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

摘要

The fluorescence signal levels produced by smart probes (SPs) largely depend on the type of fluorophore label used andthe quenching efficiency of the quencher. However, there has been little emphasis on the effects of terminal location of thefluorophore and the modifications used to attach the fluorophore to the oligonucleotide chain. To this end, we have conducteda comparative study of the fluorescent signaling characteristics of six SPs consisting of three fluorophores: 6-FAM, ATTO488and TAMRA, each of which was attached to either the 3 end or the 5 end of the oligonucleotide probe to make two SPseach. In each case, the fluorophore was paired with guanosine quenchers and we determined the fluorescence signalingcharacteristic of each SP by hybridizing it with the complementary target and measuring the fluorescence signal produced.Our results showed that the melting temperatures (Tm’s) of each two SPs consisting of the same fluorophore were differentbefore hybridization with targets but were similar once hybridized. We also found that each two SPs made of the samefluorophore gave different fluorescence signal levels. Our data also revealed a subtle correlation between Tm differencesand fluorescence intensity differences for each pair of SPs consisting of the same fluorophore. We interpret the observeddifferences in Tm’s and fluorescence signals in terms of possible structural differences in the linker moieties used for couplingthe fluorophores to the 3 and 5 ends as well as possible differences in the efficiency of coupling the fluorophore during theprobe synthesis.