Linkage between proton binding and folding in RNA: A thermodynamic framework and its experimental application for investigating pKa shifting

ELLEN M. MOODY; JULIETTE T. J. LECOMTE; PHILIP C. BEVILACQUA

doi:10.1261/rna.7177505

Linkage between proton binding and folding in RNA: A thermodynamic framework and its experimental application for investigating pK_a shifting

Department of Chemistry, The Pennsylvania State University, University Park, Pennsylvania 16802, USA

Abstract

Perturbation of pK_a values can change the favored protonation states of the nucleobases at biological pH and thereby modulate the function of RNA and DNA molecules. In an effort to understand the driving forces for pK_a shifting specific to nucleic acids, we developed a thermodynamic framework that relates proton binding to the nucleobases and the helix–coil transition. Key features that emerge from the treatment are a comprehensive description of all the actions of proton binding on RNA folding: acid and alkaline denaturation of the helix and pK_a shifting in the folded state. Practical experimental approaches for measuring pK_as from thermal denaturation experiments are developed. Microscopic pk_a values (where k_a is the acid dissociation constant) for the unfolded state were determined directly by experiments on unstructured oligonucleotides, which led to a macroscopic pK_a for the ensemble of unfolded states shifted toward neutrality. The formalism was then applied to pH-dependent UV melting data for model DNA oligonucleotides. Folded-state pk_a values were in good agreement with the outcome of pH titrations, and the acid and alkaline denaturation regions were well described. The formalism developed here is similar to that of Draper and coworkers for Mg²⁺ binding to RNA, except that the unfolded state is described explicitly owing to the presence of specific proton-binding sites on the bases. A principal conclusion is that it should be possible to attain large pK_a shifts by designing RNA molecules that fold cooperatively.

Keywords

Footnotes

↵1 Table 1 provides a list of abbreviations, thermodynamic constants, and notation for equations.
↵2 Here, we use the convention of following an unfolding reaction.
↵3 “Ligand-free” refers to the bases in their standard, neutral forms. “Ligand-bound” refers to protonation of A and C at the imino nitrogen, which leads to cationic bases. Later, we also consider deprotonation of the imino protons of G and U/T, which leads to anionic bases. The iminonitrogens have pK_as closest to neutrality and thus make the most significant contribution to the helix–coil equilibrium.
↵4 This is the convention for describing the stability of oligonucleotides.
↵5 RNA oligonucleotides were chosen for unfolded-state studies. Since we are concerned with the effect of the phosphate backbone, the general trends should be applicable to DNA. Also, removal of the 2′-hydroxyl has no observable effect on the pK_a values of adenosine and cytidine within experimental error (Izatt et al. 1971).
↵6 This value was chosen because pK_as near neutrality have been observed experimentally for oligonucleotides.
↵7 The “flex point” is the region between the two linear pieces of the plot; it is defined more precisely in the first derivative plot, where it corresponds to an inflection point.
↵8 Δ G_x is also referred to as the “intrinsic energy” of the A ⁺·C base pair based on Jencks’s use of the term in describing the contribution of an interaction to stability in the absence of associated energetic penalties (Jencks 1975).
↵9 Scheme 3 may, however, be a good model for processes in which the reference state is not completely unfolded, and consequently only a single reference-state protonation is possible.
Article and publication are at http://www.rnajournal.org/cgi/doi/10.1261/rna.7177505.
- Accepted November 17, 2004.
- Received September 9, 2004.