Fluorescent End-Labeling and Encapsulation of Long RNAs for Single-Molecule FRET-TIRF Microscopy.

Area of Science:

• Biochemistry and Molecular Biology
• Biophysics
• Nucleic Acid Chemistry

Background:

• Single-molecule Förster Resonance Energy Transition (smFRET) is crucial for observing dynamic biomolecular conformational changes.
• Monitoring RNA dynamics requires precise labeling strategies that avoid artificial modifications.
• Existing methods may not be universally applicable to all RNA sequences and sizes.

Purpose of the Study:

• To develop a versatile and direct end-labeling method for RNA suitable for smFRET studies.
• To enable the observation of RNA dynamics independent of sequence or size.
• To adapt existing chemical reactions for broad applicability in nucleic acid research.

Main Methods:

• Developed a dual-end labeling strategy targeting the 5'-phosphate (via carbodiimide activation with EDC/NHS) and 3'-ribose (via periodate oxidation) of RNA.
• Utilized commercially available chemicals for labeling, avoiding the need for custom probe synthesis.
• Encapsulated dual-end labeled single RNA molecules within phospholipid vesicles for Total Internal Reflection Fluorescence (TIRF) microscopy.

Main Results:

• Successfully achieved covalent labeling of RNA termini with a FRET pair of fluorophores.
• Demonstrated a method adaptable to various RNA molecules, irrespective of size or sequence.
• Established a single-molecule imaging approach by encapsulating labeled RNA in vesicles for TIRF microscopy.

Conclusions:

• The developed dual-end labeling method provides a generalizable approach for studying RNA dynamics using smFRET.
• This technique allows for detailed, dynamic observation of RNA behavior without introducing artificial modifications.
• The 5'-EDC/NHS activation strategy has broader implications for labeling nucleic acids with a 5'-phosphate.