Cytosine-rich strands of the insulin minisatellite adopt hairpins with intercalated cytosine(+)center dot cytosine pairs

Previously, we reported the high resolution NMR structure of the hairpin G-quartet structure formed by the G-rich strand of the insulin minisatellite of repeat sequence, ((ACAG4TGTG4)(TGTC4ACAC4)) located upstream of the human insulin gene. Here, we report structural studies on the C-rich strand of this insulin minisatellite. First, we Show by high resolution NMR that (C(4)TGTC(4)) forms a hairpin dimer with intercalated C+. C pairs (referred to as the hairpin i-motif); 340 NOE distance constraints uniquely define the nature of hairpin folding and the pattern of C+. C intercalation. Second, we show by one-dimensional NMR spectroscopy and molecular modeling studies that (C(4)TGTC(4)ACA(4)TGTC(4)) forms an intramolecularly folded hairpin with intercalated C+. C pairs. Third, we demonstrate by in vitro replication studies that several such hairpin i-motifs are present in long (C(4)TGTC(4)ACA)(n) (n greater than or equal to 6) sequences, even in the presence of their complementary strands. Finally, we discuss structural and biological significance of the hairpin i-motifs formed by the C-rich strands of the insulin minisatellite. (C) 1997 Academic Press Limited.