Solution structures of the individual single strands of the fragile X DNA triplets (GCC)n·(GGC)n

Abstract

Three-dimensional structures of the fragile X triplet repeats (GCC)n and (GGC)n are derived by using one-dimensional/two-dimensional NMR. Under a wide range of solution conditions (10-150 mM NaCl, pH 6-7) (GCC)5-7 strands form exclusively slipped hairpins with a 3′ overhanging C. The slipped hairpins of (GCC)n strands show the following structural characteristics: (i) maximization of Watson-Crick G·C pairs; (ii) formation of C·C mispairs at the CpG steps in the stem; (iii) C2′-endo, anti conformations for all the nucleotides. The ability of (GCC)n strands to form hairpin structures more readily than complementary (GGC)n strands suggests preferential slippage during replication and subsequent expansion of the (GCC)n strands. In addition, the C·C mispairs at the CpG site of (GCC)n hairpins account for their exceptional substrate efficiencies for human methyltransferase. Gel electrophoresis data show that (GGC)n strands form both hairpin and mismatched duplex structures in 10-150 mM NaCI (pH 6-7) for n < 10, but for n ≥ 11 hairpin structures are exclusively present. However, (GGC)n strands remain predominantly in the duplex state for n = 4-11 under NMR solution conditions, which require DNA concentrations 100- to 1000-fold higher than in gel electrophoresis. NMR analyses of [(GGC)n]2 duplexes for n = 4-6 show the presence of Watson-Crick G·C and mismatched Ganti·Gsyn pairs. The mismatches adjacent to the CpG step introduce local structural flexibility in these duplexes. Similar structural properties are also expected in the stem of the hairpins formed by (GGC)n strands.