Single-molecule analysis of i-motif within self-assembled DNA duplexes and nanocircles

Nucleic Acids Res. 2019 Aug 22;47(14):7199-7212. doi: 10.1093/nar/gkz565.


The cytosine (C)-rich sequences that can fold into tetraplex structures known as i-motif are prevalent in genomic DNA. Recent studies of i-motif-forming sequences have shown increasing evidence of their roles in gene regulation. However, most of these studies have been performed in short single-stranded oligonucleotides, far from the intracellular environment. In cells, i-motif-forming sequences are flanked by DNA duplexes and packed in the genome. Therefore, exploring the conformational dynamics and kinetics of i-motif under such topologically constrained environments is highly relevant in predicting their biological roles. Using single-molecule fluorescence analysis of self-assembled DNA duplexes and nanocircles, we show that the topological environments play a key role on i-motif stability and dynamics. While the human telomere sequence (C3TAA)3C3 assumes i-motif structure at pH 5.5 regardless of topological constraint, it undergoes conformational dynamics among unfolded, partially folded and fully folded states at pH 6.5. The lifetimes of i-motif and the partially folded state at pH 6.5 were determined to be 6 ± 2 and 31 ± 11 s, respectively. Consistent with the partially folded state observed in fluorescence analysis, interrogation of current versus time traces obtained from nanopore analysis at pH 6.5 shows long-lived shallow blockades with a mean lifetime of 25 ± 6 s. Such lifetimes are sufficient for the i-motif and partially folded states to interact with proteins to modulate cellular processes.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Algorithms
  • Circular Dichroism
  • Cytosine / chemistry*
  • Cytosine / metabolism
  • DNA / chemistry*
  • DNA / genetics
  • Humans
  • Hydrogen-Ion Concentration
  • Kinetics
  • Microscopy, Fluorescence
  • Models, Molecular
  • Nanopores*
  • Nucleic Acid Conformation*
  • Nucleotide Motifs*


  • Cytosine
  • DNA