Bleaching-resistant, Near-continuous Single-molecule Fluorescence and FRET Based on Fluorogenic and Transient DNA...

Area of Science:

• Biophysics
• Single-molecule spectroscopy
• Molecular dynamics

Background:

• Photobleaching of fluorescent probes limits observation duration in single-molecule fluorescence measurements.
• Current limitations hinder the study of molecular dynamics at long timescales.

Purpose of the Study:

• To present a general strategy to circumvent photobleaching in single-molecule fluorescence studies.
• To enable extended observation of molecular dynamics beyond typical photobleaching limits.

Main Methods:

• Developed a method involving transient binding of fluorogenic DNA probes to complementary DNA strands on target molecules.
• Utilized replenishing fluorescent probes to maintain signal during observation.
• Adapted the method for Förster Resonance Energy Transfer (FRET) using orthogonal DNA sequences.

Main Results:

• Achieved near-continuous single-molecule fluorescence observation for over an hour.
• Extended observation timescale by two orders of magnitude compared to typical photobleaching times.
• Demonstrated adaptability to FRET for studying conformational dynamics of structures like DNA Holliday junctions.

Conclusions:

• The developed strategy effectively overcomes photobleaching limitations in single-molecule fluorescence.
• Enables extended observation of molecular dynamics, crucial for understanding complex biological processes.
• Offers a versatile approach for studying conformational dynamics of proteins and nucleic acids across various timescales.