Thioflavin T as a fluorescence light-up probe for G4 formation

Area of Science:

• Biochemistry
• Molecular Biology
• Biophysical Chemistry

Background:

• Thioflavin T (ThT) is known to fluoresce in the presence of G-quadruplex (G4) structures, such as those formed by the human telomeric motif.
• G4 structures are non-canonical DNA secondary structures with implications in various biological processes.
• Developing specific and efficient probes for G4 detection is crucial for understanding their roles.

Purpose of the Study:

• To generalize and extend the observation of ThT fluorescence with G4 structures.
• To establish ThT as a convenient and specific probe for quadruplex structures.
• To develop a high-throughput assay for detecting G4 formation.

Main Methods:

• Investigated ThT fluorescence emission in the presence of various G4-forming sequences.
• Compared ThT fluorescence with control duplexes and single strands.
• Designed and implemented a high-throughput assay using fluorescence plate readers for parallel oligonucleotide testing.
• Applied the assay to a family of aptamers not previously recognized for G4 formation.

Main Results:

• Observed a significant increase in ThT fluorescence emission with most G4-forming sequences.
• Demonstrated a limited effect of ThT on control duplexes and single strands, enabling differential detection.
• Successfully developed a high-throughput assay capable of testing hundreds of oligonucleotides in parallel.
• Showed that ThT fluorescence signal can predict G4 formation in aptamer families.

Conclusions:

• Thioflavin T serves as a reliable and specific fluorescent probe for G-quadruplex structures.
• The developed high-throughput assay enables efficient screening of numerous sequences for G4 formation.
• This method facilitates the discovery of novel G4-forming sequences, including aptamers, and aids in understanding their biological relevance.