Mechanistic approach to the problem of hybridization efficiency in fluorescent in situ hybridization - PubMed (original) (raw)

Mechanistic approach to the problem of hybridization efficiency in fluorescent in situ hybridization

L Safak Yilmaz et al. Appl Environ Microbiol. 2004 Dec.

Abstract

In fluorescent in situ hybridization (FISH), the efficiency of hybridization between the DNA probe and the rRNA has been related to the accessibility of the rRNA when ribosome content and cell permeability are not limiting. Published rRNA accessibility maps show that probe brightness is sensitive to the organism being hybridized and the exact location of the target site and, hence, it is highly unpredictable based on accessibility only. In this study, a model of FISH based on the thermodynamics of nucleic acid hybridization was developed. The model provides a mechanistic approach to calculate the affinity of the probe to the target site, which is defined as the overall Gibbs free energy change (DeltaG degrees overall) for a reaction scheme involving the DNA-rRNA and intramolecular DNA and rRNA interactions that take place during FISH. Probe data sets for the published accessibility maps and experiments targeting localized regions in the 16S rRNA of Escherichia coli were used to demonstrate that DeltaG degrees overall is a strong predictor of hybridization efficiency and superior to conventional estimates based on the dissociation temperature of the DNA/rRNA duplex. The use of the proposed model also allowed the development of mechanistic approaches to increase probe brightness, even in seemingly inaccessible regions of the 16S rRNA. Finally, a threshold DeltaG degrees overall of -13.0 kcal/mol was proposed as a goal in the design of FISH probes to maximize hybridization efficiency without compromising specificity.

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Figures

FIG. 1.

FIG. 1.

Proposed reaction scheme for FISH. P, R, and PR denote the DNA probe, the target (rRNA), and the DNA/rRNA hybrid, respectively. The subscripts f and u represent folded and unfolded conformations.

FIG. 2.

FIG. 2.

Relation between normalized fluorescence intensity of fluorescein-labeled probes (13) and Δ_G_°overall. (a) Complete set of 176 probes; (b) probes targeting sites relatively free of protein contact; (c) all 176 probes when Δ_G_°3 estimates were mixed to maximize r2. The solid diamond, circle, and square symbols denote probes Eco 1014, Eco 1184, and Eco 1274, respectively.

FIG. 3.

FIG. 3.

Fluorescence intensity as a function of probe affinity for probes from Fuchs et al. (13) targeting helix 6 (open circles, solid regression line) and helix 18 (solid squares, dashed regression line). The dotted curve represents equation 9 fitted to the data by assuming that a [PR_]/_[_R0_] ratio of 1 corresponds to 75% fluorescence and a [PR_]/_[_R0_] ratio of 0 represents 3% fluorescence in Fuchs et al.'s normalized scale.

FIG. 4.

FIG. 4.

Probe brightness as a function of probe affinity in a subregion of helix 9. The dotted curve represents equation 9 fitted to the data by assuming that [PR_]/_[_R0_] values of 1 and 0 corresponded to 1.05 and 0.1 brightness units, respectively.

FIG. 5.

FIG. 5.

Effect of increasing the magnitude of Δ_G_°overall-i on the fluorescence intensity of probes targeting sites located in helix 9 (circles), helices 37 and 38 (squares), helices 39, 43,and 45 (triangles), and helix 46 (diamonds). The data points (except solid circles) are from Table 1. Solid arrows indicate the trend for each target site. The dotted arrow shows the effect of using 30% formamide in the hybridization of E1177. Data points represented by solid circles are from Fuchs et al. (13), and the dashed line shows the equivalency to the lower limit of class 3 in Fuchs et al.'s scale.

FIG. 6.

FIG. 6.

Effect of hybridization time on brightness of probes targeting helices 39, 43, and 45. Data points show averages and standard deviations of duplicate hybridizations. Probe E146 was used as a control.

FIG. 7.

FIG. 7.

Effect of formamide concentration on brightness of probes targeting helices 39, 43, and 45. Data points show averages and standard deviations of two independent experiments. Probe E146 was used as a control.

FIG. 8.

FIG. 8.

Venn diagram representation of the relation among the factors affecting fluorescence intensity in FISH.

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