Flipping DNA caught in the act

A new fluorescence-based approach could make it easier for researchers to watch a mysterious, but essential, mechanism of DNA repair.  

DNA repair after accidental damage is essential for life. The process often requires one DNA base to rotate to the outside of the helix, a step known as base-flipping.   The mechanism is difficult to observe, but US researchers have now developed a technique that specifically detects the flipped-out base. 

Lauren O’Neil and Olaf Wiest of the University of Notre Dame, Indiana, US, built a novel bifunctional molecule that first binds to the flipped out base and then fluoresces. The molecule includes a zinc-cyclen complex designed to bind to the protruding single base, and a dansyl fluorescent dye that undergoes a characteristic spectroscopic change when it is moved from the bulk aqueous solvent into the highly charged environment surrounding the DNA double helix. 

Control experiments with specific DNA constructs representing the flipped and the unflipped state showed that the new binder-reporter construct yields a large signal change upon base-flipping, which can be detected in the low nanomolar range. An additional control using only the reporter without the binder confirmed that the observed effect is due to the binding of the zinc-cyclen to the rogue base and not to intercalation of the dye. 

Wiest now plans to gain more understanding of the process of base flipping. ’We are currently looking at the accurate determination of the equilibrium constants for base flipping and the sequence dependence,’ he told Chemistry World. Ultimately, he plans to go back to the biological problem which motivated his research: DNA repair.

Thomas Carell from the University of Munich, Germany, recognizes the potential for Wiest’s technique. ’The detection of damaged bases is of extreme importance in the field of DNA repair,’ said Carell. ’Compounds that can detect damage may be useful for the fine tuning of cytostatic drugs.’ Michael Gross