(426i) Driving Towards Selection of Folded and Highly Structured Nucleic Acid Templates

Obianyor, C., Georgia Institute of Technology
Grover, M. A., Georgia Institute of Technology
Lozoya Colinas, A., Georgia Institute of Technology
Hud, N., Georgia Institute of Technology
Nucleic acids typically exist in their thermodynamic duplex form, after the replication of their strands. However, in order to undergo a new round of replication, without the use of enzymes, these strands must be kinetically trapped in their single stranded form. This problem of strand separations is commonly referred to as the product inhibition problem. In aqueous solvents, the replication of the strands is hindered by preference for the strands to remain in their duplex form, in addition to their high denaturing temperatures. Therefore, it is important to find a plausible prebiotic mechanism to overcome the product inhibition problem, in order to understand the chemical origins of life on the early Earth, and to improve replication efficiency.

One of the methods proposed to overcome the product inhibition problem is the use of non-aqueous solvents. These non-aqueous solvents, made similarly to deep eutectic solvents (DES), are highly viscous in nature. By tuning the viscosity of water and the non-aqueous solvent mixtures, researchers have shown that the kinetics of DNA nanostructure self-assembly can be controlled1. Additionally, researchers have also demonstrated that folded-structured templates can be kinetically trapped in their single stranded form, using non-aqueous solvents. The trapping of these folded states of the DNA templates then facilitate the copying of highly structured nucleic acid templates, overcoming product inhibition2.

In our work, we aim to understand the mechanisms by which viscous solvents facilitate the formation of kinetic traps, and promote intramolecular folds. We have designed model environmental cycles in which we can drive off water from an aqueous pool containing a hydrated viscous solvent, and DNA, while simultaneously separating the template strand. The drying of the pool creates a non-aqueous environment which lowers the melting temperature of the DNA template, subsequently creating a lower energy barrier over which to denature the template. As the pool cools down from denaturing temperatures, we can use an interplay of cooling rates to favor the formation of intramolecular folds in our model cycle. On the contrary, the rehydration of the pool during cooling, drives the system in the opposite direction, i.e. towards the formation of intermolecular duplexes. Therefore, by varying the cooling rates, and viscosity of the solvent/water content, we have created a scenario in which we can switch between the kinetic forms and thermodynamic forms of a structured nucleic acid template. The formation of these intramolecular folds are monitored using gel electrophoresis techniques. Overall, our experimental results demonstrate that the formation of intramolecular structures are promoted by low temperatures, and low relative humidity. These conditions enable us maintain the kinetic folded structures of nucleic acid templates over long periods of time, a feat unattainable in aqueous solvents. In addition, we also investigate the thermodynamic response of the solvent using numerical solving methods in MATLAB. We plan to extend our work to other solvents, and investigate the impacts of the solvent on the copying of the template strand in our model cycle.

Understanding the folding of nucleic acids in response to environmental changes such as viscosity, and temperature ramp rate, will enable researchers build nanotechnology that can use directed folding and unfolding of the templates to perform targeted responses. Additionally, our system demonstrates a plausible pathway nature could have taken for non-enzymatic replication of structured nucleic acid templates, on the early earth.


  1. Gállego, I.; Grover, M. A.; Hud, N. V., Folding and imaging of DNA nanostructures in anhydrous and hydrated deep-eutectic solvents. Angewandte Chemie International Edition 2015, 54 (23), 6765-6769.

  1. He, C.; Gállego, I.; Laughlin, B.; Grover, M. A.; Hud, N. V., A viscous solvent enables information transfer from gene-length nucleic acids in a model prebiotic replication cycle. Nature Chemistry 2017, 9 (4), 318-324.