The effect of overhanging nucleotides on fluorescence properties of hybridising oligonucleotides labelled with Alexa-488 and FAM fluorophores Original Research Article

In order to rationally select and design probes for real-time PCR, we have determined the influence of the overhang region of the complementary strand on the resulting fluorescence from a hybridising probe. A series of target oligonucleotides, each with a unique 3′ overhang (4 bases), was hybridised to either 5′ fluorescein (FAM)- or Alexa-488-labelled probes, and the changes in fluorescence properties were monitored. We found that the number of guanine bases in the overhang region of the target oligonucleotides was proportional to the amount of fluorescence quenching observed for both the FAM and Alexa-488 dyes. FAM appeared to be more sensitive to guanine-induced quenching with three and four guanine bases resulting in greater than a twofold decrease in the quantum yield of the fluorophore compared to the no-overhang target. In addition, we found that adenine bases caused fluorescence quenching of the Alexa-488-labelled probe, whereas the FAM-labelled probe appeared insensitive. The quenching data, generated with the steady-state fluorescence measurements, displayed a linear correlation with that obtained using a fluorescent thermal cycler, suggesting the applicability to real-time PCR measurements. Anisotropy data from the series of duplexes correlated with the fluorescence quantum yield, suggesting that quenching was accompanied by increased dye mobility.