When a benzylcarbamoyl analogue of AP-dC (G-Clamp) was synthesized, it was found that, when incorporated into an oligo, it exhibited similar fluorescence to AP-dC. However, when base-paired against the 8-oxo-dG, its fluorescence was severely quenched. Rather remarkably, however, when base paired with dG or any of the other bases, A, C or T, there was no change in fluorescence - making it a specific probe for 8-oxo-dG.
By attaching pyrene or perylene to the 5 position of deoxyuridine through a triple bond, the fluorophore is electronically coupled to the deoxyuridine base. This electronic coupling of the base and the fluorophore makes the fluorescence sensitive to the base pairing of the dU portion of the molecule, allowing the discrimination between perfect and one base mismatched targets.
Coupling: 6 minute coupling time recommended.
Deprotection: No changes needed from standard method recommended by synthesizer manufacturer.