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This study reveals the G-quadruplex folding pathway of a DNA oligonucleotide. The folding process involves intermediates and is significantly slower to unfold, indicating high thermodynamic stability.

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Area of Science:

  • Molecular Biology
  • Biophysics
  • Structural Biology

Background:

  • G-quadruplexes are nucleic acid structures with significant biological relevance.
  • Understanding DNA G-quadruplex folding pathways is crucial for their therapeutic applications.

Purpose of the Study:

  • To investigate the thermodynamic stability and folding pathway of the DNA oligonucleotide d[TGAG3TG3TAG3TG3TA2].
  • To elucidate the structural transitions and kinetics of G-quadruplex formation.

Main Methods:

  • Circular dichroism spectroscopy
  • Stopped-flow UV spectroscopy
  • Molecular dynamics simulations
  • 1D-proton Nuclear Magnetic Resonance (NMR)
  • Fluorescence spectroscopy

Main Results:

  • The oligonucleotide forms a stable, all-parallel G-quadruplex with three stacked quartets at 25 °C in 25 mM KCl.
  • K+-induced folding exhibits three distinct relaxation times, completing within 200 s.
  • The folding pathway involves at least two intermediates, with one potentially being antiparallel before rearranging.
  • Molecular dynamics supports a folding pathway without complete intermediate unfolding.
  • Unfolding is extremely slow, characterized by a single rate-limiting relaxation time.

Conclusions:

  • The DNA oligonucleotide d[TGAG3TG3TAG3TG3TA2] forms a stable all-parallel G-quadruplex structure.
  • The folding pathway is complex, involving transient intermediates, and the G-quadruplex exhibits remarkable kinetic stability against unfolding.