Design of Fluorescently Labeled Oligonucleotide Probes

3.1.1. Fluorescently Labeled Oligonucleotide Probes

A pair of probes with sequences complementary to the target RNA and labeled with a donor fluorophore (such as BODIPY 493/503) and an acceptor fluorophore (Cy5), respectively, were used (see Subheading 3.1.5.). These probes act as both donor and acceptor when irradiated with blue light by FRET, and exhibit red fluorescence only after hybridization with complementary target RNA molecules next to each other. Equal or excessive amounts of probes should be added for quantitative hybridization. Because an excessive amount of acceptor probe results in an artifact FRET signal, the final concentration of probes, which would depend on the concentration of the target RNA molecules and the sensitivity of detector devices (a spectrof-luorometer or a C-CCD camera mounted on a microscope), should be determined experimentally.

The design of the probe (including target position, length, and label-position) directly affects the intensity of the FRET signal. This characteristic is typical of FRET probes, and there is no special requirement for the application of double-labeled probes. We recommend that some sets of probes should be designed based on the criteria described in Subheading 3.1.2.-3.1.5. and be examined for FRET efficiency by solution hybridization experiments. FRET results in an increased acceptor emission and a decrease in donor emission fluorescence. The ratio of the intensity of the emitted fluorescence, from the acceptor as FRET signals, to the intensity of the donor was close to one or more in optimized probe sets.

3.1.2. Length of Probe

Oligonucleotides with a melting temperature of 35-45°C (ca. 15 nucleotides) are suitable for this method. The lower the melting temperature of the probes, the faster the probes hybridized with the target RNA. In preliminary time course measurements, the fluorescence intensity of the acceptor reached a maximum within 15-30 min at room temperature after hybridization of the probes with the target RNA.

Fig. 2. (A) Organization of the tomato mosaic virus (ToMV) genome (6384 nucleotides), and the targeted positions of a series of fluorescence resonance energy transfer (FRET) probe sets (shown as horizontal bars) examined in this study. The circle indicates the cap structure at the 5'-end. The 126K and 183K proteins are components of the viral replication proteins. MP, movement protein; CP, coat protein. (B) Probe sets around the first start codon (aug: nt 72-74) examined in detail. A partial sequence of ToMV RNA is shown; horizontal bars indicate the target positions for each probe set. (C) FRET efficiency of each probe set hybridizing with target RNA in the solution hybridization study. Each set was comprised of a probe labeled with BODIPY 493/ 503 and a probe labeled with Cy5 (see Note 4). Excited at 480 nm, emitted fluorescence was measured with a spectrofluorometer after 15 min of hybridization at room temperature. The solid columns represent the mean value of five independent experiments and the error bars indicate the standard deviation. Probe sets (donor and acceptor pairs) used in this experiment are (further details shown in ref. 10): (1) L(55-70)D and L(71-85)A; (2) L(62-76)D and L(77-91)A; (3) L(66-80)D and L(81-94)A; (4) L(73-87)D and L(88-100)A; (5) L(88-100)D and L(101-114)A; (6) L(3391-3406)D and L(3407-342)A; (7) L(4891-4907)D and L(4908-4922)A; (8) L(4906-4920)A and L(4921-4937)A; (9) L(5679-5681)D and L(5682-5696)A; (10) L(5703-5718)D and L(5719-5732)A; (11) L(6155-6167)D and L(6168-6181)A. The number of each probe name indicates the target position in the ToMV genome. D, single-labeled donor probe; A, single-labeled acceptor probe.

3.1.3. Target Positions

A set of DNA probes should be designed to hybridize next to one another on the target RNA (mRNA, viral RNA). There are two points for determining where the target position is (1) a specific region long enough for two probes; and (2) a sequence that is unlikely to form a strong secondary structure.A clear position effect was observed with the probe sets for ToMV, which we previously developed (Fig. 2). The gene organization of ToMV RNA and the target position of the probes examined are shown in Fig. 2A,B. FRET efficiency of the probe sets (the ratio of acceptor fluorescence to donor fluorescence) in solution hybridization experiments indicated that position-2 (nucleotides [nt] 62-91 in the genomic sequence; around the start codon of the 126 kD and 183 kD replication proteins) was an suitable target (Fig. 2C). These regions are considered to be ribosome-binding sites with little secondary structure (11). The site immediately adjacent to the cap structure may not be ideal, because several translation factors bind around cap structures and hinder hybridization with exogenous probes. We have also screened for probe sets specific to OPV at the 5'-noncoding region of the viral genome. We found the optimal position (40-71 nt) was immediately upstream of the start codon (69-71 nt). Together, these results indicate that a sequence unlikely to form a secondary structure is found in a suitable position for targeting by hybridization of the FRET probe. These results seemed reasonable since single-stranded regions, such as those in loop structures, are generally suitable for hybridization to functional oligo-nucleotides (12).

