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Single-Molecule Fluorescence Visualization of DNA Polymerase Dynamics at G-Quadruplexes
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Unraveling G-Quadruplex and i-Motif Coexistence Within a Double-Stranded DNA.

Davide Auricchio1, Michele Ghezzo1, Uroš Zavrtanik2

  • 1Department of Pharmaceutical and Pharmacological Sciences, University of Padova, Padova, Italy.

Angewandte Chemie (International Ed. in English)
|June 1, 2026
PubMed
Summary

Guanine-rich DNA sequences can form G-quadruplexes (G4s) and i-Motifs (iMs). This study confirms their simultaneous formation in double-stranded DNA, revealing thermodynamic factors that enable this coexistence.

Keywords:
DSCG‐quadruplexSAXScircular dichroismi‐Motif

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Area of Science:

  • Molecular Biology
  • Biophysics
  • Genetics

Background:

  • DNA can adopt non-canonical structures like G-quadruplexes (G4s) and i-Motifs (iMs) for regulatory functions.
  • The simultaneous formation of G4s and iMs at the same genomic site within double-stranded DNA (dsDNA) has been recently suggested but remains debated.
  • These structures are implicated as synergistic blockers of replication fork progression, highlighting their functional importance.

Purpose of the Study:

  • To investigate the equilibria governing G-quadruplex and i-Motif folding within double-stranded DNA.
  • To elucidate the thermodynamic factors that enable the coexistence of G4 and iM structures in dsDNA.
  • To provide a deeper understanding of the conditions favoring simultaneous G4 and iM formation.

Main Methods:

  • Utilized an extensive biophysical approach.
  • Analyzed an optimized modular DNA system.
  • Scaled the system across constructs of increasing molecular complexity.

Main Results:

  • Corroborated the model of simultaneous G4 and iM formation within dsDNA.
  • Clarified the thermodynamic determinants driving duplex DNA denaturation.
  • Identified factors favoring the stable folding of G4 and iM structures.

Conclusions:

  • The study confirms that G-quadruplexes and i-Motifs can form concurrently within double-stranded DNA.
  • Key thermodynamic principles governing duplex stability and G4/iM folding have been elucidated.
  • Findings advance the understanding of DNA structural dynamics and their functional implications in genomic regulation.