Protein-free parallel triple-stranded DNA complex formation

A 14 nt DNA sequence 5'-AGAATGTGGCAAAG-3' from the zinc finger repeat of th e human KRAB zinc finger protein gene ZNF91 bearing the intercalator 2-meth oxy,6-chloro,9-amino acridine (Acr) attached to the sugar-phosphate backbon e in various positions has been shown to form a specific triple helix (trip lex) with a 16 bp hairpin (intramolecular) or a two-stranded (intermolecula r) duplex having the identical sequence in:the same (parallel) orientation, Intra-molecular targets with the identical sequence in the antiparallel or ientation and a non-specific target sequence were tested as controls, Appar ent binding constants for formation of the tripler were determined by quant itating electrophoretic band shifts, Binding of the single-stranded oligonu cleotide probe sequence to the target-led to an increase in the fluorescenc e anisotropy of acridine, The parallel orientation of the two Identical seq uence segments was confirmed by measurement of fluorescence resonance energ y transfer between the acridine on the 5'-end of the probe strand as donor and BODIPY-Texas Red on the 3'-amino group of either strand of the target d uplex as acceptor, There was full protection from OsO4-bipyridine modificat ion of thymines in the probe strand of the tripler, in accordance with the presumed tripler formation,which excluded displacement of the homologous du plex strand by the probe-intercalator conjugate, The implications of these results for the existence of protein-independent parallel triplexes are dis cussed.