TY - JOUR
T1 - The Complementarity of the Loop to the Stem in DNA Pseudoknots Gives Rise to Local TAT Base-Triplets
AU - Reiling-Steffensmeier, Calliste
AU - Marky, Luis A.
PY - 2016
Y1 - 2016
N2 - 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.
AB - 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.
KW - DNA secondary structures
KW - Pseudoknot
KW - Thermodynamics
KW - Triplex
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U2 - 10.1016/bs.mie.2015.07.029
DO - 10.1016/bs.mie.2015.07.029
M3 - Article
C2 - 26794363
AN - SCOPUS:84954109513
SN - 0076-6879
VL - 567
SP - 413
EP - 432
JO - Methods in Enzymology
JF - Methods in Enzymology
ER -