Loop Sequence Context Influences the Formation and Stability of the i-Motif for DNA Oligomers of Sequence (CCCXXX)₄, where X = A and/or T, under Slightly Acidic Conditions

Abstract

The structure and stability of DNA is highly dependent upon the sequence context of the bases (A, G, C, and T) and the environment under which the DNA is prepared (e.g., buffer, temperature, pH, ionic strength). Understanding the factors that influence structure and stability of the i-motif conformation can lead to the design of DNA sequences with highly tunable properties. We have been investigating the influence of pH and temperature on the conformations and stabilities for all permutations of the DNA sequence (CCCXXX)4, where X = A and/or T, using spectroscopic approaches. All oligomers undergo transitions from single-stranded structures at pH 7.0 to i-motif conformations at pH 5.0 as evidenced by circular dichroism (CD) studies. These folded structures possess stacked C:CH(+) base pairs joined by loops of 5'-XXX-3'. Although the pH at the midpoint of the transition (pHmp) varies slightly with loop sequence, the linkage between pH and log K for the proton induced transition is highly loop sequence dependent. All oligomers also undergo the thermally induced i-motif to single-strand transition at pH 5.0 as the temperature is increased from 25 to 95 °C. The temperature at the midpoint of this transition (Tm) is also highly dependent on loop sequence context effects. For seven of eight possible permutations, the pH induced, and thermally induced transitions appear to be highly cooperative and two state. Analysis of the CD optical melting profiles via a van't Hoff approach reveals sequence-dependent thermodynamic parameters for the unfolding as well. Together, these data reveal that the i-motif conformation exhibits exquisite sensitivity to loop sequence context with respect to formation and stability.

Systematic investigation of sequence requirements for DNA i-motif formation

“…The most stable was reported (30) as being the iM with only one nucleotide in the loop. Stability of the iM also depends on the type of nucleobases in the loops (26,31) and their precise sequence (32). The most stabilizing arrangement of the first and the third loops was reported to include three thymines or two thymines with a cytosine in the middle (33).…”

Evaluation of i‐Motif Formation in the Serotonin Transporter‐Linked Polymorphic Region

“…CD melting experiments were undertaken to investigate the thermal stability of i‐motifs formed by VNTR‐S and VNTR‐L at pH 5 [18,19] . Figure 4 shows CD spectra of both oligonucleotides as a function of temperature.…”

“…Where θ 286, x °C is the molar ellipticity at 286 nm at a given temperature, θ 286, 20 °C is the molar ellipticity at 286 nm at 20 °C, and θ 286, 90 °C is the molar ellipticity at 286 nm at 90 °C [19,20] …”

“…Shown in Figure 7, transition profiles for fraction of i-motif as a function of temperature were generated using the molar ellipticity at 286 nm: Where θ 286, x°C is the molar ellipticity at 286 nm at a given temperature, θ 286, 20°C is the molar ellipticity at 286 nm at 20°C, and θ 286, 90°C is the molar ellipticity at 286 nm at 90°C. [19,20] Under conditions of molecular crowding, VNTR-L formed imotif structures more readily and a greater ellipticity was observed. While this difference was reproducible between experiments, it is unclear whether this is attributable to the VNTR-L existing in a conformation that more readily forms imotifs.…”

“…CD melting experiments were undertaken to investigate the thermal stability of i-motifs formed by VNTR-S and VNTR-L at pH 5. [18,19] Figure 4 shows CD spectra of both oligonucleotides as a function of temperature. The bathochromic shift which is characteristic of i-motif formation is observed between 65-75°C for both VNTR-S and VNTR-L, thus indicating that i-motif structures formed by both oligonucleotide variants have similar thermal stabilities.…”