Drivers of i-DNA Formation in a Variety of Environments Revealed by Four-Dimensional UV Melting and Annealing

Abstract

i-DNA is a four-stranded, pH-sensitive structure formed by cytosine-rich DNA sequences. Previous reports have addressed the conditions for formation of this motif in DNA in vitro and validated its existence in human cells. Unfortunately, these in vitro studies have often been performed under different experimental conditions, making comparisons difficult. To overcome this, we developed a four-dimensional UV melting and annealing (4DUVMA) approach to analyze i-DNA formation under a variety of conditions (e.g., pH, temperature, salt, crowding). Analysis of 25 sequences provided a global understanding of i-DNA formation under disparate conditions, which should ultimately allow the design of accurate prediction tools. For example, we found reliable linear correlations between the mid-point of pH transition and temperature (-0.04 ± 0.003 pH unit per 1.0 °C temperature increment) and between the melting temperature and pH (-23.8 ± 1.1 °C per pH unit increment). In addition, by analyzing the hysteresis between denaturing and renaturing profiles in both pH and thermal transitions, we found that loop length, nature of the C-tracts, pH, temperature, and crowding agents all play roles in i-DNA folding kinetics. Interestingly, our data indicate which conformer is more favorable for the sequences with an odd number of cytosine base pairs. Then the h m l pH l f " " -DNAs from human promoter genes were measured under near physiological conditions (pH 7.0, 37 °C). The 4DUVMA method can become a universal resource to analysis the properties of any i-DNA-prone sequence.

Engineering a Facile Aptamer “Molecule-Doctor” with Hairpin-Contained I-Motif Enables Accurate Imaging and Killing of Cancer Cells

“…Additionally, with the pH decreased from 7.4 to 6.4, the UV absorption value of pH-Apt-MD at 295 nm gradually increased. It could be seen that the CD and UV results of pH-Apt-MD were consistent with the behavior of free I-M2 under different pH conditions, indicating that the increasing acidity could facilitate i-motif folding on pH-Apt-MD. − …”

Environment-Sensitive Nucleoside Probe Unravels the Complex Structural Dynamics of i-Motif DNAs

“…This observation suggests that the C-rich ONs could adopt more than one iM form differing in folding kinetics and stabilities. 36,38,40 Collectively, it is clear that the modification has negligible impact on the structures formed by the ONs.…”

“…Much like the GQ, iM also exhibits structural polymorphism, , which poses major challenges in their study as majority of tools poorly distinguish different topologies. This is further compounded by the fact that C-rich sequences can potentially form several iMs with small energy differences between them leading to a complex population equilibrium, which depends on the composition of the sequence and environmental conditions. − iM structure, stability, and recognition are usually evaluated by circular dichroism (CD), UV-thermal melting, and to some extent by NMR and X-ray crystallography techniques. ,,, However, when multiple species are present, seldom these techniques provide useful information as they fail to resolve individual structures. In this context, ultrafast time-resolved IR and fluorescence spectroscopy techniques have been used to study the folding behavior of C-rich tracts. , Notably, a combination of FRET and FCS analysis as a function of pH in bulk and at the single-molecule level supported the coexistence of partially folded structures (iMs) along with the single-stranded structure at neutral pH, which was not evident in CD spectra .…”

Ratiometric i-Motif-Based Sensor for Precise Long-Term Monitoring of pH Micro Alterations in the Nucleoplasm and Interchromatin Granules

“…Inspired by the four-dimensional UV melting and annealing (4DUVMA) approach, 48 we further characterized FAM-C5T-TAMRA and the triple-modified iMs by melting assays with fluorescence detection at different pH (Figure 1d), which gave 4DUVMA-like iM formation diagrams (Figure 1e). Consistently with the previously reported results, 48 linear correlation between pH 1/2 and the melting temperature was observed. A minor but noticeable stabilizing effect of the modification was maintained throughout the physiological and room temperature range.…”