Thermodynamic parameters for prediction of RNA duplex stability are reported. One parameter for duplex initiation and 10 parameters for helix propagation are derived from enthalpy and free-energy changes for helix formation by 45 RNA oligonucleotide duplexes. The oligomer sequences were chosen to maximize reliability of secondary structure predictions. Each of the 10 nearest-neighbor sequences is wellrepresented among the 45 oligonucleotides, and the sequences were chosen to minimize experimental errors in AGI at 37°C. These parameters predict melting temperatures of most oligonucleotide duplexes within 5°C. This is about as good as can be expected from the nearest-neighbor model. Free-energy changes for helix propagation at dangling ends, terminal mismatches, and internal G-U mismatches, and free-energy changes for helix initiation at hairpin loops, internal loops, or internal bulges are also tabulated.Stabilities of RNA duplexes and secondary structures of RNAs are often predicted by using free-energy parameters from a nearest-neighbor model (1-5). Sometimes, however, predictions are inconsistent with experimental data (6-10). One factor hindering successful predictions is that the reliability of parameters was limited by the availability of model oligonucleotides (2). Recent breakthroughs in synthesis of RNA oligoribonucleotides (11-16) permit design of oligonucleotides to provide improved parameters. This paper presents thermodynamic parameters derived from data on 45 complementary RNA duplexes. The parameters are able to predict the stabilities of RNA duplexes within the limits ofthe nearest-neighbor model. MATERIALS AND METHODSChoice of Sequences. Sequences were selected to minimize errors in the free-energy change for duplex formation at 37°C, AG97 (17,18). Thus, as much as possible, melting temperatures at 0.1 mM are near 37°C to minimize extrapolation. The oligomers were also chosen to independently represent all 10 nearest-neighbor sequences comprising Watson-Crick base pairs.Oligonucleotide Synthesis. Oligonucleotides not reported elsewhere were synthesized on solid support using phosphoramidite procedures and purified as described (11,19). Purities were confirmed by high-performance liquid chromatography for all oligomers.Thermodynamic Parameters. Absorbance vs. temperature melting curves were measured in 1 M NaCl/0.005 M Na2HPO4/0.5 mM EDTA (disodium salt), pH 7, as described (11). Concentrations were determined from the high-temperature absorbance using extinction coefficients calculated as described (20). In units of 0.1 mM-1 cm-1, calculated hightemperature extinction coefficients at 280 nm not reported elsewhere are as follows: GUGCAC, 2.77; GUCUAGAC, 3.66; GAUAUAUC, 3.05; GUAUAUAC, 3.00. Thermodynamic parameters of helix formation were obtained by two methods. (t) Individual melting curves were fit to a two-state model with sloping baselines and the enthalpy and entropy changes derived from the fits were averaged (21), and (ii) reciprocal melting temperature, tm-1, vs. log (CT) was plot...

The thermodynamic parameters (delta H degree, delta S degree, and delta G degree 37) for 16 nearest-neighbor sets and one initiation factor are presented here in order to predict stability of RNA/DNA hybrid duplexes. To determine the nearest-neighbor parameters, thermodynamics for 68 different hybrid sequences (136 single-stranded oligonucleotides) with 5-13 nucleotide length including several duplexes with identical nearest-neighbors were measured by UV melting procedure. These sequences were selected to have many different combinations of nearest-neighbor pairs, and so that the number of the 16 nearest-neighbor sequences in the oligomers were as equal as possible. The structures of the hybrids were also investigated by measuring circular dichroism spectra. Comparing delta G degree 37 values of the hybrids with DNA/DNA and RNA/RNA parameters reported previously (Breslauer, K.J., Frank, R., Blöcker, H., & Marky, L.A. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 3746-3750; Freier, S.M., Kierzek, R., Jaeger, J.A., Sugimoto, N., Caruthers, M.H., Neilson, T., & Turner, D.H. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 9373-9377), RNA/RNA double helix is the most stable of the three kinds of helixes with the same nearest-neighbor sequences. Which is more stable between DNA/DNA and RNA/DNA hybrid duplexes depends on its sequence. Calculated thermodynamic values of hybrid formation with the present parameters reproduce the experimental values within reasonable errors.

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The effect of molecular crowding on the structure and stability of biomolecules has become a subject of increasing interest because it can clarify how biomolecules behave under cell-mimicking conditions. Here, we quantitatively analyzed the effects of molecular crowding on the thermodynamics of antiparallel G-quadruplex formation via Hoogsteen base pairs and of antiparallel hairpin-looped duplex (HP duplex) formation via Watson-Crick base pairs. The free energy change at 25 degrees C for G-quadruplex formation decreased from -3.5 to -5.5 kcal mol(-1) when the concentration of poly(ethylene glycol) 200 was increased from 0 to 40 wt %, whereas that of duplex formation increased from -9.8 to -6.9 kcal mol(-1). These results showed that the antiparallel G-quadruplex is stabilized under molecular crowding conditions, but that the HP duplex is destabilized. Moreover, plots of stability (ln K(obs)) of the DNA structures versus water activity (ln a(w)) demonstrated that the ln K(obs) for G-quadruplex formation decreased linearly as the ln a(w) increased, whereas that for duplex formation increased linearly with the increase in ln a(w), suggesting that the slope approximately equals the number of water molecules released or taken up during the formation of these structures. Thus, molecular crowding affects the thermodynamics of DNA structure formation by altering the hydration of the DNA. The stabilization of the DNA structures with Hoogsteen base pairs and destabilization of DNA structures with Watson-Crick base pairs under molecular crowding conditions lead to structural polymorphism of DNA sequences regulated by the state of hydration.

The effects of counter ion on a nucleic acid duplex stability were investigated. Since a linear free energy relationship for the thermostability of oligonucleotide duplexes between those in 1 M and in 100 mM NaCl-phosphate buffer were observed regardless of whether they are DNA-DNA, RNA-RNA or RNA-DNA duplexes, simple prediction systems for [Delta] G degrees 37as well as T mvalues in 100 mM NaCl-phosphate buffer were established. These predictions were successful with an average error of only 2.4 degrees C for T mand 5. 7% for G degrees 37values. The number of Na+newly bound to a duplex when the duplex forms (-[Delta] n) was significantly influenced by the base composition, and -[Delta] n for d(GCCAGTTAA)/d(TTAACTGGC) was different for MgCl2, CaCl2, BaCl2and MnCl2(from 0.70 to 0.76 with the same order of the duplex stability). Almost no additive effects on the duplex stability was observed for NaCl and MgCl2, suggesting a competitive binding for these cations. The sequence-dependent manner of [Delta] n suggests the presence of preferential base pairs or nearest-neighbor base pairs for the cation binding, which would affect nearest-neighbor parameters.