We have several broad goals and all focus on whether and to what extent stacking forces between nearest neighbor base pairs contribute to selective physical and biological properties of DNA. The most immediate, specific goal is the quantification of free energies of stacking interactions for the ten unique neighbor pairs. Sensitive measurements of stacking energies will be made from melting temperatures of sharp subtransitions for specially designed, multicopy, tandemly repeating synthetic inserts in polymeric plasmid DNAs, that exhibit readily detectable shifts in Tm in response to small variations in stacking energy. Microcalorimetric measurements will also be performed on these constructs in an attempt to measure the dependence of domain enthalpies on neighbor frequencies. The further goals of this project will then be to investigate selected sequence domains in both natural and synthetic sequences that exhibit anomalous thermal stabilities; to examine the relationship of stacking energies to local helical parameters determined by single crystal diffraction analysis; to explore for a dependence on stacking energies of patterns of repair of mismatched base pairs, as well as of mutation patterns and evolutionary patterns of selected sequence elements. Similar thermal stability measurements of stacking energies will be made on specific neighbors, similarly assembled in synthetic inserts in the form of repeats or tracts, to determine whether such tracts exhibit anomalous stabilities that can be attributed to next neighbor and beyond interactions in the duplex. In a related study, sensitive thermal stability studies will be conducted on reassociated recombinant plasmid-duplexes containing multicopy tandem repeats of synthetic inserts with specific mispairs. The objective of these studies is to measure the energetics of selected mispairs as well as the effects of the local sequence environment. These thermodynamic studies will be accompanied by sequencing studies to determine the nature of mispair correction in these inserts after transformation of E. coli cells.
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