The expanded repertoire of PNA binding motifs compared to other nucleic acids increases the potential for its exploitation in biotechnical, diagnostic, and therapeutic applications. A major limitation in the use of both PNA and oligonucleotides to date has been the inability to target mixed-sequence duplex DNA. In an exciting recent development, a new PNA targeting strategy has been reported in which a single-stranded PNA can invade mixed-sequence duplex DNA producing a novel structure with two identical PNA:DNA duplexes in the invasion complex.

PNA containing alternating diaminopurine and 2-thiouracil bases cannot form a self-complementary duplex because of steric hindrance between these modified bases and is thus termed pseudocomplementary. It can however form a very stable duplex with a complementary DNA strand containing alternating unmodified adenine and thymine bases, and can invade duplex DNA of this sequence to produce two DNA:PNA duplexes. This double duplex invasion has been monitored by CD spectroscopy which shows that the PNA bases become involved in a helical structure upon interaction with DNA. The stoichiometry of the final complex is confirmed to be 1:1 and the binding rate increases with PNA concentration and temperature, and decrease with ionic strength. Under pseudo-first order conditions, Arrhenius analysis reveals an ionic strength independent activation enthalpy of ca. 80 kJ mol^-1 which indicates that, as for normal PNA triplex-strand-invasion, DNA basepair opening is likely the rate-limiting step. Differences in the double duplex invasion of single and multiple sites will be discussed.