Characterizing Absolute Orientations in DNA Self-Assembly of Single Molecules

Area of Science:

• Molecular Biophysics
• Nanotechnology
• Supramolecular Chemistry

Background:

• DNA self-assembly enables precise molecular arrangement for applications like molecular exciton engineering.
• Existing methods for determining dye orientation on DNA lack a crucial degree of freedom: the dye plane orientation.
• Defining absolute 3D orientations requires accounting for all rotational parameters of the dye relative to the DNA.

Purpose of the Study:

• To develop and apply a novel methodology for determining the absolute 3D orientation of single dye molecules attached to DNA.
• To specifically include the dye plane orientation in the analysis of dye-DNA arrangements.
• To investigate the precise orientation of Cy5 dyes within DNA duplexes.

Main Methods:

• Combined fluorescence-detected linear dichroism (FDLD) and defocused dipole imaging (DDI).
• Utilized DNA point accumulation for imaging in nanoscale topography (DNA-PAINT) super-resolution microscopy for precise localization.
• Integrated these techniques to resolve all degrees of freedom for single-molecule orientation.

Main Results:

• Successfully determined the absolute 3D orientations of single Cy5 dyes relative to DNA duplexes.
• Established that absorption and emission dipoles of Cy5 are perpendicular to the DNA duplex axis.
• Found the mean dye plane of Cy5 is parallel to the DNA bases, supporting intercalation.
• The results confirm Cy5 dyes intercalate between DNA base pairs.

Conclusions:

• The developed multi-modal imaging technique accurately determines absolute 3D dye orientations on DNA, including dye plane orientation.
• The findings provide direct evidence for Cy5 dye intercalation into DNA.
• This methodology opens new avenues for controlling dye arrangements and designing novel dye-DNA nanostructures.