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Related Experiment Videos

TOTO binding affinity analysis using single-molecule fluorescence spectroscopy.

Benjamin P Bowen1, Neal W Woodbury

  • 1Department of Chemical and Materials Engineering, Arizona State University, Tempe, AZ 85287, USA. bpb@asu.edu

Photochemistry and Photobiology
|January 28, 2004
PubMed
Summary
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The thiazole orange dimer (TOTO) shows no sequence preference when binding to double-stranded DNA (dsDNA). Its binding affinity is determined by the overall DNA background sequence, not specific sites, suggesting broad applicability in biological settings.

Area of Science:

  • Molecular Biology
  • Biophysics
  • Fluorescence Spectroscopy

Background:

  • Thiazole orange dimer (TOTO) is a DNA-intercalating fluorophore.
  • Understanding sequence-specific DNA binding is crucial for molecular diagnostics and therapeutics.
  • Previous studies suggested specific high-affinity binding sites for TOTO.

Purpose of the Study:

  • To investigate the sequence dependence of TOTO's double-stranded DNA (dsDNA) binding affinity.
  • To determine if TOTO exhibits preference for previously identified high-affinity sequences.
  • To assess TOTO's binding behavior under conditions relevant to biological applications.

Main Methods:

  • Single-molecule fluorescence methods were employed to measure TOTO binding.
  • Excited-state fluorescence lifetimes of single TOTO molecules bound to dsDNA were analyzed.

Related Experiment Videos

  • Competitive binding experiments were conducted using dsDNA fragments with varying sequences (poly-AT, poly-GC, and specific sites).

    • Main Results:

    • TOTO fluorescence lifetime was solely dependent on the background DNA sequence (poly-AT vs. poly-GC).
    • No significant difference in binding affinity was observed for putative high-affinity sites compared to poly-AT or poly-GC sequences.
    • TOTO demonstrated no preference for specific binding sites over general AT or GC-rich regions.

    Conclusions:

    • TOTO binding to dsDNA exhibits minimal sequence dependence under the tested conditions.
    • The background DNA sequence, rather than specific motifs, primarily influences TOTO binding affinity.
    • These findings suggest TOTO's broad utility as a DNA-intercalating dye in diverse biological applications.