Computational tests of a thermal cycling strategy to isolate more complex functional nucleic acid motifs from random sequence pools by in vitro selection.

The dual information-function nature of nucleic acids has been exploited in the laboratory to isolate novel receptors and catalysts from random DNA and RNA sequences by cycles of in vitro selection and amplification. This strategy is particularly effective because, unlike polypeptides with random amino acid sequences, nucleic acids with random base sequences are often capable of stably folding into defined three-dimensional structures. However, the pervasive base-pairing potential of nucleic acids is also known to lead to kinetic traps in their folding landscapes. That is, the same DNA or RNA sequence can often adopt alternative base-paired structures that are local energy minima, and these folds may interconvert very slowly. We have used simulations with nucleic acid folding algorithms to evaluate the effect of misfolding on in vitro selection experiments. We demonstrate that kinetic traps can prevent the recovery of novel families of complex functional motifs by two mechanisms. First, misfolding can lead to the stochastic loss of unique sequences in the first round of selection. Second, frequent misfolding can reduce the average activity of multiple copies of a sequence to such an extent that it will be outcompeted after multiple rounds of selection. In these simulations, adding thermal cycling to sample multiple folds of one sequence during a selection for a self-modifying catalytic activity can improve the recovery of rare examples of more complex structures. Although newly isolated sequences may fold poorly, they can represent footholds in sequence space that can be improved to reliably fold after a few mutations. Thus, it is plausible that thermal cycling by day-night cycles or other mechanisms on the primordial earth may have been important for the evolution of the first RNA catalysts, and a fold sampling strategy might be used to search for more effective nucleic acid catalysts in the laboratory today.