Cellular Visualization of G-Quadruplex RNA via Fluorescence- Lifetime Imaging Microscopy

Area of Science:

• Molecular Biology
• Biochemistry
• Cell Biology

Background:

• G-quadruplex (G4) structures play crucial roles in cellular processes, necessitating reliable visualization methods.
• Existing fluorescent probes for G4 detection face challenges with selectivity and cellular uptake variations.
• Distinguishing G4s from other cellular structures requires probes with high specificity and sensitivity.

Purpose of the Study:

• To develop a novel small-molecule approach for visualizing G-quadruplexes (G4s) in cellular environments.
• To create a probe that overcomes limitations of fluorescence intensity-based detection and cellular uptake variability.
• To validate a new probe for detecting RNA G4s in cellulo.

Main Methods:

• Development of a novel thiazole orange derivative, named TOR-G4.
• Utilizing fluorescence lifetime measurements to detect G4 binding, independent of probe concentration.
• Assessing probe colocalization with cellular components, specifically RNA, in cytoplasm and nucleoli.

Main Results:

• TOR-G4 exhibits a unique fluorescence lifetime upon binding to G4s, enabling sensitive discrimination from non-G4 structures.
• The probe's detection mechanism is independent of local probe concentration, addressing a key limitation of intensity-based probes.
• TOR-G4 demonstrates significant colocalization with RNA in the cytoplasm and nucleoli, validating its use for RNA G4 studies.

Conclusions:

• TOR-G4 provides a robust, lifetime-based method for visualizing G4s in cells, overcoming common challenges.
• This probe is the first validated lifetime-based tool for studying RNA G4s in cellulo.
• The findings open new avenues for exploring the biological significance of RNA G4s.