Nucleic acid oligodeoxynucleotide 'third-strands' bind within the major groove of double-stranded DNA in a sequence specific manner. This property allows for the recognition of a unique sequence(s) within genomic DNA. Unlike current in situ hybridization (ISH) probes that require denaturation of their chromosomal targets, third-strands are able to bind to non-denatured DNA targets.
Earlier work has established that third-strand technology can be used as a primary research tool in molecular biology. Third-strands with a coupled EDTA/Fe2+ moiety can selectively bind to and cleave the targeted DNA. Other work has shown that third-strands can successfully compete with DNA binding proteins for their target sequences.
The fruit fly, Drosophila melanogaster, has been chosen as the subject organism for this project, which involves solution third-strand binding studies on supercoiled plasmids. To determine what modifications to the current protocols may be needed, we have selected certain Drosophila sequences particularly suited for third-strand binding. The Drosophila histone genes are arranged in euchromatin tandem clusters with a copy number of approximately 100. We have chosen a 16 bp target within this cluster to examine the equilibrium of third-strand binding.
As a preliminary step in the development of an in vivo third-strand binding assay for selection of a Drosophila embryonic phenotype, we explored as a potential target the heterochromatin homopurine GAGA repeat, (AAGAGAG)n. Equilibrium binding data is reported here which shows that both sequences are able to selectively bind third-strands and that adjacent targets exhibit independent binding.