Development of thermodynamic models for simulating probe dissociation profiles in fluorescence in situ hybridization

Abstract

Stringency in ribosomal RNA (rRNA)-targeted fluorescence in situ hybridization (FISH) is typically adjusted with formamide, and the optimum formamide concentration at which the probe can hybridize with the target rRNA, but not with rRNAs with mismatches, is to be found experimentally. This is a difficult task when target or closest non-target organisms are not available in pure culture, or when there are numerous non-targets of concern. The objective of this work was to formulate mechanistic models capable of simulating the effect of formamide on probe dissociation. Using a previously described equilibrium model of FISH [Yilmaz and Noguera (2004) Applied and Environmental Microbiology 70(12):7126-7139] as the basis, the effect of formamide on free energy changes of probe–target duplex formation (ΔG) and folding of target region (ΔG) was simulated to be linear, and models differing in the definitions of the slopes of these relationships (m₁ and m₃) were calibrated using experimental dissociation profiles for 27 probes targeting the 16S rRNA of Escherichia coli (E. coli). A good level of predictive power was obtained when m₁ was linearly related to probe length and when m₃ was made proportional to ΔG. The effect of single mismatches on probe dissociation with formamide was also studied, although at a preliminary level. The expected changes in ΔG with the introduction of mismatches were not sufficient to capture the overall trends of mismatched dissociation profiles. In conclusion, this study offers the first theoretical method to calculate dissociation profiles for perfectly matched probes, and suggests a direction to systematically evaluate the effect of formamide on mismatched probes. Biotechnol. Bioeng. 2007;96: 349–363.