5. Faster water cycle at reduced pressure. Comparison of the critical droplet size

(a) defined by the maximal size achievable by a condensation droplet before precipitation is observed. The lifetime (b) was measured by the time between a droplet nucleation and its precipitation into the water phase. Both (a) and (b) were measured at ambient pressure (1 bar) or 0.2 bar. c) and d) Kinetics of FRET over time at the gas-water interface over similar time span at ambient pressure (c) or 0.2 bar (d). Experimental conditions were: 50 mM NaCl, 10 mM Tris, 1 mM EDTA, pH 7.5, DNA 5 μM, a temperature gradient of 12°C between 55°C and 67°C. e) Representative snapshots of the microscale water cycle at 0.2 bar. Condensation droplets were seen as shiny spheres above the gas-liquid interface. A comparison with ambient pressure is shown in Movie 4 in the Supporting Information.