Energetics, Ion, and Water Binding of the Unfolding of AA/UU Base Pair Stacks and UAU/UAU Base Triplet Stacks in RNA Carolyn E. Carr, Irine Khutsishvili, † and Luis A. Marky* Department of Pharmaceutical Sciences, University of Nebraska Medical Center, 986025 Nebraska Medical Center, Omaha, Nebraska 68198-6025, United States * S Supporting Information ABSTRACT: Triplex formation occurs via interaction of a third strand with the major groove of double-stranded nucleic acid, through Hoogsteen hydrogen bonding. In this work, we use a combination of temperature-dependent UV spectrosco- py and differential scanning calorimetry to determine complete thermodynamic profiles for the unfolding of polyadenylic acid (poly(rA))·polyuridylic acid (poly(rU)) (duplex) and poly(rA)·2poly(rU) (triplex). Our thermody- namic results are in good agreement with the much earlier work of Krakauer and Sturtevant using only UV melting techniques. The folding of these two helices yielded an uptake of ions, Δn Na + = 0.15 mol Na + /mol base pair (duplex) and 0.30 mol Na + /mole base triplet (triplex), which are consistent with their polymer behavior and the higher charge density parameter of triple helices. The osmotic stress technique yielded a release of structural water, Δn W = 2 mol H 2 O/mol base pair (duplex unfolding into single strands) and an uptake of structural water, Δn W = 2 mol H 2 O/mole base pair (triplex unfolding into duplex and a single strand). However, an overall release of electrostricted waters is obtained for the unfolding of both complexes from pressure perturbation calorimetric experiments. In total, the ΔV values obtained for the unfolding of triplex into duplex and a single strand correspond to an immobilization of two structural waters and a release of three electrostricted waters. The ΔV values obtained for the unfolding of duplex into two single strands correspond to the release of two structural waters and the immobilization of four electrostricted water molecules. ■ INTRODUCTION The overall physical and chemical properties of a nucleic acid molecule depend on base pairing, base stacking, ion binding, and hydration. 1-4 Experimental and theoretical investigations have indicated that nucleic acid double helices are heavily hydrated. 5-7 For instance, X-ray and NMR investigations have shown that water molecules create an ordered structure called “the spine of hydration” in the minor groove of A-T base pairs in B-DNA. 8-12 Hydration of the major groove and other DNA conformations has also been reported. 13-18 However, in spite of extensive investigations on the hydration of nucleic acids, the details of hydration as it relates to conformation, sequence, and nucleotide composition remain unknown. The reason for this is the presence of two distinct types of hydrating water molecules: structural (around polar and nonpolar groups) and electrostricted (around charges). 19 These two types of water are difficult to detect and differentiate, complicating the measurement and analysis of their physical properties, especially their molar volume at the surface of a nucleic acid. Furthermore, the overall hydration of a nucleic acid molecule is closely associated with its number and type of hydrated counterions. For instance, in the interaction of divalent ions with nucleic acids, their close association involves the overlapping of their hydration shells, resulting in a release of water molecules; 20-22 the magnitude of the effect is determined by the position of this cation relative to the surface of DNA. A larger dehydration effect takes place in the formation of inner-sphere counterion-DNA complexes relative to the formation of outer-sphere complexes because of changes in their molar volumes. Polyadenylic acid (poly(rA)) and polyuridylic acid (poly- (rU)) form a double helix in an equimolar (1:1) mixture. Both temperature and salt affect the type of complex in solution, and depending on the experimental solution conditions the different types of species (single strand, duplex, and/or triplex) can be interconverted. The work of Krakauer and Sturtevant shows a phase diagram of the different helix-to-coil transitions or reactions with poly(rA) and poly(rU). 23 There are four types of reactions: reaction I is the simple unfolding of a duplex poly(rA)·poly(rU) into single strands and can be achieved by an increase in temperature at lower salt conditions. Reaction II is the unfolding of the 1:2 triplex poly(rA)·2poly(rU), which is the melting of the triplex in a single step to form single strands and occurs at low temperature and high salt. Reaction III is the unfolding of the triplex into a duplex and a single strand poly(rA)·poly(rU) + poly(rU), which refers to the removal of the third or Hoogsteen strand and occurs at lower salt Received: June 11, 2018 Published: June 22, 2018 Article pubs.acs.org/JPCB Cite This: J. Phys. Chem. B XXXX, XXX, XXX-XXX © XXXX American Chemical Society A DOI: 10.1021/acs.jpcb.8b05575 J. Phys. Chem. B XXXX, XXX, XXX-XXX Downloaded via UNIV OF SUSSEX on July 9, 2018 at 06:30:28 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.