We have developed a new quencher-free stemless linear probe involving multiple perylenes incorporated through D-threoninol; each perylene is separated by intervening natural nucleotides. Without a substrate, the flexible linear probe does not emit fluorescence due to the self-quenching of the weakly interacting fluorophores. Upon hybridization with the target, intercalation of each dye between the base pairs results in emission of strong fluorescence. The maximum signal-background ratio attained was 180, and the response rate was significantly faster than that of a classic hairpin-forming molecular beacon.

A stemless linear probe was designed that robustly detects mRNA in cells with high sensitivity. The probe is modified at some positions with base surrogates prepared from D-threoninol, with anthraquinone moieties near the 5'- and 3'-termini, and with perylene moieties. Even in cell lysate that involves various proteins and enzymes, background emission was very low. When the probe was hybridized with RNA, chromophores are intercalated between the base pairs, resulting in a remarkable light-up signal. The signal-to-background ratio was as high as 1600 under our standard buffer conditions. In the HeLa cell lysate, the linear probe had sufficient signal-to-background ratio (S/B=40) for reliable mRNA detection. No degradation was observed after a 24 h incubation in HeLa cell lysate. In cells, a probe designed to target DsRed resulted in distinct blue fluorescence only in cells transfected with plasmid encoding DsRed; no fluorescence was observed in control cells.

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As temless linear probe was designed that robustly detects mRNAi nc ells with high sensitivity.T he probe is modified at some positions with base surrogates prepared from d-threoninol, with anthraquinone moieties near the 5'-and 3'termini, and with perylene moieties.E ven in cell lysate that involves various proteins and enzymes,b ackground emission was very low.W hen the probe was hybridized with RNA, chromophores are intercalated between the base pairs,resulting in ar emarkable light-up signal. The signal-to-background ratio was as high as 1600 under our standardbuffer conditions. In the HeLa cell lysate,t he linear probe had sufficient signalto-background ratio (S/B = 40) for reliable mRNAd etection. No degradation was observed after a24hincubation in HeLa cell lysate.Incells,aprobe designed to target DsRed resulted in distinct blue fluorescence only in cells transfected with plasmid encoding DsRed;n of luorescence was observed in control cells.