Author ORCID Identifier
Doctor of Philosophy
Soma Dhakal, Ph.D.
Ashton Cropp, Ph.D.
Indika U. Arachchige, Ph.D.
Julio Alvarez, Ph.D.
Dexian Ye, Ph.D.
Although the canonical right-handed B-DNA is the predominant form of DNA in cells, DNA can adopt other secondary structures such as hairpins, G-quadruplexes (G4), and intercalated motifs (i-motif). These secondary structures are typically formed in the regions with repeat sequences. Among many secondary structures DNA can adopt, G-quadruplexes and i-motifs (tetraplex structures formed in the G-rich and C-rich sequences, respectively) caught significant research interest in recent years due to the prevalence of G-rich and C-rich sequences in the human genome. While G-quadruplexes are extensively studied, little is known about i-motifs – especially their formation and stability under cellular environments. For example, i-motif has shown to remain stable and persistent in the complex intracellular environment of living HeLa cells and implicated for gene regulation and expression such as DNA transcription and replication and telomerase inhibition. Since i-motif is stabilized by intercalated cytosine+–cytosine (CH+– C) base pairs, it was originally thought to only form in acidic pH. However, recent studies have shown that the formation and stability of i-motif does not solely rely on the solution pH but also on various microenvironments such as DNA topology, ionic conditions, and crowding. Therefore, due to the limited knowledge on the formation of i-motif under cellular environments and how such environments affect its conformational states, the biological roles and potential therapeutic applications of i-motifs are still presumptive.
One of the key reasons for the aforementioned knowledge gap is because the vast majority of i-motif studies have utilized short single-stranded sequences, a condition far from the microenvironment the DNA experiences in cells. Inside cells, the i-motif forming sequences are flanked by long stretches of DNA duplexes and packed in the genome, which results in a much lesser degree of freedom for secondary structures due to the DNA’s own topological constraint. Hence, we designed self-assembled DNA nanostructures as molecular tools to study human telomeric (hTel) i-motif under cell-mimic topologically constrained environments. Using fluorescently labeled DNA nanoassemblies and implementing single-molecule fluorescence resonance energy transfer (smFRET) as a major analysis technique, we systematically investigated the effect of topological constraint in folding and conformational dynamics of the hTel i-motif. Specifically, we incorporated the i-motif–forming sequence either at the terminal position of a dsDNA or embedded within DNA duplexes/DNA nanocircles. Using these systems, we showed that the hTel sequence assumes an i-motif structure at pH 5.5 and a partially folded state at pH 7.0 but remains unstructured at high pH (≥8.0). Interestingly, the sequence undergoes equilibrium dynamics among i-motif, partially folded, and unfolded states at slightly acidic pH (pH 6.5) and the kinetics of switching is different in different nanoassemblies, suggesting a correlated effect of topological constraint on the conformational dynamics.
Besides topological constraint, the intracellular environment is inherently crowded with various biomolecules such as proteins and nucleic acids, with water accounting for only ~60% of the solution mass in the nucleus. The crowded environment limits the available volume for DNA, which can alter not only the stability and conformational dynamics of non-B DNA structures such as i-motif but also their interaction with other biomolecules such as proteins. Therefore, it is imperative to use topologically constrained and crowded environments to decipher biologically relevant properties of non-B DNA structures. Along this line, we used polyethylene glycol (PEG6000) as a molecular crowding agent to investigate the effect of crowding on the stability and conformational dynamics of nanocircle-embedded i-motif. In this part of the study, using smFRET, we showed that the molecular crowding stabilizes i-motif even at physiological pH.
Further, we demonstrated that the i-motif can compete with the formation of dsDNA under crowding even in the presence of complementary G-rich strand. Together, we developed and implemented DNA nanocircles as molecular tools to study i-motif under biomimetic environments and our findings suggest that the crowding may facilitate the formation of i-motif inside cells despite the topological constraint imposed by DNA bending.
Apart from the possible formation of i-motifs by C-rich sequences under intracellular environments, in cancer cells the C-rich sequences are also found in the form of single-stranded circles called C-circles. These C-circles are the result of alternative lengthening of telomeres (ALT) in cancer cells to avoid degradation of telomeres. Hence, the C-circles are possible markers for ALT activation in cancer cells. This is important because ALT has been taken as an important target for anticancer treatment as ~10% of cancers depend on this telomere maintenance mechanism. Although ALT inhibitors are proposed to be helpful for cancer therapeutics, no ALT inhibitors have been successfully developed because of the lack of a suitable method to probe ALT. In this regard, sensors capable of detecting C-circles would be very useful. Therefore, we have recently developed a four-way DNA sensor intended to apply for a sensitive detection of DNA C-circles. In this part of the study, we first developed a background-free ultrasensitive sensor to detect single-stranded DNA and used it to detect C-circles. Results show that the sensor successfully discriminates between the circular and linear DNA targets, which is a suitable platform not only to detect C-circles but also to investigate other conformational properties of i motifs in C-circles. Therefore, the developed FRET-based sensing approach has the potential to enable early diagnosis of ALT+ cells via C-circle detection.
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Available for download on Monday, May 11, 2026
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