Water/Oil Emulsions with Controlled Droplet Sizes for In Vitro Selection Experiments

In the early history of life, RNA might have had many catalytic functions as ribozymes that do not exist today. To explore this possibility, catalytically active RNAs can be identified by in vitro selection experiments. Some of these experiments are best performed in nanodroplets to prevent diffusion between individual RNA sequences. In order to explore the suitability for the large-scale in emulsio selection of water-in-oil emulsions made by passing a mixture of mineral oil, the emulsifier ABIL-EM90, and a few percent of an aqueous phase through a microfluidizer, we used dynamic light scattering to characterize the size of aqueous droplets dispersed throughout the oil. We found that seven or more passes through the microfluidizer at 8000 psi with close to half molar inorganic salts and 10% polyethylene glycol produced droplets with sizes below 100 nm that were ideal for our purposes. We also identified conditions that would produce larger or smaller droplets, and we demonstrate that the emulsions are stable over weeks and months, which is desirable for different types of in vitro selection experiments.