Upon binding of a decamer bis-PNA (H-Lys-TTCCTCTCTT-(eg1)(3)-TTCTCTCCTT-Lys
NH(2)) to a complementary target in a double-stranded DNA fragment, three d
istinct complexes were detected by gel mobility shift analysis. Using in si
tu chemical probing techniques (KMnO4 and DMS) it was found that all three
complexes represent bona fide sequence-specific PNA binding to the designat
ed target, bur the complexes were structurally different. One complex that
preferentially formed at higher FNA concentrations contains two bis-PNA mol
ecules per DNA target, whereas the other two complexes are genuine tripler
invasion clamped structures. However, these two latter complexes differ by
the path relative to the DNA target of the flexible ethylene-glycol linker
connecting the two PNA oligomers that comprise a bis-PNA. We distinguish be
tween one in which the linker wraps around the non-target DNA strand, thus
making this strand part of the tripler invasion complex and another complex
that encompass the target strand only. The implications of these results a
re discussed in terms of DNA targeting by synthetic ligands. (C) 2001 Acade
mic Press.