Drying droplets could have been compartments during the origin of life

Primary source information:

Title: Impact of wet-dry cycling on the phase behavior and compartmentalization properties of complex coacervates

Authors: Hadi M. Fares, Alexander E. Marras, Jeffrey M. Ting, Matthew V. Tirrell, and Christine D. Keating.

Journal: Nat Commun 11, 5423 (2020)

Think about combining oil and water: droplets of oil might become interspersed in the water, but the oil and water do not mix and will soon separate into distinct regions.  Similarly, when oppositely charged polymers are dissolved in water at high enough concentrations, those polymers can form dense droplets that do not mix with the surrounding solution (Fig 1).  Droplet formation is dependent on electrostatic interactions between the charged polymers, and the strength of these electrostatic interactions depends on the polymer concentration and the salt concentration.

Figure 1: Mixtures of positively and negatively charged polymers can form dense droplets that do not mix with the surrounding solution.  The droplets themselves, and the surrounding solution, are each distinct liquid phases.  By changing the concentrations of the polymers (or by changing the concentration of salts), the entire system can reversibly transition to a mixed state where the polymers are dissolved in the bulk solution (1 phase).

Charged polymers may have formed billions of years ago on Earth, before the formation of the first cells.  Cycles of dehydration and rehydration are helpful for forming polymers via condensation reactions, and so many researchers favor the hypothesis that life originated in shallow pools on land. Would these charged polymers still be able to form droplets during cycles of dehydration and rehydration?  And what roles might these droplets have had during the origin of life on Earth?  These are big questions that Fares et al. seek to understand with their research (Fig 2).

Figure 2: On the early Earth, shallow pools may have gone through cycles of dehydration and rehydration over the course of days or months.  These environments are productive hotspots for chemical reactions that might have been important for the origin of life.  Researchers want to understand how droplets fare during these wet/dry cycles, and what roles these droplets might have in origin of life chemistry.

First, the researchers selected polymers that plausibly could have been available on the early Earth, and then measured a phase diagram for these polymers (Fig 3, top). We often use phase diagrams to determine whether a compound (for example, water) will be a solid, liquid, or gas at a given temperature and pressure. In exactly the same way, these researchers created a phase diagram to learn at what salt and polymer concentrations would their charged polymers mix into the surrounding solution (1 phase), and at what concentrations would the polymers form droplets (2 phase). The researchers observed that dehydration would change a sample’s position within the phase diagram (Fig 3, bottom): for example, a sample that had droplets initially might mix (2 phase –> 1 phase) during the course of dehydration. But these changes are completely reversible! As soon as the researchers rehydrated their samples, the droplets re-formed. So during periods of cyclic dehydration/rehydration on the early Earth, droplets readily dissolve and re-form.

Figure 3: (Top) Phase diagram shows where charged polymers form droplets (pink 2phase region), and where the polymers dissolve into the bulk solution (black 1 phase) as a function of NaCl and polymer concentration. (Bottom) During dehydration, the researchers observe that the droplets can disappear, and then re-form upon rehydration.  Time = 0min is shown as a black square on the phase diagram, and Time =  160min is shown as a white square.  Note: “Charge concentration” weights the polymer concentration by the number of charges on each polymer.  Note 2: the size of the pink circles within the 2 phase region are related to the size and number of droplets in the sample.

Next, the researchers added RNA molecules, and observed that most of the RNA enters the droplets (Fig 4)! As the researchers dehydrated their sample, they observed that the concentration of RNAs inside the droplets remained constant. Next, the researchers intentionally disabled the florescence of RNAs within a defined spatial region (Fig 5, white circle). This technique allowed the researchers to measure the diffusion of new RNAs into that region. They observed that RNA diffusion into the droplets increased as the sample dried. So as the environment became more dehydrated, the surrounding solution became more similar to the droplets. Upon rehydration, the droplets and surrounding solution returned to their initial state.

Figure 4: (Left) Upon adding fluorescent RNA to the droplet sample, the researchers observed that most of the RNA molecules partitioned into the droplets.  As the sample dehydrated, the concentration of RNA molecules inside the droplets remained constant, but the concentration of the RNAs outside the droplets increased as the surrounding solution evaporated.  (Right) Microscope image confirms that after rehydration, the concentration of florescent RNAs in the droplets is again much higher than the surrounding solution.   Note: the partition coefficient is the ratio of RNA concentrations inside:outside the droplets.
Figure 5: During florescence recovery after photobleaching (FRAP), the researchers disabled RNA florescence within a small region (white circle), then watched as the remaining fluorescent RNAs diffused into that region from the outside.  Using these observations, the researchers quantified diffusion of the RNAs, and they observed that diffusion increased as the sample dried.  This means that RNAs enter/exit the droplets more readily as the sample dehydrates.

These researchers have used a variety of techniques to understand the properties of charged polymer droplets during cycles of dehydration and rehydration. Their results suggest that droplet properties do change during dehydration, but upon rehydration the droplets return to their initial state. Additionally, the droplets are able to sequester high concentrations of RNA, which might have been a critical step towards the origin of life.

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