Improved Fluorescence in situ Hybridization of Individual Microbial Cells Using Polynucleotide Probes: The Network Hypothesis

Abstract

Fluorescence in situ hybridizations using polynucleotide transcript probes (poly-FISH) usually exhibit a ring-shaped halo or corona-like fluorescence signal, whereas hybridizations with oligonucleotide probes (oligo-FISH) result in a uniform and evenly distributed fluorescence throughout the cell. The superiority of poly-FISH in comparison to oligo-FISH regarding the signal intensity and the detection of cells with a low ribosome content suggested a further investigation of the possibilities of polynucleotide probes. Poly-FISH has previously only been described for bacterial cells. In the present study it could also be successfully applied to several yeast species. In addition to that the properties of polynucleotide probes were analyzed by using varying probe lengths and concentrations. This led to the formulation of a hypothesis to explain the characteristic "halo" signal observed with polynucleotide probes. This "network hypothesis" suggests the formation of a network of probes based on the secondary structure of the single stranded RNA probes. Due to the limited permeability of the cell envelope, only part of the probe is linked to its intracellular target site, while the remaining part is located-outside the cell and can form a network by hybridizing with single stranded probes, resulting in the ring-shaped fluorescence signal around the cell. The hypothesis was supported by a number of control experiments including in silico and in vitro analysis of the secondary structure of the probes and hybridizations with probes of defined secondary structures. The network concept provides a new basis for a wider application of poly-FISH.