Cystosine-rich strands of the insulin minisatellite adopt hairpins with intercalated Cytosine+·Cytosine pairs

Abstract

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,ACAG4TGTG4TGTC4ACAC4located 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 (C4TGTC4) 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 (C4TGTC4ACA4TGTC4) 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 (C4TGTC4ACA)n (n ⩾ 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.