ELECTRON-MICROSCOPY MAPPING OF OLIGOPURINE TRACTS IN DUPLEX DNA BY PEPTIDE NUCLEIC-ACID TARGETING

Biotinylated homopyrimidine decamer peptide nucleic acids (PNAs) are s hown to form sequence-specific and stable complexes with complementary oligopurine targets in linear double-stranded DNA. The noncovalent co mplexes are visualized by electron microscopy (EM) without chemical fi xation using streptavidin as an EM marker. The tripler stoichiometry o f the PNA-DNA complexes (two PNA molecules presumably binding by Watso n - Crick and Hoogsteen pairing with one of the strands of the duplex DNA) is indicated by the appearance of two streptavidin 'beads' per ta rget site in some micrographs, and is also supported by the formation of two retardation bands in a gel shift assay. Quantitative analysis o f the positions of the streptavidin 'beads' revealed that under optimi zed conditions PNA-DNA complexes are preferably formed with the fully complementary target. An increase in either the PNA concentration or t he incubation time leads to binding at sites containing one or two mis matches. Our results demonstrate that biotinylated PNAs can be used fo r EM mapping of short targets in duplex DNA.