These optimized FRET probe sets could clearly discriminate between two closely related tobamoviruses in solution hybridization experiments. Although the degree of sequence homology between two viruses was relatively high (Fig. 3A), no crossreactions were observed (Fig. 3B). The probe sequences used as specific probe sets for discrimination were as follows: (1) for ToMV, L(62-76)DD (5'-BODIPY 493/503-g-BODIPY 493/503-ccattgtagttgta-3') and L(77-91)A (5'-gctgtttgtgtgtat-Cy5-3'); (2) for OPV: O(40-55)DD (5'-BODIPY 493/ 503-t-BODIPY 493/503-gcaaatgttgtttgt-3') and O(56-71)A (5'-cattgtagttgtatgt-Cy5-3').

3.1.4. Label Position of Probe

This FRET enhancement method requires doubling the label-number of a donor probe with fluorophores (see Note 4). Because FRET efficiency is substantially affected by the labeled-position (see Note 5), it is important for the donor and acceptor molecules to be closely aligned. We also examined the

Fig. 3. (A) Target positions of fluorescence resonance energy transfer (FRET) probe sets specific for tomato mosaic virus (ToMV) or Obuda pepper virus (OPV). The number of each probe name indicates the target position in the ToMV or OPV genome. Partial sequences of ToMV and OPV genomic RNA are aligned. The numbers at both ends of sequences are the nucleotide numbers from the 5'-end and asterisks indicate matches between the two viruses. Boxes indicate the positions of the donor and acceptor probes for ToMV- or OPV-specific FRET probes. (B) Specificity of the FRET probe sets for two tobamoviruses. Two sets of FRET probes (specific for ToMV or OPV) were hybridized with ToMV or OPV RNA (purified in vitro transcripts) in a 1X SSC buffer. Excited at 480 nm, emitted fluorescence of the acceptor (665 nm) was measured 30 min after hybridization using a spectrofluorometer (F-4500, Hitachi). Fluorescence in the absence of RNA (-) was also estimated. This represents the direct fluorescence of a nonhybridized acceptor probe. The solid columns represent the mean values of five independent experiments and the error bars indicate standard deviations. DD, doublelabeled donor probe; A, single-labeled acceptor probe.

Fig. 3. (A) Target positions of fluorescence resonance energy transfer (FRET) probe sets specific for tomato mosaic virus (ToMV) or Obuda pepper virus (OPV). The number of each probe name indicates the target position in the ToMV or OPV genome. Partial sequences of ToMV and OPV genomic RNA are aligned. The numbers at both ends of sequences are the nucleotide numbers from the 5'-end and asterisks indicate matches between the two viruses. Boxes indicate the positions of the donor and acceptor probes for ToMV- or OPV-specific FRET probes. (B) Specificity of the FRET probe sets for two tobamoviruses. Two sets of FRET probes (specific for ToMV or OPV) were hybridized with ToMV or OPV RNA (purified in vitro transcripts) in a 1X SSC buffer. Excited at 480 nm, emitted fluorescence of the acceptor (665 nm) was measured 30 min after hybridization using a spectrofluorometer (F-4500, Hitachi). Fluorescence in the absence of RNA (-) was also estimated. This represents the direct fluorescence of a nonhybridized acceptor probe. The solid columns represent the mean values of five independent experiments and the error bars indicate standard deviations. DD, doublelabeled donor probe; A, single-labeled acceptor probe.

effect of the label-positions of the FRET probes in a variety of probe combinations (9). This tendency was more remarkable in double-labeled donor probes; although the results suggest a special interaction between the two donor fluorophores; most combinations labeled at positions near the ends produced relatively high and similar levels of FRET efficiency (Table 1). With these combinations, the effect of the labeled-positions is not so apparent. Based on this, we usually designed double-labeled donor probes labeled at the 5'-end

Table 1

Effect of Label-Position of FRET Probes

Table 1

Effect of Label-Position of FRET Probes

Acceptor probe

Double-labeled donor probe

Relative FRET efficiency

5'-gctgtttgtgtgt-A-3'

5'-D-D-ccattgtagttgta-3'

100

5'-gctgtttgtgtgt-A-3'

5'-D-g-D-cattgtagttgta-3'

96.8

5'-gctgtttgtgtgt-A-3'

5'-D-gc-D-attgtagttgta-3'

68.5

5'-gctgtttgtgtgt-A-3'

5'-D-gcc-D-ttgtagttgta-3'

38.7

5'-gctgtttgtgtgt-A-3'

5'-D-gcca-D-tgtagttgta-3'

86.6

5'-gctgtttgtgtgt-A-3'

5'-gccattgtagttgta-3'

0

ToMV-specific probes were labeled with BODIPY 493/503 (for double-labeled donor) or Cy5 (for acceptor) at various positions. Comparison of FRET efficiency in solution hybridization experiment. Relative value of acceptor fluorescense measure 15 min after hybridization with target RNA (in vitro ToMV transcripts). "D" and "A" indicated the substitution nucleotide by a linker spacer with a "donor" or an "acceptor fluorophore," respectively. FRET efficiency was found to be substantially affected by the labeled (substituted)-position.

