An RNA model system consisting of an oligomer binding to a 4-nt overhang at the 5' end ofa hairpin stem provides thermodynamic parameters for helix-helix interfaces. In a sequence-dependent manner, oligomers bind up to 1000-fold more tightly adjacent to the hairpin stem than predicted for binding to a free tetramer at 370C. For reaction (19, 20). Synthetic details will be provided elsewhere.Measurement of Thermodynamic Parameters. The buffer for melting experiments was 1 M NaCl/10 mM sodium cacodylate/0.5 mM EDTA. Absorbance versus temperature curves were measured at 280 nm with a heating rate of 10C/min. These curves were analyzed by fitting to a two-state model with sloping base lines through use of a nonlinear least-squares program (21).Predictions of Secondary Structure. Sets of 20 secondary structures were generated with the program of Zuker (12), kcal/mol, and internal loops of three were given a AG'7 for loop closure of 5.1 + 0.3 = 5.4 kcal/mol (23). (iii) Stacking and pairing of terminal mismatches were included in calculating free energies of internal loops in the following manner. Single mismatches were given AG7 = 0.8 kcal/mol. For larger internal loops, terminal GA, UU, and other mismatches adjacent to COG pairs were given favorable free energy increments of -2.7, -2.5, and -1.5 kcal/mol, respectively (24). These mismatches adjacent to A-U pairs were given AG' values of -2. 9218The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

UV melting and imino proton NMR studies show that the stabilities and structures of tandem GA mismatches in RNA are dependent upon the closing base pairs around these mismatches. Internal loops of sequence 5'XGAY3'3'YAGXS' and 5'XAGY3'3'YGAX5' in the middle of octanucleotides have a range of stabilities over 5 kcal/mol when XY is a Watson-Crick or GU pair. The order of stabilities for these internal loops is 5'-GGAC-3' > UGAG, CGAG > AGAU > UGAA > GGAU. The motifs GGAC, UGAG, and CGAG are stabilizing, while the other GA motifs are destabilizing. The GAGC motif is more stable than CAGG and CGAG, but less stable than GGAC. Chemical shifts for imino protons suggest that the G imino proton of each GA mismatch in 5'-GGAC-3', 5'-GAGC-3', and 5'-CAGG-3' [SantaLucia, J., Jr., Kierzek, R., & Turner, D. H. (1990) Biochemistry 29, 8813-8819] is involved in a hydrogen bond to the base A, whereas in other 5'-XGAY-3' sequences, it is not involved in a hydrogen bond to the base A.

Thermodynamic parameters derived from optical melting studies are reported for duplex formation by a series of oligoribonucleotides containing G.U mismatches. The results are used to determine nearest-neighbor parameters for helix propagation by G.U mismatches. Surprisingly, the [formula; see text] nearest-neighbor free energy increment in unfavorable in the contexts [formula; see text], and [formula; see text] but favorable in the context [formula; see text]. This is a non-nearest-neighbor effect. In contrast, the [formula; see text] free energy increment is favorable and independent of context. Circular dichroism and imino proton NMR spectra of several sequences do not reveal an obvious structural basis for this dichotomy. For example, all the G.U mismatches have two slowly exchanging imino protons. The imino resonances for the G.U mismatches in GGAGUUCC, GUCGUGAC, and CCUGUAGG, however, broaden at lower temperature than the imino resonances for the interior Watson-Crick base pairs. In contrast, the imino resonances for the G.U mismatches in GGAUGUCC remain sharp at high temperature. The improved parameters for G.U mismatches should improve predictions of RNA structure from sequence.

Thermodynamic parameters from UV melting studies are reported for the helix-helix interfaces of coaxially stacked helixes in RNA. The model system consists of a short oligomer binding to a four-nucleotide overhang at the end of a hairpin stem, creating the helix-helix interface. Interfaces containing Watson-Crick base pairs are approximately 1 kcal/mol more stable than the corresponding nearest neighbor interaction in an uninterrupted helix. Thus the sequence dependence of stability for coaxially stacked interfaces is similar to that for regular helixes. This provides experimental evidence for an assumption that has been shown to improve predictions of RNA secondary structure [Walter, A. E., Turner, D. H., Kim, J., Lyttle, M. H., Muller, P., Mathews, D. H., & Zuker, M. (1994) Proc. Natl. Acad. Sci. U.S.A. (in press)]. The results should also be useful for modeling three-dimensional structures of RNA.

An effect of 5'-phosphorylation on the stability of triple helical DNA containing pyrimidine:purine:pyrimidine strands has been demonstrated by both gel electrophoresis and UV melting. A 5'-phosphate on the purine-rich middle strand of a triple helix lowers the stability of triple helix formation by approximately 1 kcal/mol at 25 degrees C. The middle strand is involved in both Watson-Crick and Hoogsteen base pairing. In contrast, a 5'-phosphate on the pyrimidine-rich strands, which are involved in either Watson-Crick or Hoogsteen base pairing, has a smaller effect on the stability of triple helix. The order of stability is: no phosphate on either strand > phosphate on both pyrimidine strands > phosphate on purine strand > phosphate on all three strands. Differential stability of triple helix species is postulated to stem from an increase in rigidity due to steric hindrance from the 5'-phosphate. This result indicates that labelling with 32P affect equilibrium in triplex formation.