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Albany 2019: 20th Conversation - Abstracts

category image Albany 2019
Conversation 20
June 11-15 2019
Adenine Press (2019)

Thermodynamic Stability and Molecular Dimensions of Pseudoknots

Pseudoknots are prevalent RNA structures that play significant roles in the biological function of RNA. In this work, we have investigated the unfolding thermodynamics and solution molecular dimensions of two pseudoknots with stem loop complementarity to model the frameshift occurrence of ribosomal pseudoknots.

We used a combination of UV and DSC melting techniques and SAXS to determine the thermodynamic stability and solution molecular dimensions of two pseudoknots with sequences: d(TCTCTT11AAAAAAAAGAGAT5TTTTTTT) (PsK11-DNA ) and r(UCUCU-U11AAAAAAAAGAGAU5UUUUUUU) (PsK11-RNA), “T11” and “U11” are the loop sequences complementary to one stem. We used three buffer conditions: 10 mM Sodium Phosphate (NaPi), 10 mM Sodium Cacodylate (NaCac) and 10 mM Sodium Cacodylate with 10 mM MgCl2 (NaCac & Mg2+).

Both pseudoknots form intramolecularly with PsK11-DNA in the “B” conformation and PsK11-RNA in the “A” conformation, determined by their circular dichroism spectra. These pseudoknots were slightly more stable in the NaPi buffer by 0.9 kcal/mol. However, the addition of Mg 2+ to the NaCac buffer significantly increased their stability, by 10.8 (PsK11-DNA) and 8.7 kcal/mol (PsK11-RNA). Overall, PsK11-RNA is more stable by 5 kcal/mol, due to its higher TM and unfolding enthalpy. However, this is reduced to 3 kcal/mol in the presence of Mg 2+. The TM-dependencies on salt yielded similar ion uptakes of ~3 mol Na+ /mol in both buffers and ~0.1 mol of Na+ /mol in NaCac & Mg 2+. The TM-dependencies on osmolyte (ethylene glycol) yielded lower water uptakes for PsK11-DNA by 13 mol H,2O/mol, in NaPi and NaCac. However, in the presence of Mg 2+ PsK11-DNA has a greater water uptake by 10 mol H2O/mol.

The SAXS data indicate PsK11-DNA has a length of ~80 Å and width of ~32 Å, while PsK11-RNA has a length of ~65 Å and width of ~30 Å; both molecules have a thickness of about 14 Å. Thus, PsK11-RNA is more compact.

The main conclusion is that the higher compactness of the RNA pseudoknot is consistent with its greater thermodynamic stability.

Supported by Grant MCB-1122029 from the National Science Foundation.

Calliste Reiling-Steffensmeier
Luis A. Marky

Department of Pharmaceutical Sciences
College of Pharmacy
University of Nebraska Medical Center
Omaha, NE 68198-6025

Ph: (402) 559-4628
Fx: (402) 559-9543
Email: lmarky@unmc.edu