Author(s): ReilingSteffensmeier C, Marky LA
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Abstract Pseudoknots belong to an RNA structural motif that has significant roles in the biological function of RNA. An example is ribosomal frameshifting; in this mechanism, the formation of a local triplex changes the reading frame that allows for differences in the translation of mRNAs. In this work, we have used a combination of temperature-dependent UV spectroscopy and differential scanning calorimetry (DSC) to determine the unfolding thermodynamics of a set of DNA pseudoknots with the following sequence: d(TCTCTTnAAAAAAAAGAGAT5TTTTTTT), where "Tn" is a thymine loop with n=5 (PsK-5), 7 (PsK-7), 9 (PsK-9), or 11 (PsK-11). All four oligonucleotides form intramolecular pseudoknots, and the increase in the length of this loop yielded more stable pseudoknots due to higher transition temperatures and higher unfolding enthalpies. This indicates formation of one and three TAT/TAT stacks in PsK-9 and PsK-11, respectively. We have flipped one AT for a TA base pair in the core stem of these pseudoknots, preventing in this way the formation of these base-triplet stacks. The DSC curves of these pseudoknots yielded lower unfolding enthalpies, confirming the formation of a local triplex in PsK-9 and PsK-11. Furthermore, we have investigated the reaction of PsK-5 and PsK-9 with their partially complementary strands: directly by isothermal titration calorimetry and indirectly by creating a Hess cycle with the DSC data. Relative to the PsK-5 reaction, PsK-9 reacts with its complementary strand with less favorable free energy and enthalpy contributions; this indicates PsK-9 is more stable and more compact due to the formation of a local triplex. © 2016 Elsevier Inc. All rights reserved.
This article was published in Methods Enzymol
and referenced in Journal of Bioengineering & Biomedical Science