Comprehensive Analysis of Stability and Variability of DNA Minimal I-Motif Structures

Abstract

Cytosine-rich DNA sequences form i-motif structures associated with various cellular functions including gene regulation. DNA sequences containing consecutive C residues are widely deemed essential for i-motif formation; however, some sequences lacking C-tracts have been reported to form minimal i-motif structures. We systematically investigated the variability in the minimal i-motif-forming DNA sequence comprising two TCGTTCCGT sequence units, which forms two C:C⁺ pairs and two G:C:G:T base tetrads. A comprehensive analysis of structural stability by DNA thermal melting temperature measurements revealed that oligonucleotides disrupting the formation of the base tetrad or its stacking interactions with a C:C⁺ pair prevent stable i-motif formation, and modifications to the sequence context and length of the lateral loops are difficult. This study further demonstrated that spermine effectively restores the stability reduction caused by creating a bulge, long loop, or dangling end within the minimal i-motif structure, which is less pronounced in the C-rich i-motif. The results suggest that the formation of minimal i-motifs with various sequences is facilitated in polyamine-rich environments, such as the nucleus of mammalian cells. These findings are valuable for identifying potential i-motif-forming sites lacking C-tracts in genomes and provide insights into the electrostatic interactions between i-motif structures and biological polyamines.

Keywords: DNA i-motif; base stacking; bulge; spermine; thermal stability.

Scheme 1

(A) Structure and base–base interactions of the minimal i-motif determined through NMR spectroscopy (PDB ID: 5OGA). (B) The base overlap between C:C⁺ pairs, between the C:C⁺ pair and G:C:G:T tetrad, and between the tetrad and the first T residues of loops.

Scheme 2

(A) Base–base interactions and nucleotide numbering from the 5′ end of the sequence of mini-iM. (B) Slipped base interactions resulting from an insertion of X in the sequence of mini-iM: (a,b) G insertions between C and G, (c) T insertion between T and C, (d,e) C insertions between C and G, and (f) T insertion between two consecutive C residues.

Figure 1

(A) CD spectra of mini-iM in solutions with varying pH: 5.0 (red), 5.9 (black), 7.0 (green), 7.6 (light blue), 8.0 (blue), and 9.0 (purple). (B) Dependence of the T_m of mini-iM on solution pH. (C) Melting curves of mini-iM (black) and oligonucleotides with different substitutions: C replaced with T at position 1 (red) or 13 (blue); T replaced with C at position 6 (green) or 18 (purple); and T replaced with G at position 3 (dashed red line), 8 (dashed green line), 15 (dashed blue line), or 20 (dashed purple line).

Figure 2

T_m values of oligonucleotides forming the i-motifs with varying loop sequences, designed based on the sequence of mini-iM (A,B) or iM (C). Data for oligonucleotides with a T_m below 10 °C are labeled as “nd”, and dashed horizontal lines represent the level of the T_m of mini-iM (36.2 °C, in panels (A,B)) or iM (40.2 °C, in panel (C)).

Figure 3

T_m values of oligonucleotides containing nucleotide insertions in the sequence of mini-iM or iM. (A) Insertions of T, A, and S (abasic nucleotide) between C and G. (B) Insertions of A, G, and S in a nucleotide upstream of a C residue involved in C:C⁺ pairing. (C) Insertions of T and S between consecutive C residues in the sequence of iM. (D–F) Insertions of T (black), G (green), and C (blue) that induce the formation of a slipped G:C:G:T, G:C:G:C, or G:T:G:T tetrad, resulting in an additional unpaired nucleotide as part of either a dangling end (de) or a loop. Data for oligonucleotides without insertions are indicated as white bars. Data for oligonucleotides with a T_m below 10 °C are labeled as “nd”, and dashed horizontal lines represent the level of the T_m of oligonucleotides without insertions.

Figure 4

T_m values of oligonucleotides in the presence of 0.1 mM spermine (red), overlaid with the data obtained in the absence of spermine from Figure 2 and Figure 3. (A–G) Data for minimal i-motif-forming sequences from Figure 2A,B and Figure 3A,B,D–F, respectively. (H,I) Data for C-rich i-motif-forming sequences from Figure 2C and Figure 3C, respectively. Data for oligonucleotides with a T_m below 10 °C are labeled as “nd”, and dashed horizontal lines represent the level of the T_m of mini-iM (46.1 °C, in panels (A–D)) or iM (39.7 °C, in panels (H,I)).