Comparative Study of Thermal Stability and On/Off Fluorescent Signaling Characteristics of Self-Quenching Smart Probes

The fluorescence signal levels produced by smart probes (SPs) largely depend on the type of fluorophore label used and the quenching efficiency of the quencher. However, there has been little emphasis on the effects of terminal location of the fluorophore and the modifications used to attach the fluorophore to the oligonucleotide chain. To this end, we have conducted a comparative study of the fluorescent signaling characteristics of six SPs consisting of three fluorophores: 6-FAM, ATTO488 and TAMRA, each of which was attached to either the 3′ end or the 5′ end of the oligonucleotide probe to make two SPs each. In each case, the fluorophore was paired with guanosine quenchers and we determined the fluorescence signaling characteristic of each SP by hybridizing it with the complementary target and measuring the fluorescence signal produced. Our results showed that the melting temperatures (T_m’s) of each two SPs consisting of the same fluorophore were different before hybridization with targets but were similar once hybridized. We also found that each two SPs made of the same fluorophore gave different fluorescence signal levels. Our data also revealed a subtle correlation between T_m differences and fluorescence intensity differences for each pair of SPs consisting of the same fluorophore. We interpret the observed differences in T_m’s and fluorescence signals in terms of possible structural differences in the linker moieties used for coupling the fluorophores to the 3′ and 5′ ends as well as possible differences in the efficiency of coupling the fluorophore during the probe synthesis.