Evaluating Linker Architecture in RNA-Detecting Riboglow Probes and Effects on Fluorescence Turn-On

Area of Science:

• Molecular Biology
• Biochemistry
• Chemical Biology

Background:

• Riboglow probes are RNA-targeting molecules featuring a fluorophore and RNA-binding moiety linked chemically.
• Probe fluorescence changes upon binding to target RNA sequences.
• The linker's architecture modulates fluorescence properties like intensity and lifetime.

Purpose of the Study:

• To systematically investigate how linker composition affects Riboglow probe performance.
• To optimize linker design for enhanced RNA detection and multiplexing capabilities.
• To assess the utility of optimized Riboglow probes in live-cell imaging.

Main Methods:

• Synthesis and characterization of Riboglow probes with varied linker compositions (glycine, polyethylene glycol).
• Assessment of fluorescence intensity and lifetime changes upon RNA binding.
• Evaluation of probe performance in live mammalian cells.

Main Results:

• Glycine linkers demonstrated superior fluorescence 'turn-on' compared to polyethylene glycol linkers.
• Increasing polyglycine linker length amplified fluorescence changes upon RNA binding.
• Linker composition influenced fluorescence lifetime contrast, enabling RNA multiplexing.
• Riboglow probes successfully visualized RNA in live mammalian cells.

Conclusions:

• The chemical linker is a critical determinant of Riboglow probe sensitivity and specificity.
• Polyglycine linkers offer advantages for RNA detection and multiplexing.
• Optimized Riboglow probes are suitable for live-cell RNA visualization.
• Findings guide the rational design of future RNA-sensing probes.