A comprehensive model for the diffusion and hybridization processes of nucleic acid probes in fluorescence in situ hybridization

Fluorescent in situ hybridization (FISH) has been extensively used in the past decades for the detection and localization of nucleic acid sequences or of the microorganisms themselves within samples. However, a mechanistic approach of the whole FISH process is still missing, and the main limiting steps for the hybridization to occur remain unclear. In here, FISH is approached as a particular case of a diffusion-reaction kinetics, where molecular probes move from the hybridization solution to the target RNA site within the cells. Based on literature models, the characteristic times taken by different molecular probes to diffuse across multiple cellular barriers, and the reaction time associated with the formation of the duplex molecular probe-RNA were estimated. Structural and size differences at the membrane level of bacterial and animal cells were considered. For bacterial cells, the limiting step for diffusion is likely to be the peptidoglycan layer (characteristic time of 2700-4524 s), whereas for animal cells the limiting step should be the diffusion of the probe through the bulk (1.8-5.0 s) followed by the diffusion through the lipid membrane (1 s). The information provided here may serve as a basis to optimize FISH protocols.