Thermodynamic examination of trinucleotide bulged RNA in the context of HIV-1 TAR RNA

Abstract

RNA structures contain many bulges and loops that are expected to be sites for inter-and intra-molecular interactions. Nucleotides in the bulge are expected to influence the structure and recognition of RNA. The same stability is assigned to all trinucleotide bulged RNA in the current secondary structure prediction models. In this study thermal denaturation experiments were performed on four trinucleotide bulged RNA, in the context of HIV-1 TAR RNA, to determine whether the bulge sequence affects RNA stability and its divalent ion interactions. Cytosine-rich bulged RNA were more stable than uracil-rich bulged RNA in 1 M KCl. Interactions of divalent ions were more favorable with uracil-rich bulged RNA by ;2 kcal/mol over cytosine-rich bulged RNA. The UCU-TAR RNA (wild type) is stabilized by 1.7 kcal/mol in 9.5 mM Ca 2+ as compared with 1 M KCl, whereas no additional gain in stability is measured for CCC-TAR RNA. These results have implications for base substitution experiments traditionally employed to identify metal ion binding sites. To our knowledge, this is the first systematic study to quantify the effect of small sequence changes on RNA stability upon interactions with divalent ions.

Non-Enzymatic Template-Directed Recombination of RNAs

“…In case of ligation within bulges or internal loops, short (1–2 nts) dangling ends of RNA fragments, converged by the template, seem to form spatial structures optimal for ligation to occur. Our results on preferable formation of new phosphodiester bonds within bulge loops 3 nts in size is in accordance with existing data that bulges of this sort do not tense the ribose phosphate backbone of RNA significantly [ 29 , 30 ].…”

Quantifying the Temperature Dependence of Glycine—Betaine RNA Duplex Destabilization

“…Values of K obs were determined over the range of GB concentrations where 0.2 < θ < 0.8 (27). RNA duplex unfolding enthalpy values, $\mathrm{normal\Delta }{H}_{\text{obs}}^{\mathrm{o}}$, at specific GB concentrations were determined from the slopes of van’t Hoff plots (ln K obs as a function of 1/ T ) (25, 28). …”

Improved Model to Predict the Free Energy Contribution of Trinucleotide Bulges to RNA Duplex Stability

“…Despite the natural prevalence of trinucleotide bulges, few studies have characterized the thermodynamic parameters of this motif. 20 , 21 In fact, the current predictive model is based on free energy values for only six trinucleotide bulges, and it attributes a 3.2 kcal/mol free energy penalty to all trinucleotide bulges, regardless of sequence. 9 The aim of this study was to determine whether a sequence-independent model is the most accurate approach for predicting the free energy contribution of trinucleotide bulges.…”