Identification of multiple genomic DNA sequences which form i-motif structures at neutral pH

Abstract

i-Motifs are alternative DNA secondary structures formed in cytosine-rich sequences. Particular examples of these structures, traditionally assumed to be stable only at acidic pH, have been found to form under near-physiological conditions. To determine the potential impact of these structures on physiological processes, investigation of sequences with the capacity to fold under physiological conditions is required. Here we describe a systematic study of cytosine-rich DNA sequences, with varying numbers of consecutive cytosines, to gain insights into i-motif DNA sequence and structure stability. i-Motif formation was assessed using ultraviolet spectroscopy, circular dichroism and native gel electrophoresis. We found that increasing cytosine tract lengths resulted in increased thermal stability; sequences with at least five cytosines per tract folded into i-motif at room temperature and neutral pH. Using these results, we postulated a folding rule for i-motif formation, analogous to (but different from) that for G-quadruplexes. This indicated that thousands of cytosine-rich sequences in the human genome may fold into i-motif structures under physiological conditions. Many of these were found in locations where structure formation is likely to influence gene expression. Characterization of a selection of these identified i-motif forming sequences uncovered 17 genomic i-motif forming sequence examples which were stable at neutral pH.

Figure 1.

ODN stability with increasing cytosine tract length. ODNs (2.5 μM) were annealed in 10 mM sodium cacodylate with 100 mM sodium chloride at the indicated pH. + pH 5.5 T_m1; Δ pH 5.5 T_m2 (10 cytosine tract only); o pH 5.5 T_a; pH 7.4 T_m1; × pH 7.4 T_m2; and ■ pH 7.4 T_a.

Figure 2.

The effects of cytosine tract length (A) and loop length (B) on hysteresis. ODNs (2.5 μM) were annealed in 10 mM sodium cacodylate with 100 mM sodium chloride at the indicated pH. (A) × Hysteresis at pH 5.5; o Secondary hysteresis at pH 5.5 (10 cytosine tract only); ■ Hysteresis at pH 7.4; Secondary hysteresis at pH 7.4. (B) Hysteresis at pH 5.5 (▪) and at pH 7.4 (•) in ODNs containing tracts of 5 cytosines with increasing lengths of thymine loops.

Figure 3.

The thermal difference spectra calculated between 95 and 4°C of each of the oligonucleotides (ODNs) at pH 5.5 (A) and 7.4 (B). ODNs (2.5 μM) were annealed in 10 mM sodium cacodylate with 100 mM sodium chloride at the indicated pH.

Figure 4.

Room temperature native PAGE of the model ODNs (10 μM in 10 mM sodium cacodylate at pH 7.4) with increasing tract lengths at pH 7.4. Acrylamide gels (20%) were buffered with 50 mM tris, 50 mM HEPES at pH 7.4. ODNs were annealed at pH 7.4 as described above. The samples were loaded onto the gel for electrophoresis at 50 V for 5 h. Lane contents left to right are as follows: (1) C₁T₃; (2) C₂T₃; (3) C₃T₃; (4) C₄T₃; (5) C₅T₃; (6) Base pair ladder standard; (7) C₆T₃; (8) C₇T₃; (9) C₈T₃; (10) C₉T₃; (11) C₁₀T₃; (12) Base pair ladder standard.

Figure 5.

Circular dichroism of (A) human telomeric i-motif; (B) C₂T₃; (C) C₅T₃ and (D) C₁₀T₃. pH 4.0; pH 4.5; pH 5.0; pH 5.5; pH 6.0; pH 6.5; pH 7.0; pH 7.5; pH 8.0. All ODN concentrations were 10 μM in 10 mM sodium cacodylate buffer with 100 mM sodium chloride buffer at the required pH.

Figure 6.

The relationship between total loop length (the sum of all loop bases) and transitional pH. Transitional pH was calculated from fitting the CD data for all pH at 288 nm and identifying the inflection point.

Figure 7.

The relationship between total loop length (the sum of all loop bases) and melting temperature (T_m). T_m was calculated from the maxima of the first derivative of UV-melt data.