Targeting duplex DNA with chimeric α,β-triplex-forming oligonucleotides.

Area of Science:

• Synthetic biology
• Molecular biology
• Biochemistry

Background:

• Triplex-directed DNA recognition is typically restricted to polypurine sequences.
• This limitation hinders the application of DNA triplexes in various biological contexts.
• Synthetic biology offers tools to engineer novel DNA-binding molecules.

Purpose of the Study:

• To design and characterize chimeric α,β-triplex-forming oligonucleotides (TFOs).
• To investigate the DNA recognition capabilities of these chimeric TFOs.
• To explore the potential of chimeric TFOs in targeting double-stranded DNA, including base pair inversions.

Main Methods:

• Design of short chimeric α,β-TFOs.
• Hybridization studies with fluorescently labeled duplex hairpins.
• Native gel electrophoresis, on-array thermal denaturation, and fluorescence-quenching experiments.
• Circular Dichroism (CD) spectroscopy.
• EcoRI endonuclease protection assay.

Main Results:

• Chimeric TFOs recognize double-stranded DNA through a combination of reversed Hoogsteen and non-canonical hydrogen bonding.
• Chimeric TFOs form highly stable α,β-triplexes in the presence of magnesium ions.
• CD spectra indicate anti-parallel purine triplex formation with GA or GT third strands.
• Chimeric TFOs successfully demonstrated targeting of double-stranded DNA in an endonuclease protection assay.
• First-time report of chimeric TFOs recognizing base pair inversions in a DNA duplex.

Conclusions:

• Chimeric α,β-TFOs represent a significant advancement in synthetic biology for DNA recognition.
• These novel oligonucleotides overcome previous sequence limitations, enabling broader applications.
• The ability to recognize base pair inversions opens new avenues for targeted DNA manipulation and diagnostics.