STUDIES OF DNA DUMBBELLS .1. MELTING CURVES OF 17 DNA DUMBBELLS WITH DIFFERENT DUPLEX STEM SEQUENCES LINKED BY T4 ENDLOOPS - EVALUATION OF THE NEAREST-NEIGHBOR STACKING INTERACTIONS IN DNA

Seventeen DNA dumbbells were constructed that have duplex sequences ranging in length from 14 to 18 base pairs linked on the ends by T4 Single-strand loops. Fifteen of the molecules have the core duplexes with the sequences 5'G-T-A-T-C-C-(W-X-Y-Z)-G-G-A-T-A-C3', where (W-X-Y-Z) represents a unique combination of A.T, T.A, G.C, and C.G base pairs. The remaining two molecules have the central sequences (W-X-Y-Z) = A-C and A-C-A-C-A-C. These duplex sequences were designed such that the central sequences include different combinations of the 10 possible nearest-neighbor (n-n) stacks in DNA. In this sense the set of molecules is complete and serves as a model system for evaluating sequence-dependent local stability of DNA. Optical melting curves of the samples were collected in 25, 55, 85, and 115 mM [Na+], and showed, regardless of solvent ionic strength, that the transition temperatures of the dumbbells vary by as much as 14-degrees for different molecules of the set. Results of melting experiments analyzed in terms of a n-n sequence-dependent model allowed evaluation of nine independent linear combinations of the n-n stacking interactions in DNA as a function of solvent ionic strength. Although there are in principle 10 possible different n-n interactions in DNA, these 10 are not linearly independent and therefore can not be uniquely determined. For molecules with ends, there are 9 linearly independent combinations, as opposed to circular or semiinfinite repeating copolymers where only 8 linear combinations of the 10 possible n-n interactions are linearly independent. The n-n interactions are presented as combinations of the deviations from average stacking for the 5'-3' base-pair doublets, delta-G(i), and reveal several interesting features: (1) Titratable changes in the values of delta-G(i) with changing salt environment are observed. In all salts the most stable unique combination is delta-G4 = (delta-G(GpC) + delta-G(CpG))/2, and the least stable is the GpG/CpC stack, delta-G2 = delta-G(GpG)/CpC. (2) The chi-2 values of the fits of the evaluated delta-G(i)'s to experimental data increased with decreasing [Na+], suggesting that significant interactions beyond nearest neighbors become more pronounced, particularly at 25 mM Na+. (3) In 85 and 115 mM Na+, where the n-n approximation seems to be most valid, the absolute value of delta-G(i) for any n-n stack or average of two n-n stacks is not more than approximately 220 cal/mole, indicating that deviations from average stacking due to n-n interactions represent about 15% of the total stability of a base pair. The overall thermodynamic stability of DNA is predominantly determined by the sequence content (%G.C). Even though the contribution of n-n interactions to overall stability are intrinsically small, reliable predictions of DNA transition temperatures de novo from sequence can be significantly compromised by cumulative errors in the delta-G(i)'s. (4) Comparisons of our set of n-n linear combinations evaluated in 115 mM Na+ with various published sets evaluated from melting experiments of long restriction fragments, synthetic polymers, and short oligomers, and those obtained from a reanalysis of published melting data of synthetic polymers, are presented. The analysis reveals a major consensus agreement between n-n free energies evaluated from melting data of restriction fragments and long synthetic repeating copolymers. In contrast, only a minor consensus agreement is obtained between our n-n set and these values or those obtained from melting analysis of a combination of short oligomers and long polymers or those theoretically calculated. Results of these comparisons suggest the values of n-n interactions evaluated from DNA melting curves depend on the length of the melted duplex regions of the DNA molecules that comprise the sample set.