The effect of the neutral cytidine protonated analogue pseudoisocytidine on the stability of i-motif structures

Abstract

Incorporation of pseudoisocytidine (psC), a neutral analogue of protonated cytidine, in i-motifs has been studied by spectroscopic methods. Our results show that neutral psC:C base pairs can stabilize i-motifs at neutral pH, but the stabilization only occurs when psC:C base pairs are located at the ends of intercalated C:C⁺ stacks. When psC occupies central positions, the resulting i-motifs are only observed at low pH and psC:C⁺ or psC:psC⁺ hemiprotonated base pairs are formed instead of their neutral analogs. Overall, our results suggest that positively charged base pairs are necessary to stabilize this non-canonical DNA structure.

Figure 1

Expected base-pairs in psC-containing i-motifs and schematic representations of the structural equilibrium in C0 and CC0. (A) Tautomeric equilibrium of pseudoisocytidine. (B) Neutral psC:C base pair. (C) Hemiprotonated C:C⁺ base pair in i-motif structures. (D,E) Head-to-head and head-to-tail dimeric i-motif structures of C0 and CC0, respectively.

Figure 2

CC9 forms i-motif structure at neutral pH. 1D ¹H-NMR spectra at different pH. H₂O/D₂O 90:10, 25 mM phosphate buffer, 100 mM NaCl, T = 5 °C, [oligonucleotide] = 0.7–1 mM (A,B,D–F). CD pH titration of the 11-mer sequences were followed at the ellipticity maximum at pH 4: λ = 265 nm (C0, CC8 and CC9) and λ = 265 nm (C7 and CC0), 25 mM AcONa buffer, 100 mM NaCl, T = 5 °C, [oligonucleotide] = 20 μM (C).

Figure 3

Thermal stability of psC-containing i-motifs. CD melting curves of C0, C7, CC0, CC8 and CC9. Experimental conditions: [oligonucleotide] = 20 μM, 25 mM of buffer solution (cacodylate (pH 5.5) or phosphate (pH 7) and 100 mM NaCl.

Figure 4

Assignment of exchangeable protons and solution dimeric structure of CC9. Left: Imino and amino exchangeable protons region of NOESY spectrum (250 ms) of CC9. H₂O/D₂O 90:10, 25 mM phosphate buffer pH 7, 100 mM NaCl, T = 5 °C, [oligonucleotide] = 0.8 mM. Right: (A) Structural ensemble. (B) Detail of C:C⁺, C:psC and G:G stack. (C,D) Front and lateral view of a representative structure. Cytosines are shown in green, psC in yellow, guanines in blue and thymines in red.

Figure 5

Single psC residue at position 1 in HT sequence stabilizes i-motif structure at neutral pH. Series of NMR spectra at different temperature of: (A) HT-psC17, (B) HT-psC28, (C) HT-0 and (D) HT-psC1. [oligonucleotide] = 100 μM, 10 mM phosphate buffer, pH 7.