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 role of stacking in terminal base-pair formation was studied by comparison of the stability increments for dangling ends to those for fully formed base pairs. Thermodynamic parameters were measured spectrophotometrically for helix formation of the hexanucleotides AGGCCUp, UGGCCAp, CGGCCGp, GCCGGCp, and UCCGGAp and for the corresponding pentanucleotides containing a 5'-dangling end on the GGCCp or CCGGp core helix. In 1 M NaCl at 1 X 10(-4) M strands, a 5'-dangling nucleotide in this series increases the duplex melting temperature (Tm) only 0-4 degrees C, about the same as adding a 5'-phosphate. In contrast, a 3'-dangling nucleotide increases the Tm at 1 X 10(-4) M strands 7-23 degrees C, depending on the sequence [Freier, S. M., Burger, B. J., Alkema, D., Neilson, T., & Turner, D. H. (1983) Biochemistry 22, 6198-6206]. These results are consistent with stacking patterns observed in A-form RNA. The stability increments from terminal A.U, C.G, or U.A base pairs on GGCC or a terminal U.A pair on CCGG are nearly equal to the sums of the stability increments from the corresponding dangling ends. This suggests stacking plays a large role in nucleic acid stability. The stability increment from the terminal base pairs in GCCGGCp, however, is about 5 times the sum of the corresponding dangling ends, suggesting hydrogen bonding can also make important contributions.

The stabilizing effects of dangling ends and terminal base pairs on the core helix GCGC are reported. Enthalpy and entropy changes of helix formation were measured spectrophotometrically for AGCGCU, UGCGCA, GGCGCCp, CGCGCGp, and the corresponding pentamers XGCGCp and GCGCYp containing the GCGC core plus a dangling end. Each 5' dangling end increases helix stability at 37 degrees C roughly 0.2 kcal/mol and each 3' end from 0.8 to 1.7 kcal/mol. The free energy increments for dangling ends on GCGC are similar to the corresponding increments reported for the GGCC core [Freier, S. M., Alkema, D., Sinclair, A., Neilson, T., & Turner, D. H. (1985) Biochemistry 24, 4533-4539], indicating a nearest-neighbor model is adequate for prediction of stabilization due to dangling ends. Nearest-neighbor parameters for prediction of the free energy effects of adding dangling ends and terminal base pairs next to G.C pairs are presented. Comparison of these free energy changes is used to partition the free energy of base pair formation into contributions of "stacking" and "pairing". If pairing contributions are due to hydrogen bonding, the results suggest stacking and hydrogen bonding make roughly comparable favorable contributions to the stability of a terminal base pair. The free energy increment associated with forming a hydrogen bond is estimated to be -1 kcal/mol of hydrogen bond.

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Thermodynamic parameters of helix formation were measured spectroscopically for seven hexaribonucleotides containing a GC tetramer core and G.U or other terminal mismatches. The free energies of helix formation are compared with those for the tetramer core alone and with those for the hexamer with six Watson-Crick base pairs. In 1 M NaCl, at 37 degrees C, the free energy of a terminal G.U mismatch is about equal to that of the corresponding A.U pair. Although other terminal mismatches studied add between -1.0 and -1.6 kcal/mol to delta G0 37 for helix formation, all are less stable than the corresponding Watson-Crick pairs. Comparisons of the stability increments for terminal G.U mismatches and G.C pairs suggest when stacking is weak the additional hydrogen bond in the G.C pair adds roughly -1 kcal/mol to the favorable free energy of duplex formation.

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In order to make efficient use of T4 RNA ligase in a program involving chemical‐enzymatic oligoribonucleotide synthesis, the sequence effects of the acceptor oligomer have been refined. The reaction of the donor molecules pCp and pUpUpUpCp with a series of trinucleoside diphosphates ApApN, CpNpA and NpCpA (where N = U, C, A or G) was examined. High‐performance liquid chromatography was used for simultaneous analysis of all substrates, intermediates and products. For all trinucleoside bisphosphates tested a substantial amount of the intermediate A(5′)pp(5′)Cp or A(5′)pp(5′)UpUpUpCp was observed. This indicated that the extent of ligation was dependent upon sequence of the acceptor molecule and not upon adenylation of the donor. Con‐ versely, examination of the four nucleoside 3′,5′‐bisphosphate donors p u p, pCp, pAp and pGp with a common acceptor ApApU indicated that in the case of poor ligation little of the intermediate adenylated donor was formed.

The syntheses of N6-benzoyl-2'-0-tetrahydropyranyladenosine (2) and N4-benzoyl-2'-O-tetrahydropyranylcytidine (11) and their corresponding 5'-0-p-methoxytrityl derivatives, 3 and 12 respectively, are described. Compounds 2, 3, 11, and 12 are the protected derivatives of adenosine and cytidine required for insertion into a new oligoribonucleotide synthesis. Compound 2 was prepared from the known 2'-0-tetrahydropyranyladenosine by perbenzoylation followed by de-0-benzoylation. Compound 11 is prepared from cytidine by the following sequence: orthoacetate protection of the 2'-and 3'-hydroxyls; benzoylation of the 5'-and NJ-positions; orthoacetate ring opening provided a mixture of the 2'-and 3'-monoacetates from which the major, 3'-0-acetyl-N4,5'-0-dibenzoylcytidine (7) was isolated; dihydropyran treatment on the 2'-hydroxyl of 7 and subsequent selective deacylation studies were carried out. Specific de-0-acylation gave 11.