Doctor of Philosophy (PhD)
Luis A. Marky
Pseudoknots have been found to play important roles in the biology of RNA. These stem-loop motifs are considered to be very compact and the targeting of their loops with complementary strands is accompanied with lower favorable free energy terms. We used a combination of spectroscopic (UV, CD and fluorescence), calorimetric (DSC, PPC and ITC) and kinetic (SPR) techniques to investigate: 1) Local base-triplet formation in pseudoknots; 2) energetic contributions for the association of pseudoknots with their complementary strands; and 3) the kinetic rates as a function of targeting strand length.
We investigated a set of DNA pseudoknots with sequence: d(TCTCTTnAAAAAAAAGAGAT5TTTTTTT), where “Tn” is a thymine loop with n = 5, 7, 9, and 11. The favorable folding of each pseudoknot resulted in favorable enthalpy-entropy compensation, correlated to favorable base-pair stacking contributions and unfavorable uptakes of ions and water molecules. The increase in the length of the loop yielded higher TMs, 53°C to 59°C and folding enthalpies ranging from -60 to -110 kcal/mol, resulting in a significant stabilization, ΔG°(5) = -8.5 to -16.6 kcal/mol, which is consistent with the formation of 1-2 TAT/TAT base-triplet stacks. The PPC results yielded folding volume changes, ΔVs, ranging from 18 to 23 ml/mol, indicating the higher volume of the folded pseudoknots is due to the uptake of both water (ΔnW of -11 to -24 mol H2O/mol) and ions (Δnion of -2.5 to -4.1 mol Na+/mol).
We use ITC and DSC to determine thermodynamic profiles for the reaction of pseudoknots with partially complementary strands. We obtained favorable reaction free energies terms. However, the targeting of compact pseudoknots containing local base-triplets is less favorable due to their larger folding free energy term.
The SPR data indicated that the rate of association, kon, decreases while the rate of dissociation, koff, increases as the length of the targeting strand increases, which yielded increasing KD, app.. This indicates the affinity of the target strand to the pseudoknot decreases as the length of the target strand increases. A similar trend was obtained when dissociation constants, KD, DSC, were measured from DSC Hess cycles. However, the KD, DSC were much smaller. This apparent discrepancy between these techniques is that SPR is measuring both the initial association and initial dissociation rates of steady state equilibrium states, while DSC measures true equilibrium states of the entire molecules.
Steffensmeier, Calliste, "Stability and Kinetics of DNA Pseudoknots: Formation of T∗A•T Base-Triplets and Their Targeting Reactions" (2016). Theses & Dissertations. 130.