Multimodal Measurements of Single-Molecule Dynamics Using FluoRBT

Area of Science:

• Biophysics
• Molecular Biology
• Structural Biology

Background:

• Single-molecule methods offer direct insights into macromolecular dynamics.
• Current limitations exist in tracking multiple degrees of freedom simultaneously.
• Rotor bead tracking (RBT) is established for measuring DNA torque, twist, and extension.

Purpose of the Study:

• To extend RBT capabilities for simultaneous monitoring of additional degrees of freedom.
• To develop a multi-parameter single-molecule technique for complex molecular processes.
• To investigate DNA structural dynamics and nucleoprotein complex behavior.

Main Methods:

• Development of Fluorescence-RBT (FluoRBT) combining magnetic tweezers, infrared evanescent scattering, and single-molecule FRET.
• Real-time multiparameter measurements of molecular processes.
• Demonstration using DNA hairpin opening/closing and B-form to Z-form DNA transitions.

Main Results:

• Simultaneous measurement of DNA extension and FRET during hairpin dynamics.
• Observation of coupled FRET and torque changes during B-Z DNA transitions.
• Discovery of continuous FRET changes with applied torque and improved FRET data analysis using mechanical signals.

Conclusions:

• FluoRBT enables simultaneous, high-resolution mechanical and FRET measurements.
• This technique facilitates multidimensional investigations of DNA structures and macromolecular machines.
• Unanticipated continuous FRET changes were observed during DNA structural transitions.