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 (⌬G o 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 ⌬G o overall , hybridization kinetics, and fluorophore quenching is recommended for the development of bright probes.

Endosymbiotic Bacteria in the Parasitic Ciliate Ichthyophthirius multifiliis

“…The probes were fluorescently labeled at their 5Ј termini with Cy3 or Cy5 (Integrated DNA Technologies). The probes were designed to target regions of limited secondary structure in 16S rRNA, based on the structure of E. coli 16S rRNA (33). In each case, two complementary probes were synthesized: one complementary to the 16S rRNA sequence and a second with the same sequence as the transcribed 16S rRNA, which served as a negative control to detect nonspecific binding.…”

Improved In Situ Hybridization Efficiency with Locked-Nucleic-Acid-Incorporated DNA Probes

“…Based on the affinity concept, the probes were elongated to satisfy the ⌬G°o verall value for efficient hybridization. Consequently, over 90% of rRNA-targeted probes yielded moderate to high brightness after hybridization reached equilibrium (43). However, elongating probes in order to increase affinity may restrict the flexibility of probe design and decrease mismatch discrimination ability.…”

Comparison of Fecal Indicator Bacterial Populations in Surface Waters of the Kalamazoo River, USA

“…However, they are comparatively inadequate for bacterial quantification, since natural bacterial populations are often difficult to grow with only a small fraction of natural assemblages actually cultivable (2, 12). In contrast, FISH since its advent as a powerful molecular tool, has been effectively utilized for the enumeration of individual bacterial cells in complex environments (2,15,34), and even though relatively fast and effective, some major problems have been often associated with its optimization and application (14,28).Triplicate water samples were collected weekly from the air-water interface (~0.2 m below the surface) along the river edges in sterile 250 mL polypropylene bottles, during spring run-off through midsummer (May 15th to July 24th 2007) from two sites along the north branch of the Kalamazoo River (42°12'55.152N, −84°48'24.908W, Fig. S1).…”