Before analytical theories can be developed, tested and implemented to construct physical maps of genes and accurately predict the formation of secondary structure from primary sequence alone, the data base surrounding the sequence dependent stability of DNA, the energetics of hairpin and internal loop formation and the sequence dependence of triplex formation must be considerably expanded. The goal of this proposal is to expand the basis of thermodynamic knowledge surrounding the sequence dependence of these DNA intramolecular structures. Thermal denaturation and differential scanning calorimetry will be the experimental techniques employed. The experimental subjects will primarily be DNA dumbbells; duplex DNA's cross-linked on both ends by single strand loops. Experiments will be analyzed in terms of the nearest-neighbor sequence dependent, numerically exact, statistical thermodynamic theory of DNA melting. There are seven specific aims of the project. (1) Evaluation of the free-energies of hairpin loop formation for loops comprised of Xn (X - A,T,G,C; n - 2-14) nucleotide residues. (2) Evaluate the effects of heterogeneous loop sequence on the stability of DNA hairpins from melting studies of DNA hairpins with the four base loop sequences X-Y-Y-X (X,Y - A,T,G,C, X not equal to Y). . (3) Evaluate the influence of the sequence identity of the first base-pair of the duplex stem nucleating a hairpin loop from melting studies of DNA hairpins with the duplex sequence 5'GGATAX3'linked by Y4 end-loops (X,Y - A,T,G,C). (4) Evaluate the nearest-neighbor base-pair stacking free-energies in DNA as function of sequence domain length. (5) Evaluate the energetics of forming small (2-14 basepairs) and larger (greater than 80 base - pairs) internally melted loops in DNA between two intact helical regions. (6) Determine effects of nearest-neighbor sequence and sequence mis-matches on the stability of DNA triplexes formed from DNA dumbbells and single strands. (7) Determine effects of alkyl (dodecyl and hexadecyl) chains covalently attached to the ends of a DNA duplex on the stability and restriction enzyme cleavage of the duplex region.
