Materials 21 Probe Labeling

1. Inserted amino-linker spacer: Uni-Link™ AminoModifier (BD-Clontech, Palo Alto, CA).

2. Linker for 5'-terminus labeling: TFA aminolinker (Applied Biosystems, Foster City, CA).

3. Linker for 3'-terminus labeling: 3'-amino-modifier C7 CPG (Glen Research, Sterling, VA).

4. Amine-reactive dye for accepter: FluoroLink™ Cy5 Mono Reactive Dye (Amersham Biosciences, Piscataway, NJ).

5. Amine-reactive dye for donor: amine-reactive BODIPY 493/503 (Molecular Probes, Eugene, OR).

Fig. 1. The effect on fluorescence resonance energy transfer (FRET) of the number of labeled fluorophores of Obuda pepper virus (OPV) specific probe sets. Doublelabeled donor probes (DD) resulted in a remarkable increase in fluorescence intensity compared with ordinary FRET donor probes (D). Different combinations of OPV specific probe sets were hybridized with OPV RNA (purified in vitro transcripts). Excited at 480 nm, emitted fluorescence was measured with a spectrofluorometer 15 min after hybridization. (A) Relative fluorescence spectra. (B) Normalized fluorescence spectra (normalized at 515 nm), showing the ratio of donor fluorescence vs FRET. It is important for the interpretation of the ratio-imaging data. DD, doubel-labeled donor probe; D, single-labeled donor probe; AA, double-labeled acceptor probe; A, single-labeled acceptor probe.

Fig. 1. The effect on fluorescence resonance energy transfer (FRET) of the number of labeled fluorophores of Obuda pepper virus (OPV) specific probe sets. Doublelabeled donor probes (DD) resulted in a remarkable increase in fluorescence intensity compared with ordinary FRET donor probes (D). Different combinations of OPV specific probe sets were hybridized with OPV RNA (purified in vitro transcripts). Excited at 480 nm, emitted fluorescence was measured with a spectrofluorometer 15 min after hybridization. (A) Relative fluorescence spectra. (B) Normalized fluorescence spectra (normalized at 515 nm), showing the ratio of donor fluorescence vs FRET. It is important for the interpretation of the ratio-imaging data. DD, doubel-labeled donor probe; D, single-labeled donor probe; AA, double-labeled acceptor probe; A, single-labeled acceptor probe.

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