DEVELOPMENT OF A MECHANISTIC MODEL FOR FLUORESCENT IN SITU HYBRIDIZATION (FISH) BASED ON EQUILIBRIUM THERMODYNAMICS

The use of oligonucleotide probes for monitoring bacterial populations in environmental samples with fluorescent in-situ hybridization (FISH) requires the evaluation of hybridization conditions to balance stringency and specificity. This is a difficult task when discrimination of targeted organisms from those with only a few mismatches is necessary. Conventionally, the stringency is adjusted by altering the concentration of formamide in the hybridization buffer, so that there is minimum interference from mismatched organisms. Since there are no reliable theoretical or empirical methodologies for this optimization, probe design has been restricted to experimental trial and error procedures. In this study, we developed a thermodynamics based mathematical model to predict formamide dissociation profiles of probes used in FISH protocols. A hybridization reaction scheme including not only the binding of the DNA probe to the RNA target (Reaction 3), but also the self-folding of both the probe (Reaction 1) and the target region (Reaction 2) was adopted. The free energies of each reaction in this system were predicted for hybridizations without formamide using the computational tools mfold and Hyther™ and adjusted to varying formamide concentrations using an experimentally determined factor (i.e. m-value) that linearly relates free energy to the formamide percentage. Then normalized denaturation curves were calculated using the rules of equilibrium chemistry. Hybridization of the probe Nso 190 to its perfect complementary DNA oligonucleotide showed that the model could accurately predict the dissociation curve when the secondary structures of the probe and the target were insignificant. The model was then calibrated for FISH using seven linear probes with a pure culture of Escherichia coli. Best fitting m-values of 0.33 kJ/mol-% for Reaction 2 (m2) and 0.39 kJ/mol-% for Reaction 3 (m3) adequately described the effect of formamide on hybrid stability, yielding theoretical curves with ≤5 % error at the melting formamide concentration. Additional analyzes showed that the accuracy level could increase by determining local m-values representing the effect of formamide on specific regions of the 16S-rRNA molecule.