Fluorescence Anisotropy Analysis of the Interaction between Doxorubicin and DNA Origami Nanostructures

Abstract Owing to doxorubicin’s high DNA binding affinity, doxorubicin-loaded DNA origami nanostructures (DOX-DONs) are promising nanocarriers against cancer. However, understanding the interactions between doxorubicin (DOX) and DNA origami nanostructures (DONs) is important to ensure the quality of DOX-DONs. This interaction is often taken for granted and the influence of DOX loading conditions is poorly characterized. Exploiting the inherent fluorescence of DOX, steady-state and time-resolved fluorescence anisotropy spectroscopy techniques are used for characterizing non-destructively the binding between DOX and DONs, and the purity of formed complexes. The difference in fluorescence anisotropy between free DOX and DOX-DONs confirms the DOX-DON complex formation. Further, at loading ratios of DOX to DNA base pairs > 0.5, homo-Förster resonance energy transfer (homo-FRET) between closely packed DOX molecules is observed. Moreover, time-resolved anisotropy reveals DOX aggregation on DONs at high loading ratios > 1. For loading ratios > 0.1, spin-filtration to remove excess free DOX is efficient and necessary, though at loading ratios > 1 some DOX aggregates remain attached to the DONs. In summary, fluorescence anisotropy analysis provides more detailed information and insight into DOX-DONs compared to regularly used fluorescence intensity-based characterization methods, and these results can help designing more efficient and safer DNA intercalator-based nanocarriers. Competing Interest Statement The authors have declared no competing interest. Footnotes ↵* E-mail: ekaterina.lisitsyna{at}tuni.fi, E-mail: timo.laaksonen{at}helsinki.fi, E-mail: veikko.pentti.linko{at}ut.ee