Highly sequence selective DNA detections using molecular beacons (MBs) are important tools in biotechnological applications. A novel MB system comprising a quencher-free linear beacon with only a single fluorophore can discriminate between fully matched and single base-mismatched sequences.
In order to develop other quencher-free linear beacons, we have designed a UFO nucleotide bearing a 2-ethynylfluorenone moiety covalently attached to the C-5 position of deoxyuridine.
The fluorescence of UFO ODNs exhibited a dependence on the nature of its FBs. When the FBs were dT or dC, the total discrimination factors (relative area ratios of the fluorescence intensity between the perfectly matched and mismatched targets) for the duplex formations were high. In particular, the UFO DNA probe with dT-FBs behaves more sensitively than any other combinations of FBs. We also found that when a short strand (five-nucleotide), which is a completely natural strand containing a deoxyguanine (dG) nucleobase, was used as an internal quencher, the intrinsic fluorescence of a fluorescent DNA probe was reduced by 24%. These results suggest that this type of probe is an extremely powerful tool for the detection of single nucleotide alterations, and moreover, they exhibit highly dA-selective fluorescence emission.