Targeting of a DNA Pseudoknot is Hindered Due to Formation of Base-Triplet Stacks

Abstract

The formation of a triplex in RNA pseudoknots has been shown to be involved in −1 ribosomal frameshifting. We are using DNA pseudoknots to determine the role of triplex stacking in their overall stability and whether pseudoknots can be disrupted using complementary targeting strands. We used a combination of UV spectroscopy, differential scanning calorimetry (DSC), and isothermal titration calorimetry (ITC) to investigate the unfolding of two DNA pseudoknots, PsKn-1 and PsKn-2, with sequences: d(TCTCTTnA8GAGAT5T7), where T5 and Tn (n = 5, 9) are loops, respectively. These two pseudoknots are also targeted with their partially complementary strands with sequence: d(CTT7AnAGAGA), where n = 5, 9 respectively.Both pseudoknots unfold through biphasic intramolecular transitions. PsKn-2 is more stable and unfolds with a higher enthalpy of 46 kcal/mol, due to the stacking of three TAT base triplets. The targeting reaction of PsKn-1 with its complementary strand yielded favorable free energy contributions, indicating the complementary strand was able to disrupt the pseudoknot. However, the targeting reaction of PsKn-2 with its complementary strand did not take place, consistent with its higher stability. This confirms the formation of three TAT base triplets (two TAT/TAT base triplet stacks). Different targeting reactions using longer complementary stands will be presented in order to determine if these strands can disrupt the more stable pseudoknot. Furthermore, the overall thermodynamic data will be compared with targeting kinetic data. Supported by Grant MCB-1122029 from the NSF and GAANN-Fellowship from the Department of Education.