Assembly Dependent Fluorescence Enhancing Nucleic Acids in Sequence-Specific Detection of Double-Stranded DNA

Area of Science:

• Molecular Biology
• Biochemistry
• Nucleic Acid Chemistry

Background:

• Triplex-forming oligonucleotides (TFOs) are crucial for targeting specific DNA sequences.
• Thiazole orange (TO) dyes are fluorescent probes used in nucleic acid detection.
• Optimizing probe design is essential for sensitive and specific molecular diagnostics.

Purpose of the Study:

• To investigate the impact of thiazole orange derivative positioning within TFOs on fluorescence properties.
• To develop novel TFO-based probes (AFENA-TINA-TFOs) with improved fluorescence for nucleic acid detection.
• To evaluate the performance of these probes in detecting DNA and RNA targets under physiological conditions.

Main Methods:

• Synthesis of TFOs with varying thiazole orange derivative positions.
• Assembly-dependent fluorescence enhancing nucleic acids (AFENA) probe development.
• Incorporation of twisted intercalating nucleic acid (TINA) monomers into TFOs.
• Fluorescence intensity and quantum yield measurements of TFO complexes with DNA and RNA.

Main Results:

• AFENA probes exhibited low fluorescence in single-stranded states (Φ_F =0.9–1.5%) but significantly enhanced fluorescence upon DNA triplex formation (Φ_F =23.5–34.9%).
• TINA monomer insertion improved fluorescence and eliminated guanosine-induced self-aggregation, promoting triplex formation at pH 7.2.
• AFENA-TINA-TFOs demonstrated effective binding to duplex DNA in the presence of complementary RNA at 37°C.

Conclusions:

• The strategic placement of TO dyes and incorporation of TINA monomers significantly enhance TFO-based fluorescent probes.
• AFENA-TINA-TFOs offer a promising platform for sensitive and specific detection of nucleic acid targets, including RNA, in complex biological environments.
• This work advances the development of nucleic acid probes for molecular diagnostics and research applications.