Design of molecular beacons for AmpliDet RNA assay. Characterization of binding stability and probe specificity

AmpliDet RNA is a real-time diagnostic method, the specificity of which is defined mainly by the molecular beacon (MB). MBs can be characterized according to the stability of their stem-and-loop structures and that of the probe–target duplex via the free energies accompanying their formation. By the application of thermodynamic models, we propose a prediction method for these ¿G0 parameters, which was compared to experimental analysis. The average absolute discrepancies for ¿G041 and for the melting temperatures of MB secondary structures were 0.30±0.26 kcal/mol and 2.15±1.5 °C, respectively. ¿G041 of probe–target interaction was predicted with a discrepancy of 1.2±1.0 kcal/mol. To characterize specificity, we formulated a model system with several MBs of highly similar sequence, but different lengths, and template RNAs carrying different types of mutations. We demonstrated the ability to detect a point mutation, or to tolerate one, irrespective of mismatch type. Of the nucleotide analogues tested, universal pyrimidine was found to increase MB tolerance substantially toward polymorphism. In the present study MBs were characterized under AmpliDet RNA conditions, with respect to probe stability, binding strength, and specificity, which led us to propose a design method, useful for probe design for AmpliDet RNA and adaptable to microarrays.