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Related Experiment Videos

Analysis of various sequence-specific triplexes by electron and atomic force microscopies

D I Cherny1, A Fourcade, F Svinarchuk

  • 1Institute of Molecular Genetics, Moscow, Russia. dtcherny@img.ras.ru

Biophysical Journal
|April 9, 1998
PubMed
Summary

Direct visualization of DNA interactions with triple helix-forming oligonucleotides (TFOs) and peptide nucleic acids (PNAs) was achieved using electron and atomic force microscopy. These techniques revealed structural changes, including height increments and kinks, confirming sequence-specific binding.

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Area of Science:

  • Molecular Biology
  • Biophysics
  • Nanotechnology

Background:

  • Sequence-specific DNA binding is crucial for gene regulation and therapeutic applications.
  • Triple helix-forming oligonucleotides (TFOs) and peptide nucleic acids (PNAs) are key molecules for targeting specific DNA sequences.
  • Direct visualization of these interactions at the nanoscale is essential for understanding their mechanisms.

Purpose of the Study:

  • To directly visualize the sequence-specific interactions of TFOs and bis-PNAs with double-stranded DNA.
  • To characterize the structural alterations induced by TFO and bis-PNA binding using high-resolution microscopy.
  • To provide direct evidence of nanoscale DNA modifications upon binding of these agents.

Main Methods:

  • Electron Microscopy (EM) and Atomic Force Microscopy (AFM) were employed for high-resolution imaging.

Related Experiment Videos

  • Biotinylated TFOs and bis-PNAs were synthesized and incubated with plasmid DNA.
  • Streptavidin was used as a marker for detecting biotinylated TFO-DNA complexes.

    • Main Results:

    • AFM revealed a ~0.4 nm height increment in the DNA double helix at TFO-bound sites.
    • Stable TFO-DNA triplexes formed protruding structures, with apparent heights of ~1.5 nm.
    • Bis-PNA binding induced kinks in the DNA, often accompanied by a ~35% increase in apparent height.

    Conclusions:

    • This study presents the first direct visualization of sequence-specific TFO and PNA interactions with DNA.
    • EM and AFM successfully detected and characterized nanoscale structural changes induced by these molecules.
    • The findings validate the use of these techniques for studying DNA-binding agents and their effects.