Single-molecule dynamic structural biology with vertically arranged DNA on a fluorescence microscope.

Area of Science:

• Biophysics
• Molecular Biology
• Nanotechnology

Background:

• Understanding DNA-protein interactions is crucial for fundamental biological processes like DNA replication, transcription, and repair.
• Dynamic nanoscale observations are needed to elucidate the mechanisms of these interactions.
• Existing methods often lack the required spatial and temporal resolution for dynamic studies.

Purpose of the Study:

• To introduce a novel method, graphene energy transfer with vertical nucleic acids (GETvNA), for observing DNA-protein interactions at the nanoscale.
• To achieve high spatial (Ångström scale) and temporal (subsecond) resolution in studying dynamic DNA conformational changes.
• To investigate specific DNA structural features and protein binding events.

Main Methods:

• Utilizing the vertical orientation of double-stranded DNA on graphene surfaces.
• Employing Förster Resonance Energy Transfer (FRET) from a probe dye to graphene.
• Achieving nanoscale spatial resolution and subsecond temporal resolution for dynamic observations.

Main Results:

• Successfully measured DNA bending induced by structural anomalies (adenine tracts, bulges, abasic sites) and protein binding (endonuclease IV).
• Observed the translocation of O⁶-alkylguanine DNA alkyltransferase on DNA with single base-pair resolution.
• Detected preferential binding of the O⁶-alkylguanine DNA alkyltransferase to adenine tracts.

Conclusions:

• The GETvNA method provides unprecedented dynamic insights into nucleic acid and DNA-protein interactions.
• This technique achieves resolution comparable to traditional structural biology methods but in a dynamic context.
• GETvNA is poised for broad application in studying dynamic biological processes involving DNA and proteins.