Fluorogenic thiazole orange TOTFO probes stabilise parallel DNA triplexes at pH 7 and above

Area of Science:

• Biochemistry
• Molecular Biology
• Oligonucleotide Chemistry

Background:

• DNA triplexes are important for various applications but are limited by instability at neutral pH.
• Current methods to stabilize DNA triplexes are often insufficient for broad applicability.

Purpose of the Study:

• To develop a method for stabilizing DNA triplexes at neutral pH and above.
• To enhance the binding affinity and stability of triplex-forming oligonucleotides (TFOs).

Main Methods:

• Introduction of thiazole orange (TO) intercalators onto thymine nucleobases in TFOs.
• Synthesis and characterization of TO-modified TFOs.
• Assessment of triplex stability using melting temperature (Tm) measurements and binding assays.

Main Results:

• TO modification significantly enhanced triplex stability, with additive effects observed for multiple TO units.
• A TFO with three TO units increased triplex melting temperature by 50 °C at pH 7.
• TO intercalation enabled TFO binding to non-polypurine sequences using 5-(1-propynyl)cytosine (pC).

Conclusions:

• Thiazole orange modification effectively stabilizes DNA triplexes across a wide pH range and sequence contexts.
• These 'TOTFO' probes offer a simple yet powerful solution to overcome triplex instability.
• The enhanced stability and binding properties of TOTFOs can expand triplex applications in diagnostics and cell imaging.