Background: Pseudoknots have been found to play important roles in RNA function, examples include ribosomal frameshifting and in the 5' UTR of mRNA as riboswitches. In RNA frameshifting, there is a local formation of base-triplet stacks within the pseudoknot, increasing the stability of the terminal stem. The interaction of this triplex structure with the ribosome might help with the high-efficiency of frameshifting. The main objective of this work is to mimic RNA pseudoknots using DNA oligonucleotides for the control of gene expression. Specifically, we have designed a pair of DNA pseudoknots with different length in one of the loops to mimic the formation of a local triple helix, shown within RNA pseudoknots of the human telomerase.
Methods: We have used a combination of temperature-dependent UV spectroscopy and calorimetric techniques to determine the thermodynamics for the unfolding of the pseudoknots and their targeting with complementary strands. The unfolding data is then used to create thermodynamic (Hess) cycles that correspond to each of the targeting reactions. The resulting data is then compared with the thermodynamic enthalpy data obtained directly from isothermal titration calorimetry.
Results: UV melting curves of each pseudoknot show transitions with TMs independent of strand concentration, which confirms their intramolecular formation. Analysis of the differential scanning calorimetry (DSC) curves shows the pseudoknot with the longer thymine loop (PsK-9 ) to be more stable, by -5.7 kcal/mol, and to unfold with a higher enthalpy of 27.4 kcal/mol. The targeting of each pseudoknot yielded favorable reaction free energy contributions that were enthalpy driven. However, the disruption reaction of PsK-9 took place with a less favorable free energy term, by 0.7 kcal/mol, and less favorable enthalpy term, by 4.5 kcal/mol.
Conclusion: The thermodynamic unfolding data showed that PsK-9 is more stable or more compact, due to the involvement of three loop thymines of PsK-9 in forming three T*AT base-triplets, or two T*AT/ T*AT base-triplet stacks, in the stem of this pseudoknot. The targeting thermodynamic data indicated that each complementary strand is able to disrupt the pseudoknots. However, the disruption of PsK-9 takes place with a less favorable free energy contribution, confirming the formation of a short and local triplex.
Keywords: Targeting nucleic acids, intramolecular DNA structures, pseudoknots, antisense, thermodynamics, ITC, DSC