A Method for the Sensitive Detection of Base Pairing and Stacking Energies for Damaged and Noncanonical Sequences from Single Molecule Unfolding Experiments.

Noncanonical and damaged bases occur with significant frequency and recognizing them is an important step in many nuclear processes including DNA repair, homologous recombination, and transcription. Yet while both the pairing and stacking energies of the canonical bases are well known in theory and experiment, the energetics of noncanonical bases are not well understood. Incorporating noncanonical bases into DNA hairpins affords the opportunity to quantify the changes in stacking and pairing interactions during forced hairpin unfolding. Though the measurement accuracy of this technique is quite high, technical challenges are significant and the overall throughput is challenging. In this chapter, we present a method for measuring the pairing and stacking energies of such noncanonical base pairs by incorporating the hairpin defect in the lower stem and performing optical tweezers force-unfolding experiments. During unfolding, this stem frays apart before the upper half, enhancing detection and facilitating rapid and sensitive characterization of these defects.