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 brightnes

SUBMITTER: Yilmaz LS 

PROVIDER: S-EPMC1352245 | biostudies-literature | 2006 Jan

REPOSITORIES: biostudies-literature