Making all parts of the 16S rRNA of Escherichia coli accessible in situ to single DNA oligonucleotides

Abstract

rRNA accessibility is a major sensitivity issue limiting the design of working probes for fluorescence in situ hybridization (FISH). Previous studies empirically highlighted the accessibility of target sites on rRNA maps by grouping probes into six classes according to their brightness levels. In this study, a recently proposed mechanistic model of FISH, based on the thermodynamics of secondary nucleic acid interactions, was used to evaluate the accessibility of the 16S rRNA of Escherichia coli to fluorescein-labeled oligonucleotides when thermodynamic and kinetic barriers were eliminated. To cover the entire 16S rRNA, 109 probes were designed with an average thermodynamic affinity (DeltaGo (overall)) of -13.5 kcal/mol. Fluorescence intensity was measured by flow cytometry, and a brightness threshold between classes 3 and 4 was used as the requirement for proof of accessibility. While 46% of the probes were above this threshold with conventional 3-h hybridizations, extending the incubation period to 96 h dramatically increased the fraction of bright probes to 86%. Insufficient thermodynamic affinity and/or fluorophore quenching was demonstrated to cause the low fluorescence intensity of the remaining 14% of the probes. In the end, it was proven that every nucleotide in the 16S rRNA of E. coli could be targeted with a bright probe and, therefore, that there were no truly inaccessible target regions in the 16S rRNA. Based on our findings and mechanistic modeling, a rational design strategy involving DeltaGo(overall), hybridization kinetics, and fluorophore quenching is recommended for the development of bright probes.

FIG. 1.

Representation of the brightness values for 109 probes after 3 h of hybridization on the secondary structure of E. coli 16S rRNA. The scale used is equivalent to that of Fuchs et al. (17). The arrows indicate the bordering nucleotides between adjacent domains (i.e., positions 566, 912, and 1396).

FIG. 2.

Effect of hybridization time on probe brightness for selected probes.

FIG. 3.

Representation of the brightness values for 109 probes after 96 h of hybridization on the secondary structure of E. coli 16S rRNA. The scale used is equivalent to that of Fuchs et al. (17). The arrows indicate the bordering nucleotides between adjacent domains (i.e., positions 566, 912, and 1396).

FIG. 4.

(a) Relationship between affinity and brightness (96 h) for the original set of 109 probes. (b) Effect of increasing affinity on dim probes. (c) Effect of 5′ end elongation on presumably quenched probes. The dashed line indicates theoretical hybridization according to the previously described mechanistic model (50) for a probe concentration of 250 nM, when the lower and upper limits of brightness were taken as 0 and 100 CBU, respectively. Horizontal and vertical lines divide the plots into quadrants (Q1 through Q4). The numbers indicate probes shown in Fig. 5. Open circles, default data points; solid circles, probes not enhanced in brightness after initial elongation; solid squares, unsuccessful extensions; open squares, successful extensions. The error bars indicate standard deviations of the means.

FIG. 5.

Fluorescence intensities of probes with Cy3 labeling (striped columns) and fluorescein labeling (gray columns) as normalized by using the brightness of EUB338. The bars indicate the averages from two independent experiments, and the error bars indicate standard deviations. The numbers match the probes indicated in Fig. 4.

FIG. 6.

Frequency distributions of probes for the six brightness classes. The data sets included are the data sets from experiments with 3 h (gray bars) and 96 h (striped bars) of hybridization and the data set of Fuchs et al. (dotted bars) (17).

FIG. 7.

Putative kinetic accessibility map of 16S rRNA of E. coli based on the ratio of brightness with 3-h hybridizations to brightness with 96-h hybridizations. Light areas represent high 3-h/96-h ratios, while dark areas represent low 3-h/96-h ratios. Only the data from probes yielding brightness values of >40 CBU for 96-h hybridizations were used. Residues targeted by multiple probes were assigned the average 3-h/96-h ratio for all the probes. The arrows indicate the bordering nucleotides between adjacent domains (i.e., positions 566, 912, and 1396).