Viscous Solvents Enable a Model Prebiotic Nucleic Acid Replication Cycle

Increased control of molecular self-assembly is important for the further development of nucleic acid-based technologies. While nucleic acid technology research has been carried out almost exclusively in aqueous buffer, alternative solvents could increase our ability to direct nucleic acid assembly. Deep eutectic solvents (DES), closely related to ionic liquids, are characterized by significantly lower melting points than their separate components. Recently, DES have been shown to preserve nucleic acid base pairing and duplex formation, offering a relatively unexplored medium for directing nucleic acid assembly.

In this work, we show that DES provide a highly effective means for tuning nucleic acid thermodynamics and kinetics. The power of alternative solvents to alter a nucleic acid assembly pathway is illustrated by our ability to circumvent the “strand inhibition problem” by thermally cycling nucleic acids in a DES. Strand inhibition describes the propensity of a long nucleic acid polymer to form a duplex with its complementary strand rather than to serve as a template for assembly of oligonucleotides. This longstanding problem in the field of prebiotic chemistry has limited demonstration of a prebiotic nucleic acid replication mechanism.

We address the problem of nucleic acid strand inhibition by employing highly viscous solvents to control the annealing rates of long templates and shorter oligonucleotides. In a viscous environment, nucleic acid mobility is slowed in a length-dependent manner, so that long nucleic acid polymers (such as template and copy strands) are kinetically trapped. Meanwhile, short oligonucleotides can diffuse quickly and bind to their complementary targets on the templates. Template duplex formation is further slowed by the formation of intramolecular secondary structure on the template single strands. Based on these thermodynamic and kinetic effects, we show that thermal cycling in a viscous environment can be used to copy a gene-length region (over 300 nucleotides) of a longer mixed sequence template, using both DNA and RNA systems.