Mechanistic Approach to the Problem of Hybridization Efficiency in Fluorescent In Situ Hybridization

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 (⌬G°o verall ) 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 ⌬G°o verall 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 ⌬G°o verall of ؊13.0 kcal/mol was proposed as a goal in the design of FISH probes to maximize hybridization efficiency without compromising specificity.Fluorescent in situ hybridization (FISH) has proven to be a powerful molecular method for identification, visualization, and quantification of organisms of interest in microbial communities (reviewed in reference 2). Originally introduced by DeLong et al. (11), the use of FISH expanded to the study of bacterial populations in environmental applications (for reviews, see references 1, 2, and 34) and medical applications (22). Despite the wide use of FISH for more than a decade, the design and use of FISH probes remain a highly empirical procedure. Since the rRNA primary chains contain regions of variable conservation, it is straightforward to determine a target site on the rRNA with the desired level of specificity based on sequence comparisons (1, 34). However, a limitation that is difficult to overcome is the inability to predict if the signal intensity of cells hybridized with the fluorophore-labeled probe is high enough to be detectable (2,13,34).Low fluorescent responses in hybridized samples can be related to a variety of factors, such as low ribosome content of cells, difficulty in permeating cell walls, and the inaccessibility of target sites due to either the secondary and tertiary structures of the rRNA (i.e., RNA-RNA interactions) or the effect of ribosomal proteins (i.e., RNA-protein interactions) (2). The former two depend solely on the studied organism and can be potentially circumvented by changes in the experimental protocol, such as using a different fixative to better permeate the cell wall (e.g., the methods of Rolle...

Spatiotemporal dynamics of bacterial populations in the anoxic Cariaco Basin

“…For samples analyzed after the CAR-108 cruise, we replaced EPS549 with EPS682 due to higher hybridization efficiency of EPS682 (Gibbs free energy change 5 212.5 kcal mol 21 ) compared with EPS549 (Gibbs free energy change 5 29.0 kcal mol 21 ). Gibbs free energy change is a proxy for probe hybridization efficiency and was calculated according to Yilmaz and Noguera (2004). Usually, a threshold value of 213.0 kcal mol 21 Gibbs free energy change is recommended for effective probe hybridization.…”

Making All Parts of the 16S rRNA of Escherichia coli Accessible In Situ to Single DNA Oligonucleotides

“…While studies addressing the significance of the former factor in FISH have just started (5), the latter factor has been investigated extensively (7,(16)(17)(18)(19)50). The ability of probes to reach their target sites within the three-dimensional structure of the ribosome has been regarded as the major factor for hybridization efficiency (3).…”

Rapid Quantitative Detection of Salmonella enterica Using Fluorescence In Situ Hybridization with Filter-cultivation (FISHFC) Method

“…Amann et al (1995) reported that 23S rRNA was often used as a hybridization target molecule, because its sequences showed higher phylogenetic specificity than 16S rRNA sequences. Yilmaz and Noguera (2004) reported that fluorescence intensity was negatively correlated to the Gibbs free energy change (ΔG) in hybridization between the DNA probe and rRNA. Sawabe et al (2009) showed that greater brightness from the hybridized probes was obtained when V. parahaemolyticus was hybridized with probes in which ΔG ranged from −17 to −22; therefore, SAL343 was designed accordingly.…”

“…The oligonucleotide probe was optimized for Gibbs's free energy of DNA-RNA hybridization (Yilmaz and Noguera, 2004 Alexa 488 ® ) and images were captured using a digital camera D50 (Nikon). Micro-colony diameter was determined from digital images using ImageJ ver.…”