Abstract
RNA molecules fold into three-dimensional secondary structure motifs as a result of intramolecular binding of complementary regions of the RNAs. Secondary structure facilitates RNA function, including many processes critical to regulation of gene expression. As such, disruption of RNA structure can yield greater insight into the cellular functionality of RNA molecules. Disruption can be accomplished through binding RNA targets to a complementary DNA oligonucleotide, a technique which is also utilized in antisense gene therapies. However, it is difficult to design single oligonucleotides which quickly and stably bind and unfold secondary structure targets. We aimed to determine if rates of secondary structure unfolding can be accelerated by binding multiple oligonucleotides to targets. We began by adapting X-Probe architecture to design a new system of secondary structure targets, oligonucleotide opening strands, and fluorescent probes for reaction monitoring. Using this system, we measured the hybridization of single and multiple oligonucleotides of varying toehold lengths to DNA hairpins. Our results indicate that while multiple opening strand binding can successfully increase hairpin opening rates, some combinations of opening strands can also result in reduced rates of hairpin opening. For multiple oligonucleotide binding, larger total number of toehold nucleotides corresponded to larger equilibrium bound fractions of hairpin, but not to faster rates of hairpin opening. Lastly, we also noted significantly increased rates of hairpin opening in 5xPBS as compared to salty 1xTE.