The long-term goal of this project is to develop a broad understanding of the capacity of single strands of RNA or DNA to interact with segments of nucleic acid duplexes through intermolecular triple helix formation under essentially physiological conditions. This effort stems from our recent finding that triple helices of complex sequence can form. The target duplex sequences that are required for such triplex formation are homopurine-homopyrimidine; such sequences are widely distributed in nature, especially in eukaryotes and their viruses, where they occur in frequencies that exceed random expectation. Such triplex formation is of interest because it constitutes a potential mechanism for the control of gene expression at the level of the genome itself by RNA effector molecules. An understanding of the structural, equilibrium, and specificity aspects of such third-strand binding can also lead to the development of pharmacological agents for the control of expression of specific genes with a role in disease, highly specific agents for cleaving nucleic acid duplexes that can be of value in the sequencing of large genomes, and fundamental information on the physical chemistry of an important class of nucleic acid complexes that were previously thought to be restricted to very simple sequences. Although the ultimate goals of this program are biological ones, the experimental approach is physical-chemical. Experiments with deoxyribo and ribo oligomers and polymers of varying sequence complexity will be performed to determine the factors affecting the minimum length of a homopurine-homopyrimidine segment of a genomic duplex that can serve as a target for third-strand binding, the kinetics of the process, the specificity and fidelity of third- strand binding, the degree of sequence complexity amenable to third-strand binding, the stereochemical details of nucleic acid triplexes, the extent to which homopurine-homopyrimidine segments can tolerate discontinuities without interfering with third-strand binding, and related thermodynamic properties. Much of the information obtained through these studies of intermolecular third- strand binding should prove relevant as well to intramolecular triplex formation arising by disproportionation of homopurine- homopyrimidine genomic segments to triplex and covalently-linked single strand.
