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. 2000 Aug;66(8):3603-7.
doi: 10.1128/AEM.66.8.3603-3607.2000.

Unlabeled helper oligonucleotides increase the in situ accessibility to 16S rRNA of fluorescently labeled oligonucleotide probes

B M Fuchs  1 , F O Glöckner, J Wulf, R Amann
Affiliations

Unlabeled helper oligonucleotides increase the in situ accessibility to 16S rRNA of fluorescently labeled oligonucleotide probes

B M Fuchs et al. Appl Environ Microbiol. 2000 Aug.

Abstract

Target site inaccessibility represents a significant problem for fluorescence in situ hybridization (FISH) of 16S rRNA with oligonucleotide probes. Here, unlabeled oligonucleotides (helpers) that bind adjacent to the probe target site were evaluated for their potential to increase weak probe hybridization signals in Escherichia coli DSM 30083(T). The use of helpers enhanced the fluorescence signal of all six probes examined at least fourfold. In one case, the signal of probe Eco474 was increased 25-fold with the use of a single helper probe, H440-2. In another case, four unlabeled helpers raised the FISH signal of a formerly weak probe, Eco585, to the level of the brightest monolabeled oligonucleotide probes available for E. coli. The temperature of dissociation and the mismatch discrimination of probes were not significantly influenced by the addition of helpers. Therefore, using helpers should not cause labeling of additional nontarget organisms at a defined stringency of hybridization. However, the helper action is based on sequence-specific binding, and there is thus a potential for narrowing the target group which must be considered when designing helpers. We conclude that helpers can open inaccessible rRNA regions for FISH with oligonucleotide probes and will thereby further improve the applicability of this technique for in situ identification of microorganisms.

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Figures

FIG. 1
FIG. 1
Fluorescence conferred by probes and probe-helper combinations (probes are in boldface; addition of helper is indicated by +Hxxx) on whole fixed cells of E. coli DSM 30083T. Relative fluorescence intensities are standardized to the brightest probe, Eco1482. The secondary structure according to Gutell (11) of the respective target region on the 16S rRNA is shown. Different colors indicate the different brightness classes I through VI (10). The binding sites of helpers are shown in gray.
FIG. 2
FIG. 2
Influence of helper oligonucleotides on probe binding at different theoretical temperatures. The lines show the sigmoid melting curves calculated from the respective data points. The values in parentheses are excluded from the fitting procedure. a.u., arbitrary units. The error bars indicate standard deviations calculated from triplicate measurements. (A) E. coli hybridized with Eco603 (solid circles and solid line), Eco603-H621 (open circles and dotted line), and Eco603-H621L (triangles and dashed line). (B) E. coli hybridized with Eco711 (solid circles and solid line), Eco711-H729 (open circles and dotted line), and Eco711-H745 (triangles and dashed line). (C) A. brasilense hybridized with Eco603-H621azo (inverted triangles and dashed line) and Eco603-H621 (open circles and dotted line) and E. coli hybridized with Eco603x-H621L (triangles and dashed-dotted line), Eco603-H621azo (solid circles and solid line), and Eco603-H621azoL (squares and dashed–double-dotted line).
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