ToMV-specific probes were labeled with BODIPY 493/503 (for double-labeled donor) or Cy5 (for acceptor) at various positions. Comparison of FRET efficiency in solution hybridization experiment. Relative value of acceptor fluorescense measure 15 min after hybridization with target RNA (in vitro ToMV transcripts). "D" and "A" indicated the substitution nucleotide by a linker spacer with a "donor" or an "acceptor fluorophore," respectively. FRET efficiency was found to be substantially affected by the labeled (substituted)-position.

and the position between the "first and second" or "second and third" nucleotides, and an acceptor labeled at the 3'-end.

3.1.5. Fluorophore for Labeling

BODIPY 493/503 is recommended as the standard donor fluorophore because it has a stable, strong, and narrow-width fluorescence spectrum, and is not susceptible to crosstalk with the acceptor fluorescence. We have shown previously that a combination of a donor probe double-labeled with BODIPY 493/503 and an acceptor probe labeled with Cy5 (see Note 6) gave the best data on fluorophores (9).

In a comparison of seven donor fluorophores, BODIPY 493/503 doublelabeled probes produced the greatest enhancement of FRET (Fig. 4). The BODIPY dye BODIPY FL (BODIPY 503/512) had almost the same effect (data not shown). Fluorophores other than TRITC and Alexa 488 improved FRET. Alexa 488, which had the strongest FRET efficiency in experiments using a single-labeled donor probe, negatively affected enhancement when used with double-labeling donor probes.

3.1.6. Synthesis of Fluorescently Labeled Oligonucleotide Probes

Synthesize amine-modified oligonucleotides with inserted amino-linker spacers by an automatic DNA/RNA synthesizer, and label the products with fluorescent derivatives at designated positions. The oligonucleotide modified by amino-linkers can subsequently be labeled with the amine-reactive dye for

Bodipy 493/503

Alexa 488

FITC

Oregon Green 488

Rhodamine Green

Cy3 TRITC

Ratio of acceptor fluorescences (with RNA / without RNA)

Fig. 4. Comparison of donor fluorophores for improved fluorescence resonance energy transfer (FRET) efficiency. Specific probe sets for tomato mosaic virus (see Fig. 1) were double- or single-labeled with a variety of fluorophores. Cy5-labeled oligo-nucleotide was used as an acceptor probe. FRET efficiency was measured with target viral RNA (in vitro transcripts). The enhancement ratios of acceptor fluorescence (665 nm) with RNA to that without RNA were determined. The solid columns represent the ratio of the mean values of five independent experiments and the error bars indicate standard deviations. The donor probes labeled with various fluorophores used in this experiment are Alexa 488, BODIPY 493/503, FITC, Oregon Green 488, Rhodamine Green, Cy3, and TRITC.

the acceptor (FluoroLink Cy5 Mono Reactive Dye, Amersham Biosciences) and for donor (amine-reactive BODIPY 493/503, Molecular Probes). Use the linker for 5'-terminus labeling and the linker for 3'-terminus labeling for respective 5' and 3' termini labeling. The labeled oligonucleotides should be purified with reverse-phase high-performance liquid chromatography.

Bodipy 493/503

Alexa 488

FITC

Oregon Green 488

Rhodamine Green

Cy3 TRITC

Ratio of acceptor fluorescences (with RNA / without RNA)

Fig. 4. Comparison of donor fluorophores for improved fluorescence resonance energy transfer (FRET) efficiency. Specific probe sets for tomato mosaic virus (see Fig. 1) were double- or single-labeled with a variety of fluorophores. Cy5-labeled oligo-nucleotide was used as an acceptor probe. FRET efficiency was measured with target viral RNA (in vitro transcripts). The enhancement ratios of acceptor fluorescence (665 nm) with RNA to that without RNA were determined. The solid columns represent the ratio of the mean values of five independent experiments and the error bars indicate standard deviations. The donor probes labeled with various fluorophores used in this experiment are Alexa 488, BODIPY 493/503, FITC, Oregon Green 488, Rhodamine Green, Cy3, and TRITC.